WO2018192405A1 - 双靶点显像分子探针及其制备方法和应用 - Google Patents

双靶点显像分子探针及其制备方法和应用 Download PDF

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WO2018192405A1
WO2018192405A1 PCT/CN2018/082782 CN2018082782W WO2018192405A1 WO 2018192405 A1 WO2018192405 A1 WO 2018192405A1 CN 2018082782 W CN2018082782 W CN 2018082782W WO 2018192405 A1 WO2018192405 A1 WO 2018192405A1
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tate
rgd
peg
nota
dual
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PCT/CN2018/082782
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French (fr)
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朱朝晖
要少波
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中国医学科学院北京协和医院
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Priority to US16/605,134 priority Critical patent/US20200155714A1/en
Priority to EP18787253.6A priority patent/EP3613441B1/en
Priority to JP2019560737A priority patent/JP6786731B2/ja
Publication of WO2018192405A1 publication Critical patent/WO2018192405A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/082—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the peptide being a RGD-containing peptide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B6/00—Apparatus for radiation diagnosis, e.g. combined with radiation therapy equipment
    • A61B6/02—Devices for diagnosis sequentially in different planes; Stereoscopic radiation diagnosis
    • A61B6/03—Computerised tomographs
    • A61B6/037—Emission tomography
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/06—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules
    • A61K51/065—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules conjugates with carriers being macromolecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/083—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the peptide being octreotide or a somatostatin-receptor-binding peptide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/78—Detectors specially adapted therefor using more than one detector
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N2030/77—Detectors specially adapted therefor detecting radioactive properties

Definitions

  • the invention relates to the field of radioactive probes for diagnosis and treatment of diseases, in particular to a TATE-targeting integrin ⁇ v ⁇ 3 (integrin ⁇ v ⁇ 3 ) and/or somatostatin receptor (SSTR)-positive diseases.
  • RGD dual-target radioactive molecular probes and methods of preparation, and the use of the compounds as targeting molecules for diagnosis and therapy.
  • Integrin ⁇ v ⁇ 3 is an extracellular matrix receptor which is a heterodimeric transmembrane glycoprotein composed of two subunits, ⁇ and ⁇ .
  • Integrin ⁇ v ⁇ 3 is one of the important molecular markers of tumors, which is highly expressed on the surface of neovascular endothelial cells and on the surface of certain tumor cells, such as neuroblastoma, osteosarcoma, glioblastoma, breast cancer and Prostate cancer, but not expressed or expressed in established blood vessels and normal tissues, due to its high expression during tumor growth and metastasis, making integrin ⁇ v ⁇ 3 a target for diagnosis and treatment.
  • Somatostatin acts through the somatostatin receptor (SSTR), a family of G-protein-coupled receptors with seven transmembrane segments. Glycoprotein.
  • SSTR1 ⁇ 5 There are five different molecular subtypes in the SSTR gene, namely SSTR1 ⁇ 5.
  • SSTR is widely distributed, and many normal cells including endocrine cells and lymphocytes express SSTR, but in tumors such as gastrointestinal pancreatic neuroendocrine tumors (gastrinoma, insulinoma, glucagonoma), carcinoid, pituitary Expression in adenoma, pheochromocytoma, paraganglioma, medullary thyroid carcinoma is more common than normal tissue.
  • SSTR plays an important role in the occurrence and development of various human tumors.
  • Somatostatin receptors are found in a variety of neuroendocrine tumors (NET), such as gastrointestinal pancreatic neuroendocrine tumors (gastrinoma, insulinoma, glucagonoma), carcinoid, pituitary adenoma, chromaffin cells Tumor, paraganglioma, medullary thyroid carcinoma, etc.
  • NET neuroendocrine tumors
  • the peptide-based radionuclide tracer of arginine-glycine-aspartate (RGD) was developed in the 1980s and can express high integrin ⁇ on the surface of neovascular endothelial cells and on the surface of tumor cells.
  • the v ⁇ 3 subunit binds, thereby serving the purpose of displaying tumors and suggesting the degree of tumor neovascularization.
  • SPECT (/CT) imaging or PET (/CT) imaging with 99m Tc or 18 F labeled RGD peptide has been used in clinical applications in Western countries such as Europe and the United States. There are also a few units in China that have preliminary clinical applications. Strong safety and good efficacy are valuable for the diagnosis, staging and efficacy evaluation of various tumors including breast cancer, liver cancer and glioma.
  • the positron-emitting radionuclide-labeled polypeptides 68 Ga-DOTA-RGD and 68 Ga-DOTA-TATE have higher sensitivity and specificity to the receptor, due to the integrin ⁇ v ⁇ 3 and SSTR2 receptors, respectively, in most malignant
  • the expression of tumors and benign neuroendocrine tumors is high, and the high resolution of PET imaging can quantify the absorption of tumors and organs and has better superiority than SPECT.
  • the positron-like radionuclide The 68 Ga 3+ is produced by a generator and has the advantages of simple production process and low cost. Therefore, PET/CT imaging with 68 Ga labeled drugs is necessary and will have higher clinical application value. It can further improve the detection rate of tumors, and has higher clinical significance in tumor staging and prognosis evaluation, tumor-directed surgery and therapeutic evaluation.
  • the object of the present invention is to design and synthesize a TATE-RGD dual-target radioactive molecular probe containing two polypeptides of TATE and RGD for binding two targets on tumor cells, and a preparation method thereof.
  • PEG polyethylene glycol
  • PEG polyethylene glycol
  • the radiolabeled probe is concentrated to the lesion by TGF and/or RGD polypeptide tumor cell receptor targeting, using the nucleus Medical positron emission tomography, imaging diagnosis and treatment of SSTR2 and / or integrin ⁇ v ⁇ 3 high expression lesions.
  • the present invention provides a tumor targeting polypeptide compound having a dual target comprising a TATE cyclic peptide structure, an RGD cyclic peptide structure, and a NOTA chelating group, the TATE cyclic peptide structure, the RGD cyclic peptide structure, and The NOTA chelating group is linked or directly linked to the same glutamic acid molecule by a PEG segment having a degree of polymerization of 1-5; the structure of the polypeptide compound can be simplified as NOTA-PEG n -Glu ⁇ PEG m -TATE ⁇ -PEG P -RGD, wherein m, n and p each take an integer from 0 to 5.
  • the TATE cyclic peptide structure, the NOTA chelating group and the RGD cyclic peptide structure are each linked to the same glutamic acid molecule by a PEG segment having a degree of polymerization of 2-5.
  • the TATE cyclic peptide structure and the RGD cyclic peptide structure are respectively linked to the two carboxyl terminals of the same glutamic acid molecule via a PEG 4 molecular segment to form a stable amide bond;
  • the NOTA chelating group is linked to the amino terminus of the same glutamic acid molecule via a PEG 4 molecular segment;
  • the polypeptide compound is referred to as NOTA-3PEG 4 -TATE-RGD, and its specific structure is as follows ( I):
  • the present invention further provides a TATE-RGD dual-target radioactive molecular probe, which is a radionuclide-labeled polypeptide complex, which is matched with a tumor targeting polypeptide compound having dual targets according to the present invention. body.
  • the TATE-RGD dual-target radioactive molecular probe is a 68 Ga-labeled polypeptide complex
  • the polypeptide complex has the dual target tumor target of the present invention.
  • the polypeptide compound is a ligand, and the TATE cyclic peptide structure, the NOTA chelating group and the RGD cyclic peptide structure of the dual target tumor targeting polypeptide compound respectively pass the PEG segment with a degree of polymerization of 2-5 and the same valley. Amino acid linkage.
  • the TATE-RGD dual-target radioactive molecular probe is a 68 Ga-labeled polypeptide complex, and the polypeptide complex is targeted by the tumor having dual targets according to the present invention.
  • the polypeptide compound NOTA-3PEG 4 -TATE-RGD is a ligand, and the dual-target radioactive molecular probe is succinctly represented as 68 Ga-NOTA-3PEG 4 -TATE-RGD.
  • Another object of the present invention is to provide a method for preparing the polypeptide compound and the dual-target radioactive molecular probe, which is simple and convenient, and has higher product stability.
  • n is an integer from 0 to 5;
  • n is an integer from 0 to 5;
  • step b) reacting the second product obtained in step b) with NOTA-PEG m -NHS under DIPEA conditions to obtain Boc-protected glutamic acid by linking the NOTA group and the TATE peptide via the PEG m segment and the PEG n segment, respectively.
  • a third product wherein n and m each take an integer from 0 to 5;
  • step c) The third product obtained in step c) is deprotected from the protective group Boc under TFA conditions to obtain a fourth product in which the glutamic acid is linked to the NOTA group and the TATE peptide via the PEG m segment and the PEG n segment, respectively.
  • step d) reacting the fourth product obtained in step d) with the polypeptide PEG p -RGD under DIPEA conditions, wherein p takes an integer from 0 to 5, and finally obtains the tumor targeting polypeptide compound NOTA having dual targets according to the present invention.
  • p takes an integer from 0 to 5
  • a method for preparing the TATE-RGD dual-target radioactive molecular probe to prepare a 68 Ga-NOTA-3PEG 4 -TATE-RGD radiopharmaceutical comprising the following steps:
  • the NOTA-3PEG 4 -TATE-RGD was dissolved in deionized water; the gallium arsenide ( 68 Ge/ 68 Ga) generator (Ecker & Ziegler) was rinsed into the EP tube with 5 mL of 0.1 mol/L high purity hydrochloric acid solution, and collected.
  • the analytical HPLC method in the previous step was carried out using a Waters HPLC system equipped with a Waters C18 analytical column (4.6 mm ⁇ 250 mm), HPLC gradient elution conditions: 0 min, acetonitrile / water (5 / 95, v / v); 5 min, Acetonitrile / water (5 / 95, v / v); 10 min, acetonitrile / water (80 / 20, v / v); 15 min, acetonitrile / water (100 / 0, v / v); 18 min, acetonitrile / water (100 /0,v/v); 20 min, acetonitrile / water (5 / 95, v / v), eluent containing 0.1% TFA, flow rate 1 mL / min. From the radioactive HPLC profile, 68 Ga-NOTA-3PEG4-TATE-RGD had a Retention Time of 11.6 min and a radiochemical purity
  • the invention also provides the use of the TATE-RGD dual-target molecular probe for the preparation of radiopharmaceuticals for diagnosis of SSTR2 and/or integrin ⁇ v ⁇ 3 positive imaging.
  • a preferred application of the present invention is to prepare the TATE-RGD dual-target molecular probe into a colorless transparent injection for diagnosis of small cell lung cancer imaging.
  • the TATE-RGD of the present invention is a dual-target polypeptide drug, and TATE and RGD are linked to enable simultaneous binding to the somatostatin receptor and integrin ⁇ v ⁇ 3 to increase binding affinity and tumor uptake of drugs. To achieve better tumor imaging results;
  • the present invention can improve the pharmacokinetic properties of the polypeptide drug, especially the non-tumor tissue clearance rate, when a PEG molecule having the same or different degree of polymerization is introduced between the TATE and the RGD polypeptide;
  • NOTA as a chelating agent in the present invention has better in vivo and in vitro stability compared to DOTA.
  • Figure 1 is a high performance liquid chromatography (HPLC) spectrum of 68 Ga-NOTA-3PEG 4 -TATE-RGD.
  • Figure 2 shows the results of microPET imaging of H69 model of small cell lung cancer in nude mice. From left to right, the tail vein is injected with 68 Ga-NOTA-3PEG 4 -TATE-RGD (Control), 68 Ga-NOTA-3PEG 4 -TATE. -RGD+RGD(Block+RGD), 68 Ga-NOTA-3PEG 4 -TATE-RGD+TATE(Block+TATE) and 68 Ga-NOTA-3PEG 4 -TATE-RGD+RGD+TATE(Block+TATE+RGD ) The result of the imaging.
  • Figure 3 shows the results of microPET imaging of non-small cell lung cancer in nude mice A549 model. From left to right, the tail vein is injected with 68 Ga-NOTA-3PEG 4 -TATE-RGD (Control), 68 Ga-NOTA-3PEG 4 - TATE-RGD+RGD(Block+RGD), 68 Ga-NOTA-3PEG 4 -TATE-RGD+TATE(Block+TATE) and 68 Ga-NOTA-3PEG 4 -TATE-RGD+RGD+TATE(Block+TATE+ RGD) results.
  • Figure 4 shows the radioactive uptake %ID/g of each organ at different time points after injection of 68 Ga-NOTA-3PEG 4 -TATE-RGD in normal mice.
  • Figure 5 is a comparison of the imaging results of the 68 Ga-NOTA-3 PEG 4 -TATE-RGD of the present invention and the existing 68 Ga-NOTA-RGD probe in non-small cell lung cancer patients, wherein the left panel is the invention 68 The imaging results of Ga-NOTA-3PEG 4 -TATE-RGD, the right panel is the imaging result of the existing 68 Ga-NOTA-RGD, and the arrow points to the lesion.
  • Figure 6 is a comparison of the imaging results of 68 Ga-NOTA-3PEG 4 -TATE-RGD and the existing 68 Ga-NOTA-RGD probe in small cell lung cancer patients, wherein the left panel is the invented 68 Ga-NOTA- The imaging results of 3PEG 4 -TATE-RGD, the right panel is the imaging result of the existing 68 Ga-NOTA-RGD, and the arrow points to the lesion.
  • a tumor targeting polypeptide compound having a dual target comprising a TATE cyclic peptide structure, an RGD cyclic peptide structure and a NOTA chelating group, which consists of the TATE cyclic peptide structure, a NOTA chelating group and an RGD
  • the cyclic peptide structure is respectively formed by linking the same glutamic acid molecule, wherein the TATE cyclic peptide structure and the RGD cyclic peptide structure respectively connect the two carboxyl ends of the glutamic acid molecule through a PEG 4 molecular segment to form a stable amide.
  • NOTA chelating group is linked to the amino terminus of the glutamic acid molecule via a PEG 4 molecule;
  • the obtained polypeptide compound is designated as NOTA-3PEG 4 -TATE-RGD, and the specific structure thereof is as shown in the following formula (I); Show:
  • the method of preparing the NOTA-3PEG 4 -TATE-RGD comprises the following steps:
  • PEG 4 -TATE commercial tumor targeting polypeptide, dissolved in 2.6 mL of dimethylformamide (DMF)
  • DMF dimethylformamide
  • LC-MS liquid chromatography and mass spectrometry
  • TFA trifluoroacetic acid
  • the mixture was designated as NOTA-2PEG 4 -TATE.
  • the mixture obtained by lyophilization and removal of the solvent DMSO was purified by HPLC, and lyophilized to give the desired product about 5.5 mg, yield 25.6%.
  • a TATE-RGD dual-target radioactive molecular probe comprising a TATE polypeptide, an RGD polypeptide and a radionuclide 68 Ga, wherein the TATE and RGD polypeptides are linked by a PEG 4 molecule to form a TATE-3PEG 4 -RGD polypeptide and 68 Ga Linked with NOTA, the TATE-RGD dual target radioactive molecular probe is 68 Ga-NOTA-3 PEG 4 -TATE-RGD.
  • the preparation method of the 68 Ga-NOTA-3PEG 4 -TATE-RGD dual target radioactive molecular probe comprises the following steps:
  • a 5 mL syringe was used to extract 0.1 mL/L HCl 5 mL, and the ⁇ Gallium generator was rinsed, slowly rinsed, and the eluent was collected into a 1.5 mL EP tube, 1 mL per tube, for a total of 5 tubes. Radioactivity determination of each EP tube, the most deactivated tube for labeling;
  • HPLC analysis conditions column was C18 column (4.6 mm ⁇ 250 mm), mobile phase A was already (0.1% trifluoroacetic acid), mobile phase B was water (0.1% trifluoroacetic acid), flow rate was 1 mL / min: 0 ⁇ 5 min, mobile phase A 5%; 10 min, mobile phase A was 80%; 15 min, mobile phase A was 100%; 18 min, mobile phase A 100%; 20 min, mobile phase A 5%.
  • the UV detection wavelength was 210 nm and the column temperature was 20 °C.
  • Radioactive detection uses a dedicated radioactive detector for HPLC.
  • HPLC analysis showed that: 68 Ga-NOTA-3PEG 4 -TATE-RGD had a Retention Time of 11.6 min (Fig. 1) and a radiochemical purity of >99% without further purification.
  • TATE-RGD dual-target probe 68 Ga-NOTA-3PEG 4 -TATE-RGD was further prepared into a colorless transparent injection for imaging test or imaging diagnosis.
  • the specific experiments and effects are as follows:
  • the right forelimb was subcutaneously inoculated with H69 tumor-bearing mice, and the tail vein was injected with the 68 Ga-NOTA-3PEG 4 -TATE-RGD injection of Example 2 at 100-200 ⁇ Ci.
  • a static image was collected using Siemens Inveon micro PET for 10 min (Fig. 2, The arrow indicates the tumor site.
  • the results showed that when the probe of Example 2 was injected alone, there was significant radioactivity uptake at the tumor site, and the tumor was clearly visible, and the tumor uptake value was 9.78 ⁇ 2.77; while the unlabeled precursor RGD, TATE was co-injected by co-injection.
  • RGD+TATE was able to effectively reduce tumor uptake to 8.23 ⁇ 1.08, 1.41 ⁇ 0.73, and 1.05 ⁇ 0.13, respectively; when simultaneous injection of unlabeled precursor RGD and TATE, tumors reduced their radioactivity up to the background.
  • Small cell H69 tumors are predominantly expressed by somatostatin receptors, and the concentration of the dual target molecular probes of the invention in tumors is primarily inhibited by unlabeled TATE.
  • the right forelimb was subcutaneously inoculated with tumor-bearing mice of A549 tumor, and the 68 Ga-NOTA-3PEG 4 -TATE-RGD injection of Example 2 was injected into the tail vein at 100-200 ⁇ Ci. After 60 min, a static image was collected using Siemens Inveon micro PET for 10 min (Fig. 3, The arrow indicates the tumor site.
  • Non-small cell A549 tumors are mainly characterized by increased expression of integrin receptors, and the concentration of the dual-target molecular probes of the present invention in tumors is mainly inhibited by unlabeled RGD.
  • the distribution of healthy Balb/c is shown in Fig. 4.
  • the 68 Ga-NOTA-3PEG 4 -TATE-RGD of Example 2 is cleared from the blood, heart and liver faster; the kidney is more radioactive, indicating that the molecular probe is mainly excreted through the kidney.
  • Early heart, liver, and lungs have a small amount of ingestion, which decreases rapidly over time.
  • the imaging agent has a small amount of distribution in the stomach, intestines, spleen, and pancreas, and there is little brain tissue community, indicating that it cannot pass through the blood-brain barrier.
  • 68 Ga-NOTA-3PEG 4 -TATE-RGD PET/CT images of clinically diagnosed small cell lung cancer patients are shown in Figure 5.
  • Intravenous injection of 68 Ga-NOTA-3 PEG 4 -TATE-RGD 3.5 mCi of the present invention 30 minutes later using Siemens Biograph 64 PET/ CT collected the image of the trunk part, 3 min/bed, and collected a total of 5 beds.
  • the lesions were clearly displayed and the highest standard uptake value (SUV max ) was 18.2 (Fig. 5 left).
  • the existing single-target imaging agent 68 Ga-NOTA-RGD only showed an increase in uptake in the tumor region, and the SUV max value was 4.7 (Fig. 5 right).
  • the images of clinically diagnosed patients with non-small cell lung cancer are shown in Fig. 6.
  • the 68 Ga-NOTA-3PEG 4 -TATE-RGD 3.0 mCi of the present invention was intravenously injected, and images were collected using Siemens Biograph 64 PET/CT 30 min later, and 3 beds were collected at 3 min/bed. .
  • the lesions were clearly displayed and the highest standard uptake value (SUV max ) was 3.4 (Fig. 6 left).
  • the existing single-target imaging agent 68 Ga-NOTA-RGD had low tumor uptake and a SUV max value of 2.8 (Fig. 6 right).

Abstract

提供一种具有双靶点的靶向多肽化合物，其包含TATE环肽结构、RGD环肽结构和NOTA螯合基团，所述TATE环肽结构、RGD环肽结构和NOTA螯合基团分别通过聚合度为1-5的PEG链段连接或直接连接同一个谷氨酸分子；所述的多肽化合物结构可表示为NOTA-PEG n-Glu{PEG m-TATE}-PEG P-RGD，其中m、n和p分别为0-5的整数。还提供基于所述多肽化合物的TATE-RGD双靶点放射性分子探针。

Description

双靶点显像分子探针及其制备方法和应用 技术领域
本发明涉及疾病诊断和治疗的放射性探针领域，尤其涉及一种靶向整合素α vβ 3(integrinα vβ 3)和/或生长抑素受体(somatostatin receptor,SSTR)阳性疾病的TATE-RGD双靶点放射性分子探针和制备方法，以及所述化合物作为诊断和治疗的靶向分子的应用。
背景技术
肿瘤在生长过程中重要的环节是新生血管的形成，肿瘤的新生血管被多种蛋白分子调节，其中有一种关键的蛋白是整合素α vβ 3。整合素α vβ 3是一种细胞外基质受体，它是由α和β两个亚基组成的异源二聚体跨膜糖蛋白。整合素α vβ 3作为肿瘤的重要分子标志物之一，其高表达在新生血管内皮细胞表面和某些肿瘤细胞表面，如成神经细胞瘤、骨肉瘤、成胶质细胞瘤、乳腺癌和前列腺癌，而在已经形成的血管和正常组织中不表达或表达很低，由于其在肿瘤生长和转移过程中的高表达，使整合素α vβ 3成为用于诊断和治疗的靶点之一。生长抑素(somatostatin,SST)是通过生长抑素受体(somatostatin receptor,SSTR)发挥作用的，生长抑素受体属于一类G蛋白偶联的受体家族，是具有7个跨膜区段的糖蛋白。SSTR基因中存在着5种不同的分子亚型，即SSTR1～5。SSTR分布广泛，包括内分泌细胞和淋巴细胞在内的许多正常细胞都表达SSTR，但在肿瘤如胃肠胰腺神经内分泌肿瘤(胃泌素瘤、胰岛素瘤、胰高血糖素瘤)、类癌、垂体腺瘤、嗜铬细胞瘤、副神经节瘤、甲状腺髓样癌等中表达较正常组织更为普遍。SSTR在多种人类肿瘤的发生、发展等方面发挥重要作用。
生长抑素受体见于多种神经内分泌肿瘤(neuroendocrinetumor,NET)，如胃肠胰腺神经内分泌肿瘤(胃泌素瘤、胰岛素瘤、胰高血糖素瘤)、类癌、垂体腺瘤、嗜铬细胞瘤、副神经节瘤、甲状腺髓样癌等。 99mTc和 111In等放射性核素标记的生长抑素类似物，如 99mTc-HYNIC-奥曲肽和 111In-DTPA-奥曲肽，已在临床应用了较长时，在NET诊治方面发挥着重要作用；同时新的生长抑素衍生物也在不断出现或被改进，如奥曲肽、TATE等，其中TATE对SSTR2亲和力更高，而大多数NET也更多表达SSTR2。在国际领域， 68Ga-DOTA-TATE相关临床前研究已经很充分，并在德国为主导的欧洲及世界其他部分国家已经应用于临床，众多研究显示其在 胃肠胰腺内分泌肿瘤、垂体腺瘤、嗜铬细胞瘤、副神经节瘤、甲状腺髓样癌、小细胞肺癌等神经内分泌肿瘤诊断及治疗指导方面的优越性。
精氨酸-甘氨酸-天冬氨酸(RGD)的多肽类核素示踪剂是二十世纪80年代开始发展起来的，它能与新生血管内皮细胞表面及肿瘤细胞表面高表达的整合素α vβ 3亚基结合，从而起到显示肿瘤并提示肿瘤新生血管表达程度的目的。用 99mTc或 18F标记的RGD多肽进行SPECT(/CT)显像或PET(/CT)显像，在欧美等西方国家已应用于临床，国内也有少数单位进行了初步的临床应用，提示具有较强的安全性和较好的有效性，对包括乳腺癌、肝癌和神经胶质瘤等多种肿瘤的诊断、分期及疗效评估具有价值。
正电子放射性核素标记的多肽 68Ga-DOTA-RGD和 68Ga-DOTA-TATE由于其各自对受体更高的灵敏度和特异性，由于整合素α vβ 3和SSTR2受体分别在多数恶性肿瘤和偏良性的神经内分泌肿瘤中表达量较多，以及PET显像的高分辨率、可以对肿瘤及器官的吸收定量而具有比SPECT更好的优越性；同时，该正电子类放射性核素 68Ga 3+由发生器生产，具备生产工艺简单，成本低的优势，故用 68Ga标记药物的PET/CT显像具有必要性，也将产生更高的临床应用价值。可进一步提高肿瘤的检出率，并在肿瘤的分期与预后评价、肿瘤导向手术及疗效评估中具备更高的临床意义。
发明内容
本发明的目的是设计并合成一种TATE-RGD双靶点放射性分子探针及其制备方法，该分子探针含有TATE和RGD两个多肽用于结合肿瘤细胞上的两个靶点，之间通过PEG(PEG＝polyethylene glycol)分子连接，起到调节各功能基团之间的距离以及体内药代动力学特性的作用。与单靶点分子探针相比，该探针因具有更高的肿瘤摄取，能够达到更好的体内显像效果。该分子通过双功能螯合剂NOTA标记放射性核素后可得到放射性标记探针，在体内该放射性标记探针通过TATE和/或RGD多肽的肿瘤细胞受体靶向作用浓聚到病变部位，利用核医学的正电子断层显像技术，对SSTR2和/或整合素α vβ 3高表达病变进行显像诊断和治疗。
本发明的目的是通过以下技术实现的：
首先，本发明提供一种具有双靶点的肿瘤靶向多肽化合物，其结构中包含TATE环肽结构、RGD环肽结构和NOTA螯合基团，所述TATE环肽结构、RGD环肽结构和NOTA螯合基团分别通过聚合度为1-5的PEG链段连接或直接连接同一个谷氨酸分子；所述的多肽化合物结 构可简化地表示为NOTA-PEG n-Glu{PEG m-TATE}-PEG P-RGD，其中的m、n和p均取0-5的整数。
本发明优选的多肽化合物中，所述的TATE环肽结构、NOTA螯合基团和RGD环肽结构分别通过聚合度为2-5的PEG链段与同一个谷氨酸分子连接。
本发明进一步优选的多肽化合物中，所述的TATE环肽结构、NOTA螯合基团和RGD环肽结构分别通过PEG 4链段与所述的同一个谷氨酸分子连接。
本发明更进一步优选的多肽化合物中，所述的TATE环肽结构和RGD环肽结构分别通过PEG 4分子链段连接所述同一个谷氨酸分子的两个羧基端，形成稳定的酰胺键；所述的NOTA螯合基团通过PEG 4分子链段连接所述的同一个谷氨酸分子的氨基端；所述的多肽化合物记作NOTA-3PEG 4-TATE-RGD，其具体结构如下式(I)所示：
Figure PCTCN2018082782-appb-000001
本发明进一步提供一种TATE-RGD双靶点放射性分子探针，是放射性核素标记的多肽配合物，所述多肽配合物以本发明所述的具有双靶点的肿瘤靶向多肽化合物为配体。
本发明优选的TATE-RGD双靶点放射性分子探针中，所述的放射性核素选自 68Ga、 64Cu、 18F、 89Zr或 177Lu中的任意一种，进一步优选 68Ga、 64Cu或 18F。
本发明一种优选的实施方式中，所述的TATE-RGD双靶点放射性分子探针是 68Ga标记的多肽配合物，所述多肽配合物以本发明所述的具有双靶点的肿瘤靶向多肽化合物为配体，所述双靶点的肿瘤靶向多肽化合物的TATE环肽结构、NOTA螯合基团和RGD环肽结构分别通过聚合度为2-5的PEG链段与同一个谷氨酸分子连接。
本发明最优选的实施方式中，所述的TATE-RGD双靶点放射性分子探针是 68Ga标记的多 肽配合物，所述多肽配合物以本发明所述的具有双靶点的肿瘤靶向多肽化合物NOTA-3PEG 4-TATE-RGD为配体，所述的双靶点放射性分子探针简化地表示为 68Ga-NOTA-3PEG 4-TATE-RGD。
本发明的另一目的是提供一种制备所述的多肽化合物及双靶点放射性分子探针的方法，简便易行，产物稳定性更高。
本发明的上述目的是通过以下技术方案实现的：
1.一种制备所述具有双靶点的肿瘤靶向多肽化合物的方法，包括：
a)将保护的谷氨酸与多肽PEG n-TATE以摩尔比1-10:1-10的比例混合，在DIPEA和DECP作用下，利用氨基缩合反应得到TATE多肽与保护的谷氨酸通过PEG n链段连接的第一产物，其中n取0～5的整数；
b)将步骤a)得到的第一产物在哌啶条件下脱去保护基团Fmoc，得到第二产物，简单记作Glu-PEG n-TATE，其中n取0～5的整数；
c)将步骤b)得到的第二产物与NOTA-PEG m-NHS在DIPEA条件下反应，得到Boc保护的谷氨酸分别通过PEG  m链段和PEG n链段连接NOTA基团和TATE肽的第三产物，其中n和m分别取0～5的整数；
d)步骤c)得到的第三产物在TFA条件下脱去保护基团Boc，得到谷氨酸分别通过PEG  m链段和PEG n链段连接NOTA基团和TATE肽的第四产物，简单记作，NOTA-PEG m-Glu(PEG n-TATE)，其中n、m分别取0～5的整数；
e)将骤d)得到的第四产物和多肽PEG p-RGD在DIPEA条件下反应，其中p取0～5的整数，最终得到本发明所述的具有双靶点的肿瘤靶向多肽化合物NOTA-PEG n-Glu{PEG m-TATE}-PEG P-RGD。
2.制备所述的TATE-RGD双靶点放射性分子探针的方法，以制备 68Ga-NOTA-3PEG 4-TATE-RGD放射性药物为例，包括以下步骤：
将所述的NOTA-3PEG 4-TATE-RGD溶于去离子水中；用5mL 0.1mol/L高纯度盐酸溶液淋洗锗镓( 68Ge/ 68Ga)发生器(Ecker&Ziegler)至EP管中，收集其中放射性含量最高的1mL，加入1.25mol/L醋酸钠93μL将混合液pH值调至4.0～4.5；将20μg前体加入至混合液中并充分混匀，加热至100℃保持10min；反应结束后将反应液冷却至室温，并加入无菌注 射用水4mL后，通过无菌滤膜(0.22μm,13mm)过滤至无菌产品瓶中。
68Ga-NOTA-3PEG4-TATE-RGD放射性检测方法，包括：
分析性HPLC方法为使用高效液相色谱(美国Waters公司，515型泵)；紫外检测器(486型，UV吸收波长＝254nm)，放射性检测器(美国EG&G BERTHOLD公司)，放射性活度计CRC-25PET(美国Capintec公司)，Waters色谱柱Nova-Pak C18(4.6×150mm,5μm)；
进一步的，上步中分析型HPLC方法为使用Waters HPLC系统配备Waters C18分析柱(4.6mm×250mm)，HPLC梯度洗脱条件：0min，乙腈/水(5/95,v/v)；5min，乙腈/水(5/95,v/v)；10min，乙腈/水(80/20,v/v)；15min，乙腈/水(100/0,v/v)；18min，乙腈/水(100/0,v/v)；20min，乙腈/水(5/95,v/v)，洗脱剂中含有0.1％TFA，流速为1mL/min。从放射性HPLC图谱中观察， 68Ga-NOTA-3PEG4-TATE-RGD的保留时间(Retention Time)为11.6min，放化纯度(purity)>99％。
本发明还提供所述的TATE-RGD双靶点分子探针在SSTR2和/或整合素α vβ 3阳性显像诊断的放射性药物制备中的应用。
本发明优选的应用，是将所述的TATE-RGD双靶点分子探针制备成用于小细胞肺癌显像诊断的无色透明针剂。
本发明所述的TATE-RGD双靶点分子探针与现有技术相比的有益效果包括：
1.本发明TATE-RGD为双靶点多肽药物，将TATE和RGD相连，使其能够同时与生长抑素受体、整合素α vβ 3相结合，会增加结合亲和力以及肿瘤对药物的摄取，达到更好的肿瘤显像效果；
2.本发明在TATE和RGD多肽之间引入聚合度相同或不同的PEG分子时，可以改善所述多肽药物的药代动力学特性，尤其是从非肿瘤组织的清除速率；
3.本发明中使用NOTA作为螯合剂，相比于DOTA具有更好的体内和体外稳定性。
附图说明
图1为 68Ga-NOTA-3PEG 4-TATE-RGD高效液相色谱(HPLC)图谱。
图2为小细胞肺癌裸鼠H69模型microPET显像结果，图中从左至右分别为尾静脉注射 68Ga-NOTA-3PEG 4-TATE-RGD(Control)、 68Ga-NOTA-3PEG 4-TATE-RGD+RGD(Block+RGD)、 68Ga-NOTA-3PEG 4-TATE-RGD+TATE(Block+TATE)和 68Ga-NOTA-3PEG 4-TATE-RGD+RGD+TATE(Block+TATE+RGD)的显像结果。
图3为非小细胞肺癌裸鼠A549模型microPET显像结果，图中从左至右分别为尾静脉注射 68Ga-NOTA-3PEG 4-TATE-RGD(Control)、 68Ga-NOTA-3PEG 4-TATE-RGD+RGD(Block+RGD)、 68Ga-NOTA-3PEG 4-TATE-RGD+TATE(Block+TATE)和 68Ga-NOTA-3PEG 4-TATE-RGD+RGD+TATE(Block+TATE+RGD)的显像结果。
图4体现了正常小鼠注射 68Ga-NOTA-3PEG 4-TATE-RGD后不同时间点各脏器放射性摄取％ID/g。
图5为本发明的 68Ga-NOTA-3PEG 4-TATE-RGD与现有的 68Ga-NOTA-RGD探针在非小细胞肺癌患者中的显像结果比较，其中左边组图为发明的 68Ga-NOTA-3PEG 4-TATE-RGD的显像结果，右边为组图为现有的 68Ga-NOTA-RGD的显像结果，箭头指向病灶。
图6为 68Ga-NOTA-3PEG 4-TATE-RGD与现有的 68Ga-NOTA-RGD探针在小细胞肺癌患者中的显像结果比较，其中左边组图为发明的 68Ga-NOTA-3PEG 4-TATE-RGD的显像结果，右边为组图为现有的 68Ga-NOTA-RGD的显像结果，箭头指向病灶。
具体实施方式
为了更清楚地说明本发明的技术方案，以下结合附图及实例对本发明做进一步说明和阐述，但是本发明的技术方案并不限于下文所描述的某种具体实施方式和所列举的某些具体实施例。本发明以下实例中所使用的化学产品及试剂均为现有或市售产品。
实例1
一种具有双靶点的肿瘤靶向多肽化合物，其结构中包含TATE环肽结构、RGD环肽结构和NOTA螯合基团，它由所述的TATE环肽结构、NOTA螯合基团和RGD环肽结构分别连接同一个谷氨酸分子形成，其中所述的TATE环肽结构和RGD环肽结构分别通过PEG 4分子链段连接所述谷氨酸分子的两个羧基端，形成稳定的酰胺键；所述的NOTA螯合基团通过PEG 4分子连接所述的谷氨酸分子的氨基端；得到的多肽化合物记作NOTA-3PEG 4-TATE-RGD，其具体结构如下式(I)所示：
Figure PCTCN2018082782-appb-000002
制备所述NOTA-3PEG 4-TATE-RGD的方法包括以下步骤：
1)化合物Glu-PEG 4-TATE的合成：
将9.5mg Fmoc-Glu(Boc)-OH(Fmoc和Boc保护的谷氨酸)、25μL二异丙基乙胺(DIPEA)和5μL氯化二乙基磷酸(DECP)混合液加入到含有27.5mg PEG 4-TATE(市售肿瘤靶向多肽，溶于2.6mL二甲基甲酰胺(DMF))的20mL玻璃瓶中，混合溶解后在室温下搅拌2h，然后进行液相色谱和质谱联用(LC-MS)分析，结果显示得到Fmoc和Boc保护的氨基缩合多肽产物，记作中间化合物I；紧接着在中间化合物I溶液中加入0.6mL哌啶并继续在室温下搅拌1h，脱去中间化合物I的Fmoc保护基团，得到产物Glu-PEG 4-TATE。以HPLC分离纯化并经冷冻干燥后得到纯化合物约17.5mg，产率68％。
2)化合物NOTA-2PEG 4-TATE的合成：
将溶于2mL二甲亚砜(DMSO)的17.5mg步骤1)得到的化合物Glu-PEG 4-TATE和20μL DIPEA以及24.0mg NOTA-PEG 4-NHS(1,4,7-三氮杂环壬烷-N,N',N″-三乙酸活化酯，2倍当量)混合溶解。混合物在室温下搅拌20min并以HPLC监测反应进程。待上步化合物Glu-PEG 4-TATE消耗完后以LC-MS检测Boc保护的NOTA-PEG 4-TATE的生成。然后再加入0.1mL三氟乙酸(TFA)以脱去保护基团Boc得到分别通过PEG 4链段连接谷氨酸形成的目标化合物，简单记作NOTA-2PEG 4-TATE。冻干除去溶剂DMSO后的混合物以HPLC分离纯化，经冷冻干燥后得到目标产物约5.5mg，产率25.6％。
3)化合物NOTA-3PEG 4-TATE-RGD的合成：
在含有6.5mg步骤2)所得NOTA-2PEG 4-TATE(溶于1mL DMSO)的20mL玻璃反应 瓶中，加入6.0mg PEG 4-RGD(Arg-Gly-Asp环状多肽，市售)和10μL DIPEA混合溶解。混合物在室温下搅拌1h并以HPLC纯化，纯化条件：以4mL水稀释并制备型HPLC(配C18柱)分两次进样并按以下梯度在12mL/min流速下纯化，洗脱剂成分：Buffer A:0.1％TFA in H 2O；Buffer B 0.1％TFA in CH 3CN；梯度洗脱：0～20min：20～50％Buffer B。经冷冻干燥后得到产物约1.5mg，产率13.5％，纯度大于97％。经LC-MS鉴定产物：[(MHH)/2] ++＝2930.14，计算值(m/z)为2930.92(C 131H 193N 27O 43S 3)。确定为目标产物NOTA-Bn-p-SCN-PEG 4-Glu{PEG 4-Cyclo[Arg-Gly-Asp-(D-Tyr)-Lys]}-PEG 4-(D-Phe)-Cys-Tyr-(D-Trp)-Lys-Thr-Cys-Thr。
实例2
一种TATE-RGD双靶点放射性分子探针，包括TATE多肽、RGD多肽和放射性核素 68Ga，所述TATE和RGD多肽之间通过PEG 4分子连接形成TATE-3PEG 4-RGD多肽和 68Ga用NOTA连接，所述TATE-RGD双靶点放射性分子探针为 68Ga-NOTA-3PEG 4-TATE-RGD。
所述 68Ga-NOTA-3PEG 4-TATE-RGD双靶点放射性分子探针的制备方法，包括以下步骤：
(1) 68Ga的淋洗
用5mL注射器抽取0.1mol/L HCl 5mL，对锗镓发生器进行淋洗，缓慢淋洗，同时收集淋洗液至1.5mL EP管中，每管1mL，共5管。每个EP管进行放射性测定，去活度最大的一管用于标记；
(2) 68Ga-NOTA-3PEG 4-TATE-RGD的标记
取1.25mol/L的醋酸钠溶液加入至1mL 68Ga淋洗液中，将pH调节至4.0～4.5，混合均匀并加入实例1制备的NOTA-3PEG 4-TATE-RGD 20μg混合均匀，加热至100℃保持10min；反应结束后将反应液冷却至室温，并加入无菌注射用水4mL后，通过无菌滤膜(0.22μm,13mm)过滤至无菌产品瓶中；
(3)质量控制
HPLC分析条件:色谱柱为C18柱(4.6mm×250mm)，流动相A为已经(0.1％三氟乙酸),流动相B为水(0.1％三氟乙酸)，流速为1mL/min：0～5min，流动相A 5％；10min，流动相A为80％；15min，流动相A为100％；18min，流动相A 100％；20min，流动相A 5％。紫外检测波长为210nm，柱温20℃。放射性检测应用HPLC专用放射性探测器。HPLC分析 结果显示： 68Ga-NOTA-3PEG 4-TATE-RGD的保留时间(Retention Time)为11.6min(图1)，放化纯度>99％，无需进一步纯化。
(4)体外稳定性测定
68Ga-NOTA-3PEG 4-TATE-RGD标记后加入胎牛血清，分别在60和120min分别用HPLC测定其放射化学纯度。经测定，放射化学纯度分别为98％和97％。
将上述TATE-RGD双靶点探针 68Ga-NOTA-3PEG 4-TATE-RGD进一步制成无色透明针剂用于显像实验或显像诊断，具体实验及效果如下：
①小细胞肺癌H69荷瘤鼠的microPET显像
右前肢皮下接种H69肿瘤的荷瘤鼠，尾静脉注射实例2的 68Ga-NOTA-3PEG 4-TATE-RGD注射液100-200μCi，60min后使用Siemens Inveon micro PET采集10min静态图像(图2，其中箭头指示肿瘤部位)，结果显示，单独注射实例2探针时，肿瘤部位有明显的放射性摄取，肿瘤清晰可见，肿瘤摄取值为9.78±2.77；而通过共同注射过量未标记的前体RGD、TATE和RGD+TATE能够有效减少肿瘤的摄取，分别降低至8.23±1.08、1.41±0.73和1.05±0.13；当同时注射未标记前体RGD和TATE时候，肿瘤对放射性摄取几乎降低至本底。小细胞H69肿瘤以生长抑素受体表达为主，本发明双靶点分子探针在肿瘤中的浓集主要被未标记的TATE所抑制。
②非小细胞肺癌A549荷瘤鼠的microPET显像
右前肢皮下接种A549肿瘤的荷瘤鼠，尾静脉注射实例2的 68Ga-NOTA-3PEG 4-TATE-RGD注射液100-200μCi，60min后使用Siemens Inveon micro PET采集10min静态图像(图3，其中箭头指示肿瘤部位)，结果显示，单独注射实例2的探针时，肿瘤部位有明显的放射性摄取，肿瘤清晰可见，肿瘤摄取值为6.46±0.59；而通过共同注射过量未标记的前体RGD、TATE和RGD+TATE能够有效减少肿瘤的摄取，分别降低至1.75±0.53、3.80±0.48和1.35±0.26；当混合注射未标记前体RGD和TATE时候，肿瘤对放射性摄取几乎降低至本底。非小细胞A549肿瘤以整合素受体表达增高为主，本发明双靶点分子探针在肿瘤中的浓集主要被未标记的RGD所抑制。
④ 68Ga-NOTA-3PEG 4-TATE-RGD在健康小鼠体内的生物分布
健康Balb/c的分布见图4，实例2的 68Ga-NOTA-3PEG 4-TATE-RGD从血液、心脏、肝脏 中清除较快；肾脏放射性较多，说明该分子探针主要通过肾脏排泄。早期心脏、肝脏、肺有少量摄取，随时间延长，迅速减少。该显像剂在胃、肠道、脾脏、胰腺中有少量分布，脑组织社区很少，说明不能透过血脑屏障。
⑤ 68Ga-NOTA-3PEG 4-TATE-RGD在小细胞肺癌患者中的显像
临床确诊小细胞肺癌患者 68Ga-NOTA-3PEG 4-TATE-RGD PET/CT图像见图5，静脉注射本发明的 68Ga-NOTA-3PEG 4-TATE-RGD 3.5mCi，30min后使用Siemens Biograph64PET/CT采集躯干部位图像，3min/床位，共采集5个床位。病灶显示清晰，肿瘤最高标准摄取值(SUV max)为18.2(图5左)。而现有的单靶点显像剂 68Ga-NOTA-RGD仅见肿瘤部分区域摄取增高，SUV max值为4.7(图5右)
⑥ 68Ga-NOTA-3PEG 4-TATE-RGD在非小细胞肺癌患者中的显像
临床确诊非小细胞肺癌患者图像见图6，静脉注射本发明的 68Ga-NOTA-3PEG 4-TATE-RGD3.0mCi，30min后使用Siemens Biograph64PET/CT采集图像，3min/床位，共采集5个床位。病灶显示清晰，肿瘤最高标准摄取值(SUV max)为3.4(图6左)。而现有的单靶点显像剂 68Ga-NOTA-RGD肿瘤摄取低，SUV max值为2.8(图6右)。

Claims (10)

  1. 一种具有双靶点的靶向多肽化合物，其结构中包含TATE环肽结构、RGD环肽结构和NOTA螯合基团，所述TATE环肽结构、RGD环肽结构和NOTA螯合基团分别通过聚合度为1-5的PEG链段连接或直接连接同一个谷氨酸分子；所述的多肽化合物结构可简化地表示为NOTA-PEG n-Glu{PEG m-TATE}-PEG P-RGD，其中的m、n和p分别取0-5的整数。
  2. 权利要求1所述的多肽化合物，其特征在于：所述的TATE环肽结构、NOTA螯合基团和RGD环肽结构分别通过聚合度为2-5的PEG链段与同一个谷氨酸分子连接。
  3. 权利要求2所述的多肽化合物，其特征在于：所述的TATE环肽结构、NOTA螯合基团和RGD环肽结构分别通过PEG 4链段与所述的同一个谷氨酸分子连接。
  4. 权利要求1、2或3所述的任意一种多肽化合物，其特征在于：所述的TATE环肽结构和RGD环肽结构分别通过PEG 4分子链段连接所述同一个谷氨酸分子的两个羧基端，形成稳定的酰胺键；所述的NOTA螯合基团通过PEG 4分子链段连接所述的同一个谷氨酸分子的氨基端；所述的多肽化合物记作NOTA-3PEG 4-TATE-RGD，其具体结构如下式(I)所示：
    Figure PCTCN2018082782-appb-100001
  5. 一种TATE-RGD双靶点放射性分子探针，它是放射性核素标记的多肽配合物，其特征在于：所述多肽配合物以权利要求1所述的具有双靶点的靶向多肽化合物为配体。
  6. 权利要求5所述的TATE-RGD双靶点放射性分子探针，其特征在于：所述的放射性核素选自 68Ga、 64Cu、 18F、 89Zr或 177Lu中的任意一种；优选 68Ga、 64Cu或 18F；最优选 68Ga。
  7. 权利要求5所述的TATE-RGD双靶点放射性分子探针，其特征在于：它是放射性核素 68Ga标记的多肽配合物，所述多肽配合物以权利要求4所述的具有双靶点的靶向多肽化合物为 配体，所述的双靶点放射性分子探针简化地表示为 68Ga-NOTA-3PEG 4-TATE-RGD。
  8. 一种制备权利要求1所述的具有双靶点的靶向多肽化合物的方法，包括以下步骤：
    a)将保护的谷氨酸与多肽PEG n-TATE以摩尔比1-10:1-10的比例混合，在DIPEA和DECP作用下，利用氨基缩合反应得到TATE多肽与保护的谷氨酸通过PEG n链段连接的第一产物，其中n取0～5的整数；
    b)将步骤a)得到的第一产物在哌啶条件下脱去保护基团Fmoc，得到第二产物，简单记作Glu-PEG n-TATE，其中n取0～5的整数；
    c)将步骤b)得到的第二产物与NOTA-PEG m-NHS在DIPEA条件下反应，得到Boc保护的谷氨酸分别通过PEG m链段和PEG n链段连接NOTA基团和TATE肽的第三产物，其中n和m分别取0～5的整数；
    d)步骤c)得到的第三产物在TFA条件下脱去保护基团Boc，得到谷氨酸分别通过PEG m链段和PEG n链段连接NOTA基团和TATE肽的第四产物，简单记作，NOTA-PEG m-Glu(PEG n-TATE)，其中n、m分别取0～5的整数；
    e)将骤d)得到的第四产物和多肽PEG p-RGD在DIPEA条件下反应，其中p取0～5的整数，最终得到所述的具有双靶点的肿瘤靶向多肽化合物NOTA-PEG n-Glu{PEG m-TATE}-PEG P-RGD。
  9. 制备权利要求7所述的双靶点放射性分子探针的方法，包括以下步骤：
    将权利要求4所述的NOTA-3PEG 4-TATE-RGD溶于去离子水中；用5mL 0.1mol/L高纯度盐酸溶液淋洗锗镓( 68Ge/ 68Ga)发生器至EP管中，收集其中放射性含量最高的1mL，加入1.25mol/L醋酸钠93μL将混合液pH值调至4-4.5；将20μg前体加入至混合液中并充分混匀，加热至100℃保持10min；反应结束后将反应液冷却至室温，并加入无菌注射用水4mL后，通过无菌滤膜(0.22μm,13mm)过滤至无菌产品瓶中。
  10. 权利要求1所述的双靶点的靶向多肽化合物和权利要求5所述的TATE-RGD双靶点放射性分子探针在SSTR2和/或整合素α vβ 3阳性病变显像诊断的放射性药物制备中的应用；优选将所述的TATE-RGD双靶点分子探针制备成用于小细胞肺癌显像诊断的无色透明针剂。
PCT/CN2018/082782 2017-04-17 2018-04-12 双靶点显像分子探针及其制备方法和应用 WO2018192405A1 (zh)

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