Tumor high affinity peptide YQP-3 and application thereof

Abstract

The application discloses a tumor high-affinity peptide YQP-3 and application thereof, the high-affinity peptide can be specifically combined with various tumor cells, preferably breast cancer, liver cancer and colorectal cancer. The high-affinity characteristic of the targeting peptide can be used for optical imaging and nuclear medical imaging of malignant tumors. The high-affinity peptide monomer directly or indirectly coupled with a fluorescent dye can be used as a tumor-specific targeting molecular probe, and the effect of accurate positioning of a tumor boundary can be achieved, and real-time image navigation before and during operation can be brought, and the surgical accuracy can be improved.

Description

[0001] Case Analysis

[0002] This invention is a divisional application with application number 2021112337463, application date 2021-10-22, and invention title "Several high-affinity peptides for tumors and their applications". Technical Field

[0003] This invention belongs to the fields of bioengineering and pharmaceutical technology and protein and polypeptide drugs and biomedical engineering, specifically involving tumor-targeting peptides and their applications, such as in tumor diagnosis, intraoperative navigation and tumor treatment. Background Technology

[0004] Cancer has become a major threat to human health and life, making early diagnosis and effective treatment of cancer particularly important and urgent. Conventional imaging techniques for cancer diagnosis mainly include ultrasound, CT, and MRI. These techniques diagnose by displaying functional changes in tissues and have good application value, but they still have certain limitations in differential diagnosis, systemic staging, and early efficacy evaluation. Undeniably, screening and optimizing peptides targeting tumors is a new approach that can develop novel molecular imaging drugs for cancer diagnosis, staging, and surgical guidance, enabling the detection of even smaller lesions and achieving early diagnosis.

[0005] The cadherin family is a class of transmembrane glycoproteins that mediate calcium-dependent cell adhesion. Functionally, cadherin-mediated adhesion regulates cell growth and differentiation. Cadherins mediate cell-cell adhesion by forming complexes with catecholamines, maintaining tissue stability. Due to the crucial roles of cadherins in cell recognition, adhesion, and signal transduction, investigating their expression changes and functional roles in cancer development is of great significance. Altered P-cadherin expression has been detected in various human malignant tumor tissues, and its expression is closely related to the occurrence and development of malignant tumors. The effects of P-cadherin on tumors vary in different tumor types. For example, in bladder, colon, breast, and pancreatic cancers, P-cadherin promotes tumor development and metastasis.

[0006] Cyanide dyes possess advantages such as small molecular weight, low toxicity, wide tunable wavelength range, and high molar extinction coefficient, making them widely used in the field of fluorescent labeling. Modifying the structure of cyanide dyes to attach active reactive groups allows them to react with the amino or carboxyl groups of specific target molecules such as antibodies, proteins, short peptides, and small molecules to form stable covalent bonds, creating specific targeting molecular probes for in vivo fluorescent molecular imaging—an important application of near-infrared fluorescent dyes. Single-photon emission tomography / computerized tomography (SPECT / CT) is a novel nuclear medicine imaging technique developed and widely adopted in clinical practice over the past 20 years. It primarily utilizes short-half-life radionuclides to label specifically targeted ligands for tracking and imaging, displaying information such as in vivo metabolism, cell proliferation, and receptor distribution, for disease diagnosis and research on human life activities. Therefore, specifically targeted ligands are crucial for fluorescence imaging and radionuclide imaging.

[0007] Based on the above considerations, the applicant has designed a novel class of tumor-targeting peptides. These peptides specifically target P-cadherin in tumor tissue. Coupled with fluorescent dyes, they enable optical imaging to assist surgeons in precisely locating tumor boundaries during surgery using molecular imaging-guided surgical equipment, thereby achieving accurate tumor resection, reducing patient trauma, and lowering the risk of postoperative recurrence. Furthermore, these targeting peptides can also be coupled with radionuclides for radionuclide imaging, enabling early diagnosis and treatment of tumors. Summary of the Invention

[0008] The primary objective of this invention is to provide several novel, tumor-specific targeted peptides and their sequences.

[0009] Another object of the present invention is to provide methods for preparing several tumor-specific targeting fluorescent probes;

[0010] Another object of the present invention is to provide methods for preparing several tumor-specific targeted radioactive probes;

[0011] Another object of the present invention is to provide several applications of the described probes in optical and SPECT imaging.

[0012] The above-mentioned objectives of the present invention can be achieved through the following technical solutions:

[0013] Tumor-specific targeting peptides, selected from any of the following:

[0014] The tumor-targeting peptide YQP-1 has the sequence Hyp-Ser-Asp-Asn-Tyr-Thr-NH2.

[0015] The tumor-targeting peptide YQP-2 has the sequence Glu-Nle-Gly-Hyp-Ser-Asp-Asn-Tyr(3-I)-Thr-NH2;

[0016] The tumor-targeting peptide YQP-3 has the sequence Hyp-Ser-Asp-Asn-Tyr(3-I)-Thr-NH2;

[0017] The tumor-targeting peptide YQP-4 has the sequence Glu-Ile-Asp-Pro-Ser-Asp-Asn-Tyr-Thr-Tyr-Tyr-Asn-Gln-Asn-Phe-Lys-Gly;

[0018] The tumor-targeting peptide YQP-5 has the sequence Cys-Pro-Ser-Asp-Asn-Tyr-Thr-Cys 1-7, a disulfide ring;

[0019] The tumor-targeting peptide YQP-6 has the sequence Phe-Thr-Ala-Tyr-Asn-Gly-Tyr-Tyr-Asp-Gly-Gly-Phe-NH2.

[0020] Wherein: Hyp is hydroxyproline, Tyr(3-I) is iodotyrosine, and Nle is ortholeucine.

[0021] The tumor-targeting peptide YQP-X (X = 1-6) described in this invention can be synthesized by a biotechnology company or prepared using conventional techniques in the field.

[0022] The solid-phase synthesis method of polypeptide YQP-1 is as follows:

[0023] 1) Resin swelling

[0024] Weigh out RinkAmide MBHA resin and place it in the reaction column. Add an appropriate amount of dichloromethane (DCM) and gently purge with nitrogen gas for 10-30 minutes to allow the resin to fully expand. Remove the DCM solution, wash three times with DMF, and then dry.

[0025] 2) Removal of Fmoc

[0026] Add 20% hexahydropyridine DMF solution to the reaction column and deprotect it every 5 minutes, then every 8 minutes. After the reaction is complete, wash the column 6 times with DMF.

[0027] 3) Coupling

[0028] Accurately weigh three times the molar amount of Fmoc-Thr(trt)-OH and O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU) into DMF, completely dissolve them, add N,N-diisopropylethylamine (DIPEA) to activate the carboxyl group, then add the solution to the reaction column for reaction. Detect the structure after 1 hour; a positive result is sufficient. Decouple and remove Fmoc sequentially from the C-terminus to the N-terminus until the last amino acid, Fmoc-Hyp-OH, is coupled. After removing Fmoc, shrink the target resin peptide and weigh it.

[0029] 4) Pyrolysis

[0030] Prepare a lysis buffer consisting of 87.5% TFA + 5% anisole + 2.5% ethylenedithiol + 2.5% phenol + 2.5% water. Under low temperature conditions, slowly add the lysis buffer to the resin peptide. The volume of the lysis buffer should be 7-8 times the weight of the crude peptide resin. After stirring slowly for 2 hours, filter to obtain the liquid. Add ice-cold ether and stir. Then centrifuge to obtain the solid. Wash three times with ether, dry under vacuum, weigh, and determine the mass-to-charge ratio to determine the molecular weight.

[0031] 5) Purification and separation

[0032] Purification was performed using high performance liquid chromatography (HPLC). The chromatographic packing material was a 10 μm reversed-phase C18 column, and the mobile phase system was 0.1% TFA / water solution-0.1% TFA / acetonitrile solution. Gradient elution was used, and the sample was injected repeatedly for purification. The crude solution was loaded into the chromatographic column, the mobile phase was started for elution, the main peak was collected, and the acetonitrile was removed by evaporation to obtain the target peptide concentrate. The concentrate was then lyophilized to obtain the target polypeptide.

[0033] The preparation method of the cyclic polypeptide YQP-5 is as follows:

[0034] 1) Resin swelling

[0035] Weigh out Fmoc-Cys(trt)-2chlorotrityl Resin resin, place it in the reaction column, add an appropriate amount of dichloromethane (DCM), and gently purge with nitrogen gas for 10-30 minutes to allow the resin to fully expand. Remove the DCM solution, wash three times with DMF, and then dry under vacuum.

[0036] 2) Removal of Fmoc

[0037] Add 20% hexahydropyridine DMF solution to the reaction column and deprotect it every 5 minutes, then every 8 minutes. After the reaction is complete, wash the column 6 times with DMF.

[0038] 3) Coupling

[0039] Accurately weigh three times the molar amount of Fmoc-Cys(trt)-OH and O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU) into DMF, completely dissolve them, add DIEA to activate the carboxyl group, and then add the solution to the reaction column for reaction. Detect the structure after 1 hour; a positive result is sufficient. Decouple and remove Fmoc sequentially from the C-terminus to the N-terminus until the last amino acid, Fmoc-Cys(trt)-OH, is coupled. After removing Fmoc, shrink the target resin peptide and weigh it.

[0040] 4) Pyrolysis

[0041] A lysis buffer of 87.5% TFA + 5% anisole + 2.5% ethylenedithiol + 2.5% phenol + 2.5% water was prepared. Under low temperature conditions, the lysis buffer was slowly added to the resin peptide. The volume of the lysis buffer was 7-8 times the weight of the crude peptide resin. After stirring slowly for 2 hours, the liquid was filtered, ice-cold ether was added and stirred, and then the solid was obtained by centrifugation. The solid was washed three times with ether, dried, weighed, and the mass-to-charge ratio was determined to determine the molecular weight.

[0042] 5) Disulfide bond cyclization

[0043] Dissolve the sample in pure water and stir. Adjust the pH to make the polypeptide solution weakly alkaline. Add hydrogen peroxide and react for 0.5 hours before testing. A positive result is acceptable. Adjust the pH of the polypeptide solution to make it acidic.

[0044] 6) Purification and separation

[0045] Purification was performed using high performance liquid chromatography (HPLC). The chromatographic packing material was a 10 μm reversed-phase C18, and the mobile phase system was 0.1% TFA / water solution-0.1% TFA / acetonitrile solution. Gradient elution was used, and the sample was injected repeatedly for purification. The cyclized solution was loaded into the chromatographic column, and the mobile phase was started for elution. The main peak was collected, and after acetonitrile was removed, the target peptide concentrate was obtained. The concentrate was then lyophilized to obtain the target polypeptide.

[0046] The present invention relates to the application of the tumor-specific targeting peptide or its dimer or multimer in the preparation of tumor diagnostic reagents or tumor therapeutic drugs; preferably in the preparation of tumor diagnostic imaging agents; and more preferably in the preparation of precise tumor boundary localization and intraoperative image navigation imaging reagents or in the preparation of radionuclide imaging reagents.

[0047] A fluorescent molecular imaging probe having the following general formula:

[0048] MLR,

[0049] Wherein, M represents optical labeling, and the optical labeling is selected from near-infrared fluorescent dyes, organic chromophores, organic fluorophores, light-absorbing compounds, light-reflecting compounds, light-scattering compounds, and bioluminescent molecules;

[0050] L is a linking group, and L is preferably any one of Aca, PEG4, PEG6, and G6;

[0051]

[0052] R is any one of the tumor-specific targeting peptides or its dimers or multimers as described in claim 1.

[0053] The preferred structure of the fluorescent molecular imaging probe is shown in the following formula:

[0054]

[0055] Its structure contains a polypeptide R for targeting tumors, a near-infrared fluorescent dye structure MPA for optical imaging (the structure on the left above), and a linker L that increases the distance between the targeting polypeptide and the near-infrared fluorescent dye and regulates the pharmacokinetic properties in vivo.

[0056] The present invention also provides a method for preparing the polypeptide fluorescent probe, comprising:

[0057] 1) Synthesis of near-infrared fluorescent dye MPA

[0058] Glacial acetic acid, p-hydrazinobenzenesulfonic acid, methyl isopropyl ketone, and sodium acetate were mixed and reacted, and the product 2,2,3-trimethyl[3H]-indole-5-sulfonic acid was obtained after purification. Then, o-dichlorobenzene was added to a mixture of 2,2,3-trimethyl[3H]-indole-5-sulfonic acid and 1,3-propanesulfonic acid lactone to prepare 2,2,3-trimethyl-5-sulfonic acid-1-(3-sulfonic acid-propyl)-[3H]-indole. The product was then reacted with N-[(3-(anilinomethylene)-2-chl oro-1-cyclohexen-1-yl)methylene]-anilinemonohydrochloride to obtain a green carbocyanine dye. Finally, the carbocyanine dye was reacted with mercaptopropionic acid and triethylamine to prepare a water-soluble near-infrared dye MPA by liquid-phase separation and purification.

[0059] 2) Synthesis of MPA-L-YQP-X (X=1-6)

[0060] The near-infrared dye MPA obtained through separation and purification was dissolved in dimethyl sulfoxide along with the L-YQP-X (X = 1-6) polypeptide. An appropriate amount of N,N-diisopropylethylamine (DIPEA) was added, and the mixture was reacted overnight at room temperature. After the reaction was complete, the target fluorescent compound was obtained by preparative liquid chromatography purification. L-YQP-X can be synthesized in a solid-phase manner by a biotechnology company.

[0061] A radionuclide probe, characterized in that it is a tumor-specific targeting peptide or its dimer polypeptide of claim 1 labeled with a radionuclide; wherein the radionuclide is preferably selected from... 125 I, 131 I, 18 F, 99m Tc, 68 Ga 64 Cu, 67 Ga 90 Y, 111 In or 177 Lu.

[0062] As a preferred embodiment of the present invention, the present invention further provides a radionuclide probe, which is a radionuclide-labeled polypeptide complex. The polypeptide YQP-X (X = 1-6) contains tyrosine residues, wherein the ortho-position of the phenolic hydroxyl group can be radiolabeled with iodine via an electrophilic substitution reaction. 125 I / 131 I), in addition, select 18 F labels the polypeptide with polypeptide YQP-X (X = 1-6), using different nuclides for multi-molecule labeling, for the diagnosis or treatment of diseases. The structural formula is shown in (II).

[0063]

[0064] As another preferred embodiment of the present invention, the present invention further provides another radionuclide probe, which is a polypeptide or dimer polypeptide labeled with the radionuclide technetium, with the structural formula shown in (IV). Furthermore...

[0065]

[0066] Its structure contains a target-targeting peptide YQP-X (X = 1-6) or a dimer peptide, a bifunctional chelating agent for radiolabeling, 6-hydrazinopyridine-3-carboxylic acid (HYNIC), radionuclide ligands N-tris(hydroxymethyl)methylglycine (Tricine) and triphenylphosphine tris(m-sulfonate) sodium salt (TPPTS), as well as a radionuclide and a linker group. The linker group can be selected from a linker scaffold (L2E) composed of two linker groups L connected by two carboxyl groups of glutamic acid, or a linker scaffold (L2K) composed of two linker groups L connected by a carboxyl and amino group of lysine, or a linker group L that increases the distance between the target peptide and the radionuclide ligands N-tris(hydroxymethyl)methylglycine (Tricine) and triphenylphosphine tris(m-sulfonate) sodium salt (TPPTS) and regulates the pharmacokinetic properties in vivo. L is selected from Aca, PEG4, PEG6, and G6.

[0067] Among these methods, by modifying the bifunctional chelating agent, such as replacing it with bifunctional chelating agents DOTA, NOA, or DTPA, radionuclides can be selectively removed. 99m Other radionuclides besides Tc, such as 68 Ga 64 Cu, 67 Ga 90 Y, 111 In or 177 Lu is used for the diagnosis or treatment of diseases, and its structural formula is shown in (VIII). M represents a nuclide, and L is selected from Aca, PEG4, PEG6, and G6.

[0068]

[0069] The present invention also provides a method for preparing the radionuclide probe, comprising:

[0070] 1) Synthesis of HYNIC-NHS

[0071] 6-Chloronicotinic acid and 80% hydrazine hydrate were added to ethanol and heated under reflux. After the reaction was complete, the solvent was evaporated under reduced pressure. The resulting viscous substance was added to distilled water, and the pH was adjusted to approximately 5.5, precipitating a solid. This solid was filtered and dried to obtain a yellow solid. The product was identified as 6-hydroxynicotinic acid by ESI-MS mass spectrometry and 1H NMR spectroscopy. The obtained 6-hydroxynicotinic acid and p-aminobenzaldehyde were added to dimethyl sulfoxide (DMSO) and heated for 5-6 hours. After the reaction was complete, the solid was added to water and precipitated. The solid was filtered and dried. This solid, along with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS), was added to DMSO and reacted at room temperature. After the reaction was complete, the solid was added to water and precipitated. This solid was purified by silica gel column chromatography and identified as the target product by ESI-MS mass spectrometry and 1H NMR spectroscopy.

[0072] 2) Synthesis of HYNIC-Aca-YQP-1

[0073] The purified intermediate HYNIC-NHS was dissolved in DMSO, and then 2 moles of EDCI and 2 moles of NHS were added. The reaction was carried out at room temperature for 5 hours, and the reaction progress was monitored by analytical high performance liquid chromatography. After the reaction was completed, 2 moles of the targeting peptide YQP-X were added, followed by 4 moles of DIPEA. The reaction was carried out at room temperature for 3 hours. After the reaction was completed, the intermediate was separated and purified by preparative liquid chromatography and confirmed by mass spectrometry.

[0074] 3) Radioactive probe 99m Synthesis of Tc-HYNIC-Aca-YQP-1

[0075] Prepare TPPTS (triphenylphosphine tris(m-sulfonate)) solution at a concentration of 100.0 mg / mL, Tricine (trimethylglycine) solution at a concentration of 130.0 mg / mL, and succinate-sodium succinate buffer solution at a concentration of 102.4 mg / mL (77.0 mg succinate and 25.4 mg sodium succinate). Take 10 μL of each TPPTS solution, Tricine solution, and succinate-sodium succinate buffer solution, and mix them with 10 μL (1.0 mg / mL) of the (YQP-X)2-L2E-HYNIC solution in a vial. Then add 10 mL of Na... 99m TcO4 was heated in a metal bath at 100°C for 20 minutes. After the reaction was completed, the mixture was cooled to room temperature to prepare polypeptide radiopharmaceuticals. The products were analyzed and identified using an Agilent ZORBAX SB-Aq analytical column.

[0076] The application of the fluorescent molecular imaging probes and the radionuclide probes described in this invention in the preparation of reagents for tumor diagnosis, treatment or tracing, and targeted gene therapy or chemotherapy drugs.

[0077] The polypeptide compounds described in this invention can specifically target tumor sites and have good uptake and retention at the tumor sites, with a high target / non-target ratio. They are suitable for use as fluorescent tumor imaging agents, radionuclide imaging agents, and therapeutic agents, and can also be used to prepare optical imaging drugs for intraoperative tumor image navigation and precise localization of tumor boundaries.

[0078] The novel polypeptides described in this invention and the fluorescent and radionuclide probes constructed from these polypeptides have the following advantages compared with existing technologies:

[0079] 1. The YQP-X series peptides discovered in this invention are low molecular weight peptides with low synthesis costs. Furthermore, three amino acids in this series of short peptides are modified non-natural amino acids. The introduction of non-natural amino acids can greatly improve the stability of this series of peptides in vivo, making them less prone to degradation and less likely to have their targeting activity destroyed. They have a stronger potential to target the target site. In vitro and in vivo experimental results show that this series of probes has excellent stability and targeting ability in vivo and in vitro. The enhanced stability will promote the concentration and retention of this series of imaging probes at the tumor site, thereby achieving better tumor imaging results and making it more conducive to clinical application.

[0080] 2. The YQP-X series peptides have been shown to have excellent imaging effects on a variety of tumors through in vivo optical and radionuclide imaging results, including prostate cancer, breast cancer, pancreatic cancer, colorectal cancer, lung cancer, and liver cancer. Their ability to specifically target tumor sites may enable nuclear medicine diagnosis and treatment of malignant tumors, as well as optical imaging to guide surgeons in surgical navigation, achieving precise resection of lesions.

[0081] 3. In this invention, the near-infrared fluorescent dye MPA, which has better stability and water solubility, is used as an optical imaging group, thereby improving the pharmacokinetics of the drug in vivo.

[0082] 4. This invention introduces multiple water-soluble PEG4 or PEG6 molecules to further improve pharmacokinetic properties, especially clearance kinetics from non-tumor tissues.

[0083] 5. In this invention, HYNIC is used as a bifunctional chelating agent, and Tricine and TPPTS are used as synergistic ligands to achieve " 99m The Tc-HYNIC core exhibits better in vivo and in vitro stability.

[0084] The present invention will be further described below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0085] Figure 1 The structure of the prepared near-infrared dye MPA (A), the structure of the peptide YQP-1 (B), and the mass spectrum of the prepared MPA-Aca-YQP-1 (C).

[0086] Figure 2 Synthesized for Example 2 125 Structure of I-YQP-1 (A), radiometric HPLC chromatogram (B).

[0087] Figure 3 The structure of peptide YQP-1 (A), the structure of HYNIC (B), and the mass spectrometry of HYNIC-Aca-YQP-1 (C).

[0088] Figure 4 Targeted radiopharmaceutical prepared in Example 3 99m Structural diagram of Tc-HYNIC-Aca-YQP-1 (A), radiometric HPLC chromatogram (C)

[0089] Figure 5 Optical imaging of the compound MPA-Aca-YQP-1 prepared in Example 1 in MCF-7 breast cancer-bearing mice.

[0090] Figure 6 Optical imaging of compound MPA-Aca-YQP-1, prepared in Example 1, in HCT116 colorectal cancer-bearing mice.

[0091] Figure 7 Compound prepared in Example 2 125 SPECT-CT imaging of I-YQP-1 in HCT116 tumor-bearing mice with colorectal cancer.

[0092] Figure 8 The compound prepared in Example 3 99m SPECT-CT imaging of Tc-HYNIC-Aca-YQP-1 in Bel-7404 hepatocellular carcinoma-bearing mice.

[0093] Figure 9 The compound prepared in Example 3 99m SPECT-CT imaging of Tc-HYNIC-Aca-YQP-1 in HepG2 tumor-bearing mice with liver cancer.

[0094] Figure 10 The compound prepared in Example 3 99m SPECT-CT imaging of Tc-HYNIC-Aca-YQP-1 in MCF-7 breast cancer-bearing mice.

[0095] Figure 11 The compound prepared in Example 3 99m SPECT-CT imaging of Tc-HYNIC-Aca-YQP-1 in HCT116 tumor-bearing mice with colorectal cancer.

[0096] Figure 12 Optical imaging of compound MPA-Aca-YQP-2 prepared in Example 7 in breast cancer MCF-7 tumor-bearing mice.

[0097] Figure 13 The compound prepared in Example 8 99m SPECT-CT imaging of Tc-HYNIC-Aca-YQP-3 in MCF-7 breast cancer-bearing mice.

[0098] Figure 14 The compound prepared in Example 9 99m SPECT-CT imaging of Tc-HYNIC-Aca-YQP-4 in MCF-7 breast cancer-bearing mice. Detailed Implementation

[0099] The present invention will be further illustrated below through specific embodiments and application examples: the chemical substances used in the synthesis steps are all existing substances or commercially available products. The synthesis of the polypeptides of the present invention is prior art, and can be synthesized either according to the method described in the invention, or according to other existing technologies in the field, or commissioned to a biotechnology company with protein synthesis business for synthesis.

[0100] Example 1: Synthesis of the fluorescent targeting compound MPA-Aca-YQP-1

[0101] The synthesis steps are based on an invention patent previously applied for by our research group, authorized patent number: CN101440282. The main synthesis steps are as follows:

[0102] 10 mg of the solid-phase synthesized Aca-YQG-1 compound and 12.38 mg of the prepared pure dye MPA were weighed and added to 200 μL of dimethyl sulfoxide (DMSO). Then, 2.3 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) coupling agent and 3.82 mg of N-hydroxysuccinimide (NHS) were added. After mixing, 4.1 mg of N,N-diisopropylethylamine (DIPEA) was added. The mixture was reacted overnight at room temperature. After the reaction was completed, the mixture was separated and purified by preparative liquid chromatography. The preparative liquid chromatography conditions were as follows: An Agilent 1220 Infinity II series HPLC system equipped with an Agilent ZORBAX SB-C18 semi-preparative column (9.4 × 250 mm, 5 μm) was used. Gradient elution was performed for 60 min at a flow rate of 2 mL / min. Mobile phase A was ultrapure water (0.01% TFA) and mobile phase B was acetonitrile (0.01% TFA). The elution gradient was set as follows: 95% A and 5% B for 0-5 minutes, 80% A and 20% B for 15 minutes, 50% A and 50% B for 45 minutes, and 5% A and 95% B for 60 minutes. The final green product was confirmed as the expected product MPA-Aca-YQG-1 by analytical HPLC and ESI-MS mass spectrometry. (See [link to relevant documentation]). Figure 2 In the above preparation process, the L-YQG-X polypeptide synthesized in the solid phase was used to replace the Aca-YQG-1 polypeptide used in the step, thus obtaining the other 5 polypeptide compounds of the present invention with tumor-targeting optical imaging function.

[0103] Example 2 125 Synthesis of I-YQP-1

[0104] 1. First, accurately weigh 5 μg of Iodogen oxidant solid and dissolve it in 20 μL of anhydrous dichloromethane. Use a pipette to transfer the Iodogen dichloromethane solution to a 1.5 ml EP tube. Gently heat the bottom of the tube and use a nitrogen evaporator to fully evaporate the low-boiling-point dichloromethane. After the dichloromethane solvent evaporates, a thin film of Iodogen oxidant will form at the bottom of the EP tube.

[0105] 2. Add the pre-prepared phosphate buffer (pH = 7.4, 20 μL, 0.5 mg / ml) of YQP-1 peptide to an EP tube containing an oxidant membrane. Then, add a Na125I solution with a total radioactivity of 300 μCi. Finally, place the mixture on a shaker at room temperature and allow it to react for 5 min.

[0106] 3. Take a 1 μL sample of the reaction solution after the reaction, with a radioactivity of 15 μCi. Dilute to phosphate buffer (pH = 7.4, 20 μL) and inject into high-performance liquid chromatography (HPLC) to observe the reaction. The HPLC is equipped with a radioactive detector. Set the detection radionuclide type to [missing information]. 125 I.

[0107] Targeted radiopharmaceutical prepared in Example 3 99m Tc-HYNIC-Aca-YQP-1

[0108] 5 mg of the synthesized and purified intermediate (PEG4)2E-HYNIC was dissolved in 0.3 mL of DMSO, followed by the addition of 2.1 mg of EDCI and 1.25 mg of NHS. The reaction was carried out at room temperature for 5 hours, and the reaction progress was monitored by analytical high performance liquid chromatography. After the reaction was completed, 7.8 mg of the target peptide YQG-1 was added, followed by 5.6 mg of DIPEA. The reaction was carried out at room temperature for 3 hours. After the reaction was completed, the product was separated and purified by preparative liquid chromatography, and finally 6.5 mg of yellow solid was obtained, which was confirmed by mass spectrometry to be the target product.

[0109] Prepare solutions of 100.0 mg / mL TPPTS (triphenylphosphine tris(m-sulfonate)), 130.0 mg / mL Tricine (trimethylglycine), and 102.4 mg / mL succinate-sodium succinate buffer (77.0 mg succinate and 25.4 mg sodium succinate). Take 10 μL of each TPPTS solution, Tricine solution, and succinate-sodium succinate buffer, and mix them with 10 μL (1.0 mg / mL) of the (YQP-1)2-(PEG4)2E-HYNIC solution in a vial. Then add 10 mL of Na... 99mTcO4 was heated in a metal bath at 100°C for 20 minutes. After the reaction was completed, the mixture was cooled to room temperature to prepare the polypeptide radiopharmaceutical (YQP-1)2-(PEG4)2E-HYNIC- 99m Tc, the product was identified by analysis using an Agilent ZORBAX SB-Aq column. The HPLC method used was an Agilent 1220 Infinity II series HPLC system equipped with an online radiometric detector (Flow-RAM) and an Agilent ZORBAX SB-Aq column (4.6 × 250 mm, 5 μm). Gradient elution was performed for 45 minutes at a flow rate of 1 mL / min, with mobile phase A being ultrapure water (0.01% TFA) and mobile phase B being acetonitrile (0.01% TFA). The elution gradient was set as follows: 95% A and 5% B for 0-5 minutes, 70% A and 30% B for 15 minutes, 65% A and 35% B for 20 minutes, 45% A and 55% B for 25 minutes, and 5% A and 95% B for 45 minutes.

[0110] Example 4: Optical imaging of compound MPA-Aca-YQP-1 in tumor-bearing mice

[0111] Compound MPA-Aca-YQP-1 was prepared according to Example 4 and dissolved in physiological saline. 0.1 mL (approximately 10 nmol) was injected via the tail vein into the tail veins of two types of tumor-bearing nude mice (MCF-7 and HCT116), with three mice of each type. Optical signals were acquired at 1 h, 2 h, 4 h, 8 h, 10 h, and 12 h post-administration. The distribution of the probe in the mice and its enrichment in the tumor region were observed. The imaging results at 1 h are shown in the figure below. Figure 5 and 6 As shown, the imaging results of compound MPA-Aca-YQP-1 in three tumor-bearing nude mice were basically consistent. The 1-hour imaging showed that the probe had been significantly taken up in the tumor, and it remained in the tumor until 10 hours. The probe was most enriched in the tumor at 4 hours, while it was taken up and cleared quickly in other background organs. The signal from the bladder indicated that the probe was mainly metabolized by the kidneys.

[0112] Compound prepared in Example 5 125 SPECT-CT imaging of I-YQP-1 in HCT116 tumor-bearing mice with colorectal cancer

[0113] The compound was prepared according to the method in Example 2. 125I-YQP-1 was prepared as a physiological saline solution, and 0.1 mL (approximately 10 nmol) was injected into the tail vein of three nude mice bearing HCT116 colorectal cancer tumors. SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The distribution of the radionuclide probe in the mice and its enrichment in the tumor region were observed. The imaging results at 1 h are shown in the figure below. Figure 7 As shown, the probe 125 I-YQP-1 showed significant uptake at the tumor site, indicating that this probe can target colorectal cancer HCT116 tumor cells and is mainly metabolized and excreted through the kidneys.

[0114] Compound prepared in Example 6 99m SPECT-CT imaging of Tc-HYNIC-Aca-YQP-1 in tumor-bearing mice

[0115] The compound was prepared according to the method in Example 3. 99m Tc-HYNIC-Aca-YQP-1 was prepared into a physiological saline solution. 0.1 mL (approximately 10 nmol) was injected via the tail vein into the tail veins of four tumor-bearing nude mice (HepG2, Bel-7404, MCF-7, and HCT116), with three mice of each type. SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, 3 h, and 4 h post-administration to observe the distribution of the radionuclide probe in the mice and its enrichment in the tumor region. The 1-hour imaging result is shown in the image below. Figure 8 As shown in 9, 10 and 11, the probe 99m Tc-HYNIC-Aca-YQP-1 showed significant uptake at the tumor site, indicating that this probe can target tumor cells and is mainly metabolized and excreted through the kidneys.

[0116] Example 7 Optical imaging of compound MPA-Aca-YQP-2 in breast cancer MCF-7 tumor-bearing mice

[0117] The compound MPA-Aca-YQP-2 was prepared according to the method in Example 1 and diluted to a physiological saline solution. 0.1 mL (approximately 10 nmol) was injected into the tail vein of three MCF-7 breast cancer-bearing nude mice (approximately 22 g in weight). Optical signals were acquired at 1 h, 2 h, 4 h, 8 h, 10 h, and 12 h after administration. The distribution of the probe in the mice and its enrichment in the tumor region were observed. The imaging results are shown in the figure below. Figure 12As shown, the imaging results of compound MPA-Aca-YQP-2 in three tumor-bearing nude mice were basically consistent. The imaging images at 2 hours showed that the probe had been significantly taken up in the tumor, and it remained in the tumor until 10 hours. The probe was most enriched in the tumor at 4 hours, while it was taken up and cleared quickly in other background organs. The signal from the bladder indicated that the probe was mainly metabolized by the kidneys.

[0118] Compound prepared in Example 8 99m SPECT-CT imaging of Tc-HYNIC-Aca-YQP-3 in MCF-7 breast cancer-bearing mice

[0119] The compound was prepared according to the method in Example 3. 99m Tc-HYNIC-Aca-YQP-3 was prepared into a physiological saline solution. 0.1 mL (approximately 10 nmol) was injected into the tail vein of three MCF-7 breast cancer-bearing nude mice. SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, 3 h, and 4 h post-administration. The distribution of the radionuclide probe in the mice and its enrichment in the tumor region were observed. The 1-hour imaging result is shown in the figure below. Figure 13 As shown, the probe 99m Tc-HYNIC-Aca-YQP-2 showed significant uptake at the tumor site, indicating that this probe can target MCF-7 breast cancer tumor cells and is mainly metabolized and excreted through the kidneys.

[0120] Compound prepared in Example 9 99m SPECT-CT imaging of Tc-HYNIC-Aca-YQP-4 in MCF-7 tumor-bearing mice with breast cancer

[0121] The compound was prepared according to the method in Example 3. 99m Tc-HYNIC-Aca-YQP-4 was prepared into a physiological saline solution. 0.1 mL (approximately 10 nmol) was injected into the tail vein of three MCF-7 breast cancer-bearing nude mice. SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The distribution of the radionuclide probe in the mice and its enrichment in the tumor region were observed. The 1-hour imaging result is shown in the figure below. Figure 14 As shown, the probe 99m Tc-HYNIC-Aca-YQP-2 showed significant uptake at the tumor site, indicating that this probe can target MCF-7 breast cancer tumor cells and is mainly metabolized and excreted through the kidneys.

Claims

1. A tumor-specific targeting peptide, characterized in that, Selected from the following polypeptides: The tumor-targeting peptide YQP-3 has the sequence Hyp-Ser-Asp-Asn-Tyr(3-I)-Thr-NH2; Where: Hyp is hydroxyproline, and Tyr(3-I) is iodotyrosine.

2. The use of the tumor-specific targeting peptide according to claim 1 in the preparation of breast tumor diagnostic reagents.

3. The application according to claim 2, characterized in that, The application of the tumor-specific targeting peptide of claim 1 in the preparation of a breast tumor diagnostic imaging agent.

4. The application according to claim 2, characterized in that, The application of the tumor-specific targeting peptide of claim 1 in the preparation of a precise localization and intraoperative image navigation imaging reagent for breast tumor boundaries or in the preparation of a radionuclide imaging reagent.

5. A fluorescent molecular imaging probe, characterized in that, It has the following general formula: MLR, Wherein, M represents optical labeling, and the optical labeling is selected from infrared fluorescent dyes, compounds containing organic chromophores or organic fluorophores, light-absorbing compounds, light-reflecting compounds, light-scattering compounds, or bioluminescent molecules; L is a linking group; R is the tumor-specific targeting peptide as described in claim 1.

6. The fluorescent molecular imaging probe according to claim 5, characterized in that, L is selected from Aca, PEG4, PEG6, and G6.

7. The fluorescent molecular imaging probe according to claim 5 or 6, characterized in that, The fluorescent molecular imaging probe is selected from any one of the following: 。 8. A radioactive nuclide probe, characterized in that, The tumor-specific targeting peptide of claim 1 is labeled with a radionuclide.

9. The radionuclide probe according to claim 8, characterized in that, The radionuclides mentioned are selected from 125 I, 131 I, 18 F, 99m Tc, 68 Ga 64 Cu, 67 Ga 90 Y, 111 In or 177 Lu.

10. The radionuclide probe according to claim 8 or 9, characterized in that, The radionuclide probe is radiolabeled with radioactive iodine or fluorine to mark the hydrogen atom at the phenolic hydroxyl position of tyrosine in the tumor-specific targeting peptide of claim 1.

11. The radionuclide probe according to claim 8 or 9, characterized in that, The radionuclide probe comprises the tumor-specific targeting peptide, linker group, radionuclide ligand, bifunctional chelating agent for radionuclide labeling, and radionuclide as described in claim 1.

12. The radionuclide probe according to claim 11, characterized in that, The linking group is selected from Aca, PEG4, PEG6, and G6; the bifunctional chelating agent is HYNIC, DOTA, NOTA, or DTPA; and the radionuclide is selected from... 99m Tc, 68 Ga 64 Cu, 67 Ga 90 Y, 111 In or 177 Lu, the radioactive nuclide ligands are N-tris(hydroxymethyl)methylglycine and sodium triphenylphosphine tri-m-sulfonate.

13. The use of the fluorescent molecular imaging probe of claim 5 or 6, or the radionuclide probe of claim 8 or 9, in the preparation of reagents for the diagnosis or tracing of breast tumors.

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