WO2020238795A1 - 一种靶向HER2的rk多肽放射性药物及其制备方法 - Google Patents

一种靶向HER2的rk多肽放射性药物及其制备方法 Download PDF

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WO2020238795A1
WO2020238795A1 PCT/CN2020/091801 CN2020091801W WO2020238795A1 WO 2020238795 A1 WO2020238795 A1 WO 2020238795A1 CN 2020091801 W CN2020091801 W CN 2020091801W WO 2020238795 A1 WO2020238795 A1 WO 2020238795A1
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pkm
polypeptide
hynic
peg
radiopharmaceutical
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PCT/CN2020/091801
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French (fr)
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王凡
史继云
杜帅樊
贾兵
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北京大学
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Priority to US17/595,751 priority Critical patent/US20220211884A1/en
Priority to EP20814931.0A priority patent/EP3978033A4/en
Publication of WO2020238795A1 publication Critical patent/WO2020238795A1/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
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or 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
    • A61K51/06—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the present invention relates to a new type of tumor diagnostic radiopharmaceutical and a preparation method thereof, in particular to imaging diagnosis for patients with HER2-positive tumors, medication guidance and real-time efficacy monitoring for patients receiving anticancer drug trastuzumab.
  • Breast cancer is one of the most common malignant tumors in women, accounting for about 25% of women's malignant tumors, and has a trend of getting younger, seriously threatening women's life and health.
  • Human epidermal growth factor receptor 2 (human epidermal growth factor receptor 2, HER2) is a proto-oncogene. Abnormal amplification of HER2 and overexpression of its protein can lead to malignant transformation of cells, as well as the invasion, metastasis and invasion of breast cancer. Recurrence is closely related. In clinical cases, about 30% of breast cancer patients are HER2 positive.
  • trastuzumab is a humanized monoclonal antibody against HER2. It is a clinical first-line drug for the treatment of HER2-positive breast cancer. It can effectively improve the overall survival rate and is used in the treatment of early and late (metastatic) breast cancer Both showed efficacy. However, only some patients are sensitive to trastuzumab treatment, and after a period of treatment, patients treated with single antibody therapy and combined with other drugs will develop resistance to varying degrees. Therefore, it is particularly important to evaluate the changes in HER2 expression before and during treatment. Clinically, surgery or puncture biopsy is usually used to obtain pathological tissue, and then the expression level of HER2 is judged by IHC or FISH.
  • the biopsy is traumatic, and there is a high inconsistency rate of HER2 expression between the primary tumor and metastasis (6%- 48%), and the small sample size does not necessarily represent the HER2 expression status of the entire tumor. Based on this situation, molecular probes that can target the HER2 site show great advantages.
  • the KLRLEWNR sequence polypeptide has good HER2 targeting performance, and can effectively distinguish the HER2 expression of different tumor cells.
  • the optimized rk polypeptide drug (rnwelrlk) was synthesized.
  • the D-type polypeptide sequence will not be recognized by the protease in the body, which can effectively improve the metabolic stability in the body, thereby increasing the uptake of tumor tissue.
  • the rk polypeptide dimer introduces a long enough linking agent between the two polypeptides so that the two polypeptides in the dimer molecule have a long enough distance to bind to two HER2 targets at the same time, which is higher than the monomer. Affinity.
  • rk peptide drugs and trastuzumab bind to different sites of HER2 respectively, so they can be used to monitor the therapeutic effect of trastuzumab during the treatment of trastuzumab, regardless of the amount of medication, and it is accurate for patients Medication plays a key role.
  • the purpose of the present invention is to provide a novel polypeptide radiopharmaceutical targeting HER2-positive tumors.
  • the purpose of the present invention is achieved through the following technical solutions:
  • a HER2-targeted rk polypeptide radiopharmaceutical comprising an rk polypeptide dimer and a radionuclide, the radionuclide labels the rk polypeptide dimer through a chelating agent, and the rk polypeptide dimer is a combination of PKM and The rk polypeptide monomer is connected, and then two rk polypeptide monomers connected with PKM are dimerized to form a polypeptide dimer; the rk polypeptide monomer is a D-type amino acid linear 8-membered polypeptide, and its sequence is: Arg- Asn-Trp-Glu-Leu-Arg-Leu-Lys (arginine-asparagine-tryptophan-glutamic acid-leucine-arginine-leucine-lysine, abbreviated rnwelrlk);
  • the PKM stands for pharmacokinetic modifying molecule.
  • PKM is also connected between the rk polypeptide dimer and the chelating agent.
  • PKM is polyethylene glycol (PEGn) or 8-aminooctanoic acid (Aoc), and PEGn is preferably PEG 4 , PEG 6 , PEG 8 , and PEG 12 .
  • the radionuclide is any one of 99m Tc, 68 Ga, 64 Cu, 111 In, 90 Y and 177 Lu.
  • the chelating agent is any one of HYNIC, NOTA, DOTA and DTPA.
  • rk polypeptide radiopharmaceutical is a colorless and transparent liquid injection.
  • the radionuclide 99m Tc is a D-type amino acid linear 8-membered polypeptide rnwelrlk
  • the rk polypeptide dimer is to connect PEG4 or Aoc to the rk polypeptide monomer, and then connect the two A rk polypeptide dimer synthesized by dimerization of rk polypeptide monomers with PEG 4 or Aoc.
  • the radionuclide 99m Tc is labeled with a bifunctional chelating agent HYNIC.
  • the rk polypeptide radiopharmaceutical is a colorless and transparent liquid injection.
  • the drug first connects PKM to the D-type polypeptide rnwelrlk, and then dimerizes the dimerization so that the two polypeptides in the dimer molecule have a long enough distance to bind to two HER2 targets at the same time. While improving pharmacokinetic properties, it also improves tumor targeting. The combination of this bivalent form can further enhance tumor uptake of drugs and achieve better diagnostic results.
  • the drug uses a bifunctional chelating agent to label the radionuclide 99m Tc onto the rk polypeptide dimer molecule, and the labeled drug is concentrated to the tumor site through the targeting effect of the rk polypeptide in the body, using nuclear medicine single photon tomography technology , Perform imaging diagnosis of HER2-positive tumors.
  • a preparation method of rk polypeptide radiopharmaceutical includes the following steps:
  • Dissolve (PKM-rk-Dde) 2 -Glu and HYNIC-PKM-OSu in DMF add DIEA to adjust the pH to 8.5-9.0, stir overnight at room temperature, the crude product is separated and purified by YMC-Pack ODS-A semi-preparative column HPLC , Collect the fractions of the target, combine the collection solutions and freeze-dry to obtain the expected product HYNIC-PKM-(PKM-rk-Dde) 2 ; Dissolve HYNIC-PKM-(PKM-rk-Dde) 2 in a volume fraction of 2% to hydrate The DMF solution of hydrazine was reacted at room temperature for 30 minutes.
  • the crude product was separated and purified by YMC-Pack ODS-A semi-preparative column HPLC. The fractions of the target were collected, combined and lyophilized. The obtained product was confirmed by MALDI-TOF-MS analysis. It is the expected product HYNIC-PKM-(PKM-rk) 2 ;
  • TPTS triphenylphosphine sodium trisulfonate
  • tricine trimethylolglycine
  • disodium succinate succinic acid
  • HYNIC-PKM-(PKM-rk) 2 HYNIC-PKM-(PKM-rk) 2 .
  • the mass ratio of the substances is: 4-6:6-7:38-39:12-13:0.04, and the mixture is freeze-dried.
  • Add 1ml of Na 99m TcO 4 solution to the lyophilized powder heat the reaction in a water bath at 100°C for 20-25 minutes, and cool at room temperature after the reaction is completed to prepare rk polypeptide radiopharmaceutical. Analyze by HPLC for later use.
  • the HPLC method is using Agilent 1260 HPLC system equipped with YMC-Pack ODS-A semi-preparative column (250 ⁇ 10mm, IDS-5 ⁇ m, 12nm) or analytical column (250 ⁇ 4.6mm, IDS-5 ⁇ m, 12nm), gradient elution 30 Minutes, the mobile phase A is deionized water (containing 0.05% TFA) and the mobile phase B is acetonitrile (containing 0.05% TFA).
  • Step a Equipped with a semi-preparative column with a flow rate of 4 mL/min, and the elution gradient is set to 90% A and 10% B at the beginning, 50% A and 50% B at 25 minutes, and 90% A and 10% at 30 minutes B.
  • Step b equipped with analytical column, flow rate 1mL/min, elution gradient is 90% A and 10% B at the beginning, 30% A and 70% B at 20 minutes.
  • the rk polypeptide radiopharmaceutical is used for imaging diagnosis of HER2-positive tumor patients, as well as medication guidance and real-time curative effect monitoring for patients receiving anticancer drug trastuzumab.
  • the D-type polypeptide sequence will not be recognized by the protease in the body, and can effectively improve the metabolic stability in the body, thereby increasing the uptake of tumor tissue.
  • the rk polypeptide radiopharmaceutical of the present invention firstly connects four polyethylene glycol molecules (PEG4) or 8-aminooctanoic acid (Aoc) with the D-type polypeptide monomer, and then dimerizes the dimer molecule.
  • PEG4 polyethylene glycol molecules
  • Aoc 8-aminooctanoic acid
  • the two peptides in the two peptides have a long enough distance to bind two HER2 targets at the same time. This bivalent form of combination can further enhance the tumor's uptake of drugs and achieve better diagnostic results.
  • the present invention not only introduces PKM (PEG 4 or Aoc) between two rk polypeptides, but also introduces PKM between the bifunctional chelating agent HYNIC for radionuclide labeling and the rk polypeptide dimer targeted by HER2 HYNIC-PKM-(PKM-rk) 2 improves the biocompatibility of the probe and optimizes the pharmacokinetic properties, especially the clearance kinetics from non-tumor tissues.
  • PKM PEG 4 or Aoc
  • HYNIC is used as a bifunctional chelating agent, and tricine and TPPTS are used as co-ligands at the same time, so that the " 99m Tc-HYNIC core" has better in vivo and in vitro stability.
  • FIG. 1 Schematic diagram of the structure of 99m Tc-HYNIC-PKM-(PKM-rk) 2 marker. Where rnw represents rk polypeptide.
  • Figure 4 SPECT/CT imaging in NOD SCID mouse SKBR3 breast cancer model after injection of 99m Tc-HYNIC-PEG 4 -(Aoc-rk )2 0.5h, 1h, and 2h.
  • FIG. 5 (A) SPECT/CT imaging of experimental group, cold peptide blocking group and antibody blocking group in SKBR3 breast cancer model after injection of 99m Tc-HYNIC-PEG 4 -(Aoc-rk) 2 0.5h ; (B) 0.5h SPECT/CT image of 99m Tc-HYNIC-PEG 4- rk in SKBR3. Where rnw represents rk polypeptide.
  • FIG. 7 99m Tc-HYNIC-PEG 4 -(Aoc-rk) 2 in (A) SKBR3 tumor model (HER2 high expression), (B) HT29 tumor model (HER2 moderate expression), (C) BxPC3 tumor model ( HER2 low expression) SPECT/CT imaging.
  • Figure 8 In vivo distribution results in the SKBR3 breast cancer model after injection of 99m Tc-HYNIC-PEG 4 -(Aoc-rk) 2 0.5h and 1h. Where rnw represents rk polypeptide.
  • FIG. 9 SKBR3 breast cancer model injected with 99m Tc-HYNIC-PEG 4 -(PEG 4 -rk) 2 (A) 0.5h, 1h, 2h after SPECT/CT imaging; (B) 0.5h cold peptide blockade Group SPECT/CT imaging. Where rnw represents rk polypeptide.
  • FIG. 10 Imaging comparison and quantitative data analysis results of HER2 positive model (SKBR3 breast cancer model);
  • Figure 14 (A) Cell flow cytometry; (B) Representative image of SPECT/CT imaging; (C) Correlation between HER2 expression and tumor uptake;
  • HYNIC-NHS (hydrazine amide) was purchased from Noca- Biochem Company.
  • PEG4-rnwelrlk and Aoc-rnwelrlk peptide monomers were purchased from China Gil Biochemical Company.
  • Na99mTcO4 eluate was purchased from Beijing Atom High-Tech Co., Ltd.
  • a HER2-targeted rk polypeptide radiopharmaceutical comprising an rk polypeptide dimer and a radionuclide, the radionuclide labels the rk polypeptide dimer by a chelating agent, and the rk polypeptide dimer is a combination of PKM and The rk polypeptide monomer is connected, and then two rk polypeptide monomers connected with PKM are dimerized to form a polypeptide dimer; the rk polypeptide monomer is a D-type amino acid linear 8-membered polypeptide, and its sequence is: Arg- Asn-Trp-Glu-Leu-Arg-Leu-Lys (arginine-asparagine-tryptophan-glutamic acid-leucine-arginine-leucine-lysine, abbreviated rnwelrlk);
  • the PKM represents a modified pharmacokinetic molecule, and the chemical structural formula of the polypeptide is shown in
  • PKM is also connected between the rk polypeptide dimer and the chelating agent.
  • PKM is polyethylene glycol (PEG n ) or 8-aminocaprylic acid (Aoc), and PEGn is preferably PEG 4 , PEG 6 , PEG 8 , and PEG 12 .
  • the radionuclide is any one of 99m Tc, 68 Ga, 64 Cu, 111 In, 90 Y and 177 Lu.
  • the chelating agent is any one of HYNIC, NOTA, DOTA and DTPA.
  • the rk polypeptide radiopharmaceutical is a colorless and transparent liquid injection.
  • 99m Tc-HYNIC-PEG 4 -(Aoc-rk) 2 polypeptide radiopharmaceutical and its preparation method are taken as an example.
  • the rk polypeptide monomer is a D-type amino acid linear polypeptide rnwelrlk
  • the rk polypeptide dimer is the linking agent Aoc and the rk polypeptide monomer, and then the two are connected
  • the rk polypeptide dimer formed by the dimerization of rk polypeptide monomer with Aoc, the radionuclide 99m Tc is labeled with a bifunctional chelating agent HYNIC, the rk polypeptide dimer and the bifunctional chelating agent
  • PEG4 pharmacokinetic modification molecule
  • the rk polypeptide radiopharmaceutical is 99m Tc-HYNIC-PEG 4 -(Aoc-rk) 2 , and the structural formula of the marker is shown in Figure 2.
  • the rk polypeptide radiopharmaceutical It is a colorless and transparent liquid injection.
  • HYNIC-PEG 4 -COOH Dissolve Fmoc protected PEG 4 -COOH in DMF, add piperidine to make the final concentration 20%, react at room temperature for 20 minutes, add 10ml of 4°C ether to precipitate PEG 4 -COOH, 4000rpm Centrifuge at 4°C for 5 minutes, discard the supernatant, wash the precipitate 3 times with ether at 4°C, and remove the residual ether by rotary evaporation to obtain the product as NH 2 -PEG 4 -COOH; combine HYNIC-NHS and NH 2 -PEG 4 -COOH Dissolve in DMF, add DIEA to adjust the pH to 8.5-9.0, stir overnight at room temperature, the crude product is separated and purified by YMC-Pack ODS-A semi-preparative column HPLC.
  • the HPLC method used Agilent 1260HPLC system equipped with YMC-Pack ODS-A semi-preparative column (250 ⁇ 10mm, IDS-5 ⁇ m, 12nm), gradient elution for 30 minutes, flow rate 4mL/min, and mobile phase A was deionized water (containing 0.05% TFA), mobile phase B is acetonitrile (containing 0.05% TFA).
  • the elution gradient is set to 90% A and 10% B at the beginning, 50% A and 50% B at 25 minutes, and 90% A and 10% B at 30 minutes.
  • HYNIC-PEG 4 -OSu Dissolve HYNIC-PEG 4 -COOH in DMF, add NHS and EDC ⁇ HCl, stir at room temperature for 7 hours, add a volume fraction of 50% ACN aqueous solution to the reaction solution and filter, the filtrate is passed through YMC -Pack ODS-A semi-preparative column HPLC separation and purification.
  • the HPLC method used Agilent 1260HPLC system equipped with YMC-Pack ODS-A semi-preparative column (250 ⁇ 10mm, IDS-5 ⁇ m, 12nm), gradient elution for 30 minutes, flow rate 4mL/min, and mobile phase A was deionized water (containing 0.05% TFA), mobile phase B is acetonitrile (containing 0.05% TFA).
  • the elution gradient is set to 90% A and 10% B at the beginning, 50% A and 50% B at 25 minutes, and 90% A and 10% B at 30 minutes.
  • (AOC-rk-Dde) 2 -Glu preparation Dissolve AOC-rk-Dde and OSu 2 -Glu-Boc in DMF, add DIEA to adjust the pH to 8.5-9.0, stir overnight at room temperature, the crude product is subjected to YMC-Pack ODS-A semi-preparative column HPLC separation and purification.
  • the HPLC method used Agilent 1260HPLC system equipped with YMC-Pack ODS-A semi-preparative column (250 ⁇ 10mml.DS-5 ⁇ m, 12nm), gradient elution for 30 minutes, flow rate 4mL/min, and mobile phase A was deionized water (containing 0.05% TFA), mobile phase B is acetonitrile (containing 0.05% TFA).
  • the elution gradient is set to 90% A and 10% B at the beginning, 50% A and 50% B at 25 minutes, and 90% A and 10% B at 30 minutes.
  • the HPLC method was Agilent 1260 HPLC system equipped with YMC-Pack ODS-A semi-preparative column (250 ⁇ 10mm, IDS-5 ⁇ m, 12nm), gradient elution for 30 minutes , The flow rate is 4mL/min, the mobile phase A is deionized water (containing 0.05% TFA), and the mobile phase B is acetonitrile (containing 0.05% TFA).
  • the elution gradient is set to 90% A and 10% B at the beginning, 50% A and 50% B at 25 minutes, and 90% A and 10% B at 30 minutes.
  • HPLC Purification HPLC method is to use Agilent 1260HPLC system equipped with YMC-Pack ODS-A semi-preparative column (250 ⁇ 10mm, IDS-5 ⁇ m, 12nm), gradient elution for 30 minutes, flow rate 4mL/min, and mobile phase A is deionized water (Containing 0.05% TFA), mobile phase B is acetonitrile (containing 0.05% TFA).
  • the elution gradient is set to 90% A and 10% B at the beginning, 50% A and 50% B at 25 minutes, and 90% A and 10% B at 30 minutes.
  • Tc-HYNIC-PEG 4 -(Aoc-rk) 2 preparation preparation containing triphenylphosphine sodium trisulfonate (TPPTS) 5.0mg, trimethylolglycine (tricine) 6.5mg, disodium succinate 38.5mg 500 ⁇ L of a mixture of 12.7 mg of succinic acid and 50 ⁇ g of HYNIC-PEG 4 -(Aoc-rk) 2 was placed in a 10 mL vial, and the mixture was lyophilized.
  • TPTS triphenylphosphine sodium trisulfonate
  • the rk polypeptide radiopharmaceuticals were sampled for radioactive HPLC analysis.
  • the HPLC method used Agilent 1260HPLC system equipped with YMC-Pack ODS-A analytical column (250 ⁇ 4.6mm, IDS-5 ⁇ m, 12nm), gradient elution for 20 minutes, flow rate 1mL/min, and mobile phase A was deionized water (containing 0.05% TFA), mobile phase B is acetonitrile (containing 0.05% TFA).
  • the elution gradient is set to 90% A and 10% B at the beginning, and 30% A and 70% B at 20 minutes.
  • the labeling rate of 99m Tc-HYNIC-PEG 4 -(Aoc-rk) 2 >95%, and the radiochemical purity>98% after purification by Sep-Pak C18 column.
  • 99m Tc-HYNIC-PEG 4 -(Aoc-rk) 2 SPECT/CT imaging results in tumor-bearing mice are shown in Figure 4:
  • 99m Tc-HYNIC-PEG 4 -(Aoc-rk) 2 SPECT/CT imaging results in tumor-bearing mice are shown in Figure 4:
  • 99m Tc-HYNIC-PEG 4 -(Aoc-rk) 2 SPECT/CT imaging results in tumor-bearing mice are shown in Figure 4:
  • 99m Tc-HYNIC-PEG 4 -(Aoc-rk) 2 SPECT/CT imaging results in tumor-bearing mice are shown in Figure 4:
  • 99m Tc-HYNIC-PEG 4 -(Aoc- The tumor uptake of rk) 2 is clearly visible, which is significantly higher than that of the monomer, and the whole body background is clean, which can be used for early tumor diagnosis.
  • the polypeptide stays in the tumor site for a long time, and
  • the tumor uptake was significantly reduced, indicating the specific binding of the polypeptide to the HER2 site.
  • Herceptin blocking experimental group the tumor uptake was basically unchanged, indicating that the binding sites of the probe and the polypeptide are different, which can be used for real-time monitoring of the therapeutic effect of Herceptin treatment patients.
  • the SPECT/CT imaging of pancreatic cancer tumors is shown in Figure 7.
  • the results show that the uptake of 99m Tc-HYNIC-PEG 4 -(Aoc-rk) 2 is linearly correlated with the expression level of HER2, which can be used to detect the expression of HER2 in tumors happening.
  • Cell flow cytometry The above cells were digested with 0.25% EDTA/trypsin and blocked for 15 minutes. After centrifugation and washing with PBS, they were divided into experimental group and control group. The experimental group was added with PE direct-labeled anti-human HER2 antibody (1:100 dilution ), the control group was resuspended in an equal volume of PBS, incubated at 4°C for 0.5h in the dark, washed 3 times with cold PBS, analyzed by flow cytometry, and calculated the average fluorescence intensity of each antibody bound on each cell by flow cytometry (MFI).
  • MFI flow cytometry
  • SPECT/CT imaging select different tumor models with similar tumor sizes, inject 1mCi 99m Tc-HYNIC-PEG 4 -(Aoc-rk) 2 via the tail vein, and perform SPECT/CT imaging 0.5h after injection.
  • the SPECT image was reconstructed and fused with the CT image to obtain a 3D image of the mouse.
  • InVivoScope software was used to deduct the tumor site for quantitative analysis.
  • 99m Tc-HYNIC-PEG 4 -(Aoc-rk) 2 SPECT/CT imaging in breast cancer patients with the approval and informed consent of the Ethical Review Committee of Peking Union Medical College Hospital, 20 people were recruited to be suspected of mammography or ultrasound examination
  • SPECT/CT and PET/CT imaging were performed 1 week before surgery.
  • the patient received an intravenous injection of 11.1MBq (0.3mCi)/kg body weight of 99m Tc-HYNIC-PEG 4 -(Aoc-rk) 2 , 30 minutes later, SPECT/CT scan; intravenous injection of 5.6MBq (0.15mCi)/kg body weight of 18 F -FDG, PET/CT scan 60min later.
  • 99m Tc-HYNIC-PEG 4 -(PEG 4 -rk) 2 polypeptide radiopharmaceutical and its preparation method are taken as an example.
  • the rk polypeptide monomer is a D-type amino acid linear polypeptide rnwelrlk
  • the rk polypeptide dimer is the linking agent PEG4 and the rk polypeptide monomer, and then two The rk polypeptide dimer is formed by dimerization of rk polypeptide monomer connected with PEG 4 , and the radionuclide 99m Tc is labeled with a bifunctional chelating agent HYNIC.
  • the rk polypeptide dimer and bifunctional A pharmacokinetic modification molecule PEG 4 is also connected between the chelating agents.
  • the rk polypeptide radiopharmaceutical is 99m Tc-HYNIC-PEG 4 -(PEG 4 -rk) 2
  • the rk polypeptide radiopharmaceutical is a colorless and transparent liquid injection.
  • HYNIC-PEG 4 -COOH Dissolve Fmoc protected PEG 4 -COOH in DMF, add piperidine to make the final concentration 20%, react at room temperature for 20 minutes, add 10ml of 4°C ether to precipitate PEG 4 -COOH, 4000rpm Centrifuge at 4°C for 5 minutes, discard the supernatant, wash the precipitate 3 times with ether at 4°C, and remove the residual ether by rotary evaporation to obtain the product as NH 2 -PEG 4 -COOH; combine HYNIC-NHS and NH 2 -PEG 4 -COOH Dissolve in DMF, add DIEA to adjust the pH to 8.5-9.0, stir overnight at room temperature, the crude product is separated and purified by YMC-Pack ODS-A semi-preparative column HPLC.
  • the HPLC method used Agilent 1260HPLC system equipped with YMC-Pack ODS-A semi-preparative column (250 ⁇ 10mm, IDS-5 ⁇ m, 12nm), gradient elution for 30 minutes, flow rate 4mL/min, and mobile phase A was deionized water (containing 0.05% TFA), mobile phase B is acetonitrile (containing 0.05% TFA).
  • the elution gradient is set to 90% A and 10% B at the beginning, 50% A and 50% B at 25 minutes, and 90% A and 10% B at 30 minutes.
  • HYNIC-PEG 4 -OSu Dissolve HYNIC-PEG 4 -COOH in DMF, add NHS and EDC ⁇ HCl, stir at room temperature for 7 hours, add a volume fraction of 50% ACN aqueous solution to the reaction solution and filter, the filtrate is passed through YMC -Pack ODS-A semi-preparative column HPLC separation and purification.
  • the HPLC method used Agilent 1260HPLC system equipped with YMC-Pack ODS-A semi-preparative column (250 ⁇ 10mm, IDS-5 ⁇ m, 12nm), gradient elution for 30 minutes, flow rate 4mL/min, and mobile phase A was deionized water (containing 0.05% TFA), mobile phase B is acetonitrile (containing 0.05% TFA).
  • the elution gradient is set to 90% A and 10% B at the beginning, 50% A and 50% B at 25 minutes, and 90% A and 10% B at 30 minutes.
  • the HPLC method used Agilent 1260HPLC system equipped with YMC-Pack ODS-A semi-preparative column (250 ⁇ 10mm, IDS-5 ⁇ m, 12nm), gradient elution for 30 minutes, flow rate 4mL/min, and mobile phase A was deionized water (containing 0.05% TFA), mobile phase B is acetonitrile (containing 0.05% TFA).
  • the elution gradient is set to 90% A and 10% B at the beginning, 50% A and 50% B at 25 minutes, and 90% A and 10% B at 30 minutes.
  • HYNIC-PEG 4 -(PEG 4 -rk) 2 Dissolve (PEG 4 -rk-Dde) 2 -Glu and HYNIC-PEG 4 -OSu in DMF, add DIEA to adjust the pH to 8.5-9.0, and stir at room temperature Overnight, the crude product was separated and purified by YMC-Pack ODS-A semi-preparative column HPLC using Agilent 1260 HPLC system equipped with YMC-Pack ODS-A semi-preparative column (250 ⁇ 10mm, IDS-5 ⁇ m, 12nm), gradient elution 30 minutes, the flow rate is 4mL/min, the mobile phase A is deionized water (containing 0.05% TFA), and the mobile phase B is acetonitrile (containing 0.05% TFA).
  • HPLC method is to use Agilent 1260HPLC system equipped with YMC-Pack ODS-A semi-preparative column (250 ⁇ 10mm, IDS-5 ⁇ m, 12nm), gradient elution for 30 minutes, flow rate 4mL/min, of which the mobile phase A is the Ionized water (containing 0.05% TFA), mobile phase B is acetonitrile (containing 0.05% TFA).
  • the elution gradient is set to 90% A and 10% B at the beginning, 50% A and 50% B at 25 minutes, and 90% A and 10% B at 30 minutes.
  • Tc-HYNIC-PEG 4 -(PEG4-rk) 2 preparation preparation containing triphenylphosphine trisulfonate (TPPTS) 5.0mg, trimethylolglycine (tricine) 6.5mg, disodium succinate 38.5mg 500 ⁇ L of a mixture of 12.7 mg of succinic acid and 50 ⁇ g of HYNIC-PEG 4 -(PEG 4 -rk) 2 was placed in a 10 mL vial, and the mixture was lyophilized.
  • TPTS triphenylphosphine trisulfonate
  • the rk polypeptide radiopharmaceuticals were sampled for radioactive HPLC analysis.
  • the HPLC method used Agilent 1260HPLC system equipped with YMC-Pack ODS-A analytical column (250 ⁇ 4.6mm, IDS-5 ⁇ m, 12nm), gradient elution for 20 minutes, flow rate 1mL/min, and mobile phase A was deionized water (containing 0.05% TFA), mobile phase B is acetonitrile (containing 0.05% TFA).
  • the elution gradient is set to 90% A and 10% B at the beginning, and 30% A and 70% B at 20 minutes.
  • the labeling rate of 99m Tc-HYNIC-PEG 4 -(PEG 4 -rk) 2 is >95%, and the radiochemical purity is >98% after purification by Sep-Pak C 18 column.
  • the Chinese patent with publication number CN 109045313A discloses a compound 99m Tc-based on the refvffly polypeptide sequence (the sequence of D-type amino acid linear polypeptide is: Arg-Glu-Phe-Val-Phe-Phe-Leu-Tyr, referred to as ref polypeptide)
  • the present invention is improved on the basis of the compound.
  • the polypeptide selected in the present invention has better water solubility than refvffly polypeptide.
  • the target specificity of the improved compound is better than 99m Tc-HYNIC-PKM-ref. See Figure 10 for details.
  • Figure 10A is the result of imaging comparison and quantitative analysis of the same model.
  • Figure 10B is the result of quantitative analysis from Figure 10A.
  • the imaging agent 99m Tc-HYNIC-PEG 4 -(Aoc-rk) 2 of the present invention (abbreviated as 99m Tc-HPArk2 in the drawing) is relative to 99m Tc-HYNIC in CN 109045313A.
  • -PEG 4 -ref 99m Tc-HP 4 -ref for short
  • 99m Tc-HYNIC-PEG 24 -ref 99m Tc-HP 24 -ref for short
  • the tumor uptake of 99m Tc-HPArk2 is higher than that of 99m Tc-HP 24 -ref, which is higher than that of 99m There was no significant difference in Tc-HP 4 -ref.
  • the liver uptake of 99m Tc-HPArk2 is the lowest, which is lower than both 99m Tc-HP 4 -ref and 99m Tc-HP 24 -ref. Therefore, 99m Tc-HPArk2 has the highest tumor to liver ratio. This is particularly critical for imaging breast cancer tumors in situ in the thoracic cavity. Therefore, the 99m Tc-HPArk2 imaging agent of the present invention has more advantages in imaging breast cancer tumors in situ.
  • FIG. 11 The specific difference between the specific in vivo pharmacokinetic distribution of radiopharmaceuticals in the present invention and the patent document with the publication number CN 109045313 A is shown in FIG. 11.
  • the tumor uptake and contrast in FIG. 11 correspond to the imaging data in FIG. 10.
  • Figure 11 is quantitative data
  • Figure 10 is visual image data.
  • Figure 11B is the result of the calculation in Figure 11A.
  • the uptake value of the tumor and each organ was compared and analyzed. It can be seen from Figure 11 that compared with 99m Tc-HP 4 -ref and 99m Tc-HP 24 -ref, the radiopharmaceutical 99m Tc-HPArk2 of the present invention has relatively higher uptake in tumors, which helps to improve the sensitivity of tumor imaging.
  • the specific detection of the radiopharmaceutical for HER2 was performed. It has good specificity no matter at the protein level or at the cell level.
  • the radiopharmaceutical published in the patent document with publication number CN 109045313 A can image the HER2-negative EGFR-positive MDA-MB-468 breast cancer model
  • the distinguishing effect is not as good as the medicine of the present invention.
  • the medicament of the present invention is negative, the discrimination is higher, and no false positive result is obtained because of EGFR expression. It can be seen that the radiopharmaceutical of the present invention has obvious advantages in distinguishing tumors with high expression of HER2 and EGFR, reduces the generation of false positive results, and has obvious advantages.

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Abstract

本发明公开了一种靶向HER2的rk多肽放射性药物及其制备方法，包括rk多肽二聚体和放射性核素，放射性核素通过螯合剂标记rk多肽二聚体，rk多肽二聚体是将PKM与rk多肽单体相连，再将两个连接有PKM的rk多肽单体二聚化而成的多肽二聚体；rk多肽单体为D型氨基酸线性8元多肽，其序列为：Arg-Asn-Trp-Glu-Leu-Arg-Leu-Lys；PKM表示药代动力学修饰分子。该药物用于HER2阳性肿瘤患者的显像诊断，以及对接受抗癌药物曲妥珠单抗治疗患者的用药指导和实时疗效监测。

Description

一种靶向HER2的rk多肽放射性药物及其制备方法 技术领域
本发明涉及一种新型肿瘤诊断放射性药物及其制备方法，特别涉及用于HER2阳性肿瘤患者的显像诊断，以及对接受抗癌药物曲妥珠单抗治疗患者的用药指导和实时疗效监测。
背景技术
乳腺癌是女性最常见的恶性肿瘤之一，约占女性恶性肿瘤的25％，且有年轻化趋势，严重威胁了女性的生命健康。人类表皮生长因子受体2(human epidermal growth factor receptor 2，HER2)是一种原癌基因，HER2的异常扩增及其蛋白的过表达可导致细胞的恶性转化，与乳腺癌的浸润、转移和复发息息相关。在临床病例中，约30％的乳腺癌患者为HER2阳性。
有统计数据显示，乳腺癌早期患者的治愈率极高，但由于很多肿瘤早期症状不明显，确诊时已经错过了最佳治疗时期，而可以观察细胞和分子水平的分子影像学的出现使肿瘤的早期诊断成为了可能。其中，核医学成像(PET，SPECT)凭借其灵敏度高，组织穿透能力强，可进行在体定量，多种核素可供选择的优点，在临床上起到越来越重要的作用。
曲妥珠单抗是一种针对HER2的人源化单克隆抗体，是临床上治疗HER2阳性乳腺癌的一线用药，能有效提高总体生存率，在早期和晚期(转移性)乳腺癌的治疗中均显示出疗效。但是，仅有一部分患者对曲妥珠单抗治疗敏感，并且治疗一段时间后，单一抗体治疗和联合其他药物治疗的患者均会出现不同程度的耐药。因此，在治疗前和治疗期间评估HER2表达水平的变化就显得格外重要。临床上通常采用手术或者穿刺活检获得病理组织，然后通过IHC或FISH判断HER2的表达水平，但是活检有创伤，并且原发肿瘤与转移灶之间HER2表达水平有较高的不一致率(6％-48％)，以及小样本量不一定代表整个肿瘤的HER2表达状态。基于这种情况，能够靶向HER2位点的分子探针就显示出巨大的优势。
研究证实KLRLEWNR序列多肽具有很好的HER2靶向性能，能够有效地分辨出不同肿瘤细胞的HER2表达情况。本实验合成优化的rk多肽药物(rnwelrlk)，D型多肽序列不会被体内的蛋白酶识别，能够有效的提高体内代 谢稳定性，从而提高肿瘤组织的摄取。此外，rk多肽二聚体在两个多肽之间引入足够长的连接剂，使二聚体分子中的两个多肽有足够长的距离能够同时结合两个HER2靶点，比单体具有更高的亲和力。在用于放射性核素标记的双功能螯合剂HYNIC与rk多肽二聚体之间加入了PKM，优化了药代动力学性质，以达到更好的诊治效果。值得注意的是，rk多肽药物与曲妥珠单抗分别结合在HER2的不同位点，因此可用于在曲妥珠单抗治疗过程中的疗效监测，而不受用药量的影响，对患者精准用药起到关键作用。
发明内容
本发明的目的在于提供一种新型的靶向HER2阳性肿瘤的多肽放射性药物。本发明的目的是通过如下技术方案实现：
一种靶向HER2的rk多肽放射性药物，包括rk多肽二聚体和放射性核素，所述放射性核素通过螯合剂标记所述rk多肽二聚体，所述rk多肽二聚体是将PKM与rk多肽单体相连，再将两个连接有PKM的rk多肽单体二聚化而成的多肽二聚体；所述rk多肽单体为D型氨基酸线性8元多肽，其序列为：Arg-Asn-Trp-Glu-Leu-Arg-Leu-Lys(精氨酸-天门冬酰胺-色氨酸-谷氨酸-亮氨酸-精氨酸-亮氨酸-赖氨酸，简写rnwelrlk)；所述PKM表示药代动力学修饰分子。
进一步的，所述rk多肽二聚体与所述螯合剂之间还连接有PKM。
进一步的，PKM为聚乙二醇(PEGn)或者为8-氨基辛酸(Aoc)，PEGn优选PEG 4、PEG 6、PEG 8、PEG 12。
进一步的，所述放射性核素为 99mTc， 68Ga， 64Cu， 111In， 90Y和 177Lu中任意一种。
进一步的，螯合剂为HYNIC，NOTA，DOTA和DTPA中任意一种。
进一步的，所述rk多肽放射性药物为无色透明液体针剂。
该药物的优选方案：放射性核素 99mTc，所述rk多肽为D型氨基酸线性8元多肽rnwelrlk，所述rk多肽二聚体是将PEG4或Aoc与rk多肽单体连接，再将两个连接有PEG 4或Aoc的rk多肽单体二聚化而合成的rk多肽二聚体，所述放射性核素 99mTc通过一个双功能螯合剂HYNIC标记所述rk 多肽二聚体，所述rk多肽二聚体与所述双功能螯合剂之间还连接有药代动力学修饰分子PKM(PKM＝Aoc或PEG4)，所述rk多肽放射性药物为 99mTc-HYNIC-PKM-(PKM-rk) 2，所述rk多肽放射性药物为无色透明液体针剂。
该药物首先将PKM与D型多肽rnwelrlk连接，然后再将此二聚化，使二聚体分子中的两个多肽有足够长的距离能够同时结合两个HER2靶点，在增强体内稳定性、改善药代动力学性质的同时，提高肿瘤的靶向性。这种双价形式的结合可以进一步增强肿瘤对药物的摄取，达到更好的诊断效果。该药物通过双功能螯合剂将放射性核素 99mTc标记到rk多肽二聚体分子上，在体内标记药物通过rk多肽的靶向作用浓聚到肿瘤部位，利用核医学的单光子断层显像技术，对HER2阳性肿瘤进行显像诊断。
一种rk多肽放射性药物的制备方法，包括以下步骤：
a、HYNIC-PKM-COOH的制备
将Fmoc保护的PKM-COOH溶于终浓度体积分数20％哌啶的DMF溶液，室温反应15～30分钟后，加入乙醚使PKM沉淀，离心，弃掉上清，沉淀用乙醚洗涤，除去残留的乙醚，获得预期产物NH 2-PKM-COOH；将HYNIC-NHS和NH 2-PKM-COOH溶于DMF，加入DIEA调节pH值至8.5-9.0，室温搅拌过夜，粗产品经YMC-Pack ODS-A半制备柱HPLC分离纯化，收集目标物的馏分，合并收集液并冻干，获得产物经MALDI-TOF-MS质谱分析确认为预期产物HYNIC-PKM-COOH；
b、HYNIC-PKM-OSu的制备
将HYNIC-PKM-COOH溶于DMF，加入NHS和EDC·HCl，室温搅拌5～10小时，向反应液中加入体积分数50％ACN的水溶液并过滤，滤液经YMC-Pack ODS-A半制备柱HPLC分离纯化，收集目标物的馏分，合并收集液并冻干，获得产物经MALDI-TOF-MS质谱分析确认为预期产物HYNIC-PKM-OSu；
c、(PKM-rk-Dde) 2-Glu的制备
将PKM-rk-Dde和OSu 2-Glu-Boc溶于DMF，加入DIEA调节pH值至8.5-9.0，室温搅拌过夜，粗产品经YMC-Pack ODS-A半制备柱HPLC分离纯化，收集目标物的馏分，合并收集液并冻干，获得预期产物(PKM-rk-Dde) 2-Glu-Boc；将冻干产物(PKM-rk-Dde) 2-Glu-Boc溶于1ml TFA，室 温反应5min，反应液用氮气吹干，获得产物经MALDI-TOF-MS质谱分析确认为预期产物(PKM-rk-Dde) 2-Glu；其中以rk表示rk多肽单体；
d、HYNIC-PKM-(PKM-rk) 2的制备
将(PKM-rk-Dde) 2-Glu和HYNIC-PKM-OSu溶于DMF，加入DIEA调节pH值至8.5-9.0，室温搅拌过夜，粗产品经YMC-Pack ODS-A半制备柱HPLC分离纯化，收集目标物的馏分，合并收集液并冻干，获得预期产物HYNIC-PKM-(PKM-rk-Dde) 2；将HYNIC-PKM-(PKM-rk-Dde) 2溶于体积分数2％水合肼的DMF溶液，室温反应30分钟，粗产品经YMC-Pack ODS-A半制备柱HPLC分离纯化，收集目标物的馏分，合并收集液并冻干，获得产物经MALDI-TOF-MS质谱分析确认为预期产物HYNIC-PKM-(PKM-rk) 2；
e、 99mTc-HYNIC-PKM-(PKM-rk) 2的制备
配制含三苯基膦三磺酸钠(TPPTS)、三羟甲基甘氨酸(tricine)、琥珀酸二钠、琥珀酸和HYNIC-PKM-(PKM-rk) 2的混合液，混合液中上述各物质的质量比为：4～6:6～7:38～39:12～13:0.04，将混合液冻干。在冻干粉末中加入1ml Na 99mTcO 4溶液，100℃水浴加热反应20～25分钟，待反应结束后室温冷却，制成rk多肽放射性药物。经HPLC分析备用。
所述HPLC方法为使用Agilent 1260HPLC系统配备YMC-Pack ODS-A半制备柱(250×10mm,I.D.S-5μm，12nm)或分析柱(250×4.6mm,I.D.S-5μm，12nm)，梯度淋洗30分钟，其中流动A相为去离子水(含0.05％TFA)，流动B相为乙腈(含0.05％TFA)。步骤a：配备半制备柱，流速4mL/min，淋洗梯度设定为起始时90％A和10％B，25分钟时50％A和50％B，30分钟时90％A和10％B。步骤b：配备分析柱，流速1mL/min，淋洗梯度为初始时90％A和10％B，20分钟时30％A和70％B。
所述rk多肽放射性药物用于HER2阳性肿瘤患者的显像诊断，以及对接受抗癌药物曲妥珠单抗治疗患者的用药指导和实时疗效监测。
本发明的有益效果：
1、在本发明rk多肽放射性药物，D型多肽序列不会被体内的蛋白酶识别，能够有效的提高体内代谢稳定性，从而提高肿瘤组织的摄取。
2、本发明rk多肽放射性药物，首先将四个聚乙二醇分子(PEG4)或8-氨基辛酸(Aoc)与D型多肽单体连接，然后再将此二聚化，使二聚体分子中的两个多肽有足够长的距离能够同时结合两个HER2靶点，这种双价形式的结合可以进一步增强肿瘤对药物的摄取，达到更好的诊断效果。
3、本发明不仅在两个rk多肽之间引入PKM(PEG 4或Aoc)，同时在用于放射性核素标记的双功能螯合剂HYNIC与HER2靶向的rk多肽二聚体之间引入了PKM，即HYNIC-PKM-(PKM-rk) 2，改善了探针的生物相容性，优化了药代动力学性质，特别是从非肿瘤组织的清除动力学。
4、本发明中使用HYNIC作为双功能螯合剂，同时使用tricine和TPPTS作为协同配体从而使“ 99mTc-HYNIC核”具有更加良好的体内外稳定性。
附图说明
图1.(A)rk多肽，(B)HYNIC-PEG 4-rk，(C)HYNIC-PEG 4-(Aoc-rk) 2结构示意图。
图2. 99mTc-HYNIC-PKM-(PKM-rk) 2标记物结构示意图。其中的rnw表示rk多肽。
图3. 99mTc-HYNIC-PEG 4-(Aoc-rk) 2细胞结合实验。其中的rnw表示rk多肽。
图4.注射 99mTc-HYNIC-PEG 4-(Aoc-rk )20.5h，1h，2h后，在NOD SCID鼠SKBR3乳腺癌模型中SPECT/CT显像图。
图5.(A)注射 99mTc-HYNIC-PEG 4-(Aoc-rk) 20.5h后，在SKBR3乳腺癌模型中实验组，冷肽阻断组和抗体阻断组SPECT/CT显像图；(B) 99mTc-HYNIC-PEG 4-rk在SKBR3中0.5h SPECT/CT显像图。其中的rnw表示rk多肽。
图6. 99mTc-HYNIC-PEG 4-(Aoc-rk) 2在NOD SCID鼠早期SKBR3乳腺癌模型中(V＝30mm 3)延迟显像图。
图7. 99mTc-HYNIC-PEG 4-(Aoc-rk) 2在(A)SKBR3肿瘤模型(HER2高表达)，(B)HT29肿瘤模型(HER2中度表达)，(C)BxPC3肿瘤模型(HER2低表达)中SPECT/CT显像图。
图8.注射 99mTc-HYNIC-PEG 4-(Aoc-rk) 20.5h，1h后，在SKBR3乳腺癌模型中体内分布结果。其中的rnw表示rk多肽。
图9.SKBR3乳腺癌模型中注射 99mTc-HYNIC-PEG 4-(PEG 4-rk) 2(A)0.5h，1h，2h后SPECT/CT显像图；(B)0.5h冷肽阻断组SPECT/CT显像图。其中的rnw表示rk多肽。
图10.HER2阳性模型(SKBR3乳腺癌模型)的显像对比及定量数据分析结果；
图11.HER2阳性模型的生物分布数据和肿瘤与正常组织比值结果；
图12.蛋白和细胞层面对放射性药物 99mTc-HYNIC-PEG 4-(Aoc-rk) 2(简写为 99mTc-HPArk2)对HER2结合特异性检测结果；
图13. 99mTc-HYNIC-PEG 4-(Aoc-rk) 2(简写为 99mTc-HPArk2)和 99mTc-HP 4-ref对HER2阴性EGFR阳性模型的显像结果。
图14.(A)细胞流式图；(B)SPECT/CT成像的代表图像；(C)HER2表达量与肿瘤摄取的相关性；
图15.病理诊断为(A)HER2(2+)；(B)HER2(-)；(C)HER2(3+)患者的 18F-FDG PET/CT显像图(左)和 99mTc-HYNIC-PEG 4-(Aoc-rk) 2(简写为 99mTc-HPArk2)SPECT/CT显像图(右)；(D) 99mTc-HYNIC-PEG 4-(Aoc-rk) 2(简写为 99mTc-HPArk2)SPECT/CT的SUVmax与HER2表达水平的相关性(R 2＝0.863，P＝0.001)；(E) 18F-FDG PET/CT的SUVmax与HER2表达水平的相关性(R 2＝0.475，P＝0.073)。
具体实施方式
本发明实施例中所采用的材料：
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDC·HCl，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)，N-hydroxysuccinimide(NHS，N-羟基琥珀酰亚胺)，succinic acid(琥珀酸)，disodium succinate hexahydrate(琥珀酸二钠)，trisodium triphenylphosphine-3,3',3″-trisulfonate(TPPTS，三苯基膦三磺酸钠)，N,N-Dimethylform amide(DMF，N,N-二甲基甲酰胺)，tricine(三羟甲基甘氨酸)均购自美国Sigma-Aldrich公司。HYNIC-NHS(联肼尼克酰胺)购自美国Noca-biochem公司。PEG4-rnwelrlk、Aoc-rnwelrlk多肽单体购自中国吉尔生化公司。Na99mTcO4洗脱液购自北京原子高科股份有限公司。
一种靶向HER2的rk多肽放射性药物，包括rk多肽二聚体和放射性核素，所述放射性核素通过螯合剂标记所述rk多肽二聚体，所述rk多肽二聚体是将PKM与rk多肽单体相连，再将两个连接有PKM的rk多肽单体二聚化而成的多肽二聚体；所述rk多肽单体为D型氨基酸线性8元多肽，其序列为：Arg-Asn-Trp-Glu-Leu-Arg-Leu-Lys(精氨酸-天门冬酰胺-色氨酸-谷氨酸-亮氨酸-精氨酸-亮氨酸-赖氨酸，简写rnwelrlk)；所述PKM表示药代动力学修饰分子，所述多肽化学结构式如图1所示。
所述rk多肽二聚体与所述螯合剂之间还连接有PKM。PKM为聚乙二醇(PEG n)或者为8-氨基辛酸(Aoc)，PEGn优选PEG 4、PEG 6、PEG 8、PEG 12。所述放射性核素为 99mTc， 68Ga， 64Cu， 111In， 90Y和 177Lu中任意一种。螯合剂为HYNIC，NOTA，DOTA和DTPA中任意一种。所述rk多肽放射性药物为无色透明液体针剂。
以下实施例为优选的靶向HER2的rk多肽放射性药物，及其制备方法。
实施例1：
本实施例以 99mTc-HYNIC-PEG 4-(Aoc-rk) 2多肽放射性药物及其制备方法为例。
99mTc-HYNIC-PEG 4-(Aoc-rk) 2中，rk多肽单体为D型氨基酸线性多肽rnwelrlk，rk多肽二聚体是将连接剂Aoc与rk多肽单体连接，再将两个连接有Aoc的rk多肽单体二聚化而成的rk多肽二聚体，放射性核素 99mTc通过一个双功能螯合剂HYNIC标记所述rk多肽二聚体，rk多肽二聚体与双功能螯合剂之间还连接有药代动力学修饰分子PEG4，所述rk多肽放射性药物为 99mTc-HYNIC-PEG 4-(Aoc-rk) 2，标记物结构式如图2所示，所述rk多肽放射性药物为无色透明液体针剂。
99mTc-HYNIC-PEG 4-(Aoc-rk) 2制备方法如下：
HYNIC-PEG 4-COOH的制备：将Fmoc保护的PEG 4-COOH溶于DMF，加入哌啶使终浓度为20％，室温反应20分钟后，加入10ml 4℃乙醚使PEG 4-COOH沉淀，4000rpm 4℃离心5分钟，弃掉上清，沉淀用4℃乙醚洗涤3次，旋蒸除去残留的乙醚，获得产物为NH 2-PEG 4-COOH；将HYNIC-NHS 和NH 2-PEG 4-COOH溶于DMF，加入DIEA调节pH值至8.5-9.0，室温搅拌过夜，粗产品经YMC-Pack ODS-A半制备柱HPLC分离纯化。HPLC方法为使用Agilent 1260HPLC系统配备YMC-Pack ODS-A半制备柱(250×10mm,I.D.S-5μm，12nm)，梯度淋洗30分钟，流速4mL/min,其中流动A相为去离子水(含0.05％TFA)，流动B相为乙腈(含0.05％TFA)。淋洗梯度设定为起始时90％A和10％B，25分钟时50％A和50％B，30分钟时90％A和10％B。收集目标物的馏分，合并收集液并冻干，获得产物经MALDI-TOF-MS质谱分析m/z＝568.60([M+H]+)，确认为预期产物HYNIC-PEG4-COOH。
HYNIC-PEG 4-OSu的制备：将HYNIC-PEG 4-COOH溶于DMF，加入NHS和EDC·HCl，室温搅拌7小时，向反应液中加入体积分数50％ACN的水溶液并过滤，滤液经YMC-Pack ODS-A半制备柱HPLC分离纯化。HPLC方法为使用Agilent 1260HPLC系统配备YMC-Pack ODS-A半制备柱(250×10mm,I.D.S-5μm，12nm)，梯度淋洗30分钟，流速4mL/min,其中流动A相为去离子水(含0.05％TFA)，流动B相为乙腈(含0.05％TFA)。淋洗梯度设定为起始时90％A和10％B，25分钟时50％A和50％B，30分钟时90％A和10％B。收集目标物的馏分，合并收集液并冻干，获得产物经MALDI-TOF-MS质谱分析m/z＝665.67([M+H] +)，确认为预期产物HYNIC-PEG 4-OSu。
(AOC-rk-Dde) 2-Glu的制备：将AOC-rk-Dde和OSu 2-Glu-Boc溶于DMF，加入DIEA调节pH值至8.5-9.0，室温搅拌过夜，粗产品经YMC-Pack ODS-A半制备柱HPLC分离纯化。HPLC方法为使用Agilent 1260HPLC系统配备YMC-Pack ODS-A半制备柱(250×10mml.D.S-5μm，12nm)，梯度淋洗30分钟，流速4mL/min,其中流动A相为去离子水(含0.05％TFA)，流动B相为乙腈(含0.05％TFA)。淋洗梯度设定为起始时90％A和10％B，25分钟时50％A和50％B，30分钟时90％A和10％B。收集目标物的馏分，合并收集液并冻干，获得产物经MALDI-TOF-MS质谱分析m/z＝3050.70([M+H]+)，确认为预期产物(Aoc-rk-Dde) 2-Glu-Boc；将冻干产物(Aoc-rk-Dde) 2-Glu-Boc溶于1ml TFA，室温反应5min，反应液用氮气吹干，获 得产物经MALDI-TOF-MS质谱分析m/z＝2950.58([M+H]+)，确认为预期产物(Aoc-rk-Dde) 2-Glu。
HYNIC-PEG 4-(Aoc-rk) 2的制备：将(Aoc-rk-Dde) 2-Glu和HYNIC-PEG 4-OSu溶于DMF，加入DIEA调节pH值至8.5-9.0，室温搅拌过夜，粗产品经YMC-Pack ODS-A半制备柱HPLC分离纯化，HPLC方法为使用Agilent 1260HPLC系统配备YMC-Pack ODS-A半制备柱(250×10mm,I.D.S-5μm，12nm)，梯度淋洗30分钟，流速4mL/min,其中流动A相为去离子水(含0.05％TFA)，流动B相为乙腈(含0.05％TFA)。淋洗梯度设定为起始时90％A和10％B，25分钟时50％A和50％B，30分钟时90％A和10％B。收集目标物的馏分，合并收集液并冻干，获得产物经MALDI-TOF-MS质谱分析m/z＝3501.16([M+H] +)，确认为预期产物HYNIC-PEG 4-(Aoc-rk-Dde) 2；将HYNIC-PEG 4-(Aoc-rk-Dde) 2溶于体积分数2％水合肼的DMF溶液，室温反应30分钟，粗产品经YMC-Pack ODS-A半制备柱HPLC分离纯化，HPLC方法为使用Agilent 1260HPLC系统配备YMC-Pack ODS-A半制备柱(250×10mm,I.D.S-5μm，12nm)，梯度淋洗30分钟，流速4mL/min,其中流动A相为去离子水(含0.05％TFA)，流动B相为乙腈(含0.05％TFA)。淋洗梯度设定为起始时90％A和10％B，25分钟时50％A和50％B，30分钟时90％A和10％B。收集目标物的馏分，合并收集液并冻干，获得产物经MALDI-TOF-MS质谱分析m/z＝3172.75([M+H] +)，确认为预期产物HYNIC-PEG 4-(Aoc-rk) 2；化学结构式如图1所示。
99mTc-HYNIC-PEG 4-(Aoc-rk) 2的制备：配制含三苯基膦三磺酸钠(TPPTS)5.0mg，三羟甲基甘氨酸(tricine)6.5mg，琥珀酸二钠38.5mg，琥珀酸12.7mg和50μg的HYNIC-PEG 4-(Aoc-rk) 2的混合液500μL于10mL西林瓶中，将混合液冻干。在冻干粉末中加入1.0-1.5mL的Na 99mTcO 4溶液(10～35mCi)，100℃水浴加热西林瓶反应20～25分钟，待反应结束后室温冷却10分钟，制成rk多肽放射性药物，其化学结构式如图2所示。
对rk多肽放射性药物取样进行放射性HPLC分析。HPLC方法为使用Agilent 1260HPLC系统配备YMC-Pack ODS-A分析柱(250×4.6mm,I.D.S-5μm，12nm)，梯度淋洗20分钟，流速1mL/min,其中流动A相为去离子水 (含0.05％TFA)，流动B相为乙腈(含0.05％TFA)。淋洗梯度设定为起始时90％A和10％B，20分钟时30％A和70％B。 99mTc-HYNIC-PEG 4-(Aoc-rk) 2的标记率>95％，经Sep-Pak C18柱纯化后放射化学纯度>98％。
HYNIC-PEG 4-(Aoc-rk) 2与HER2结合亲和力测定结果如图3所示：高表达HER2的人乳腺癌细胞SKBR3和不表达HER2的人乳腺癌细胞MCF7作为实验样本，使用 99mTc-HYNIC-PEG 4-(Aoc-rk) 2作为HER2受体特异性结合的放射性配基，采用细胞结合实验，分别测定 99mTc-HYNIC-PEG 4-(Aoc-rk) 2与SKBR3、MCF7的结合力，并设置过量HYNIC-PEG 4-(Aoc-rk) 2封闭组，验证 99mTc-HYNIC-PEG 4-(Aoc-rk) 2与SKBR3结合的特异性。实验结果显示 99mTc-HYNIC-PEG 4-(Aoc-rk) 2与SKBR3、MCF7和SKBR3封闭组的结合分别为4.49％，1.14％，1.24％每10 5个细胞，存在明显的统计学差异，表明HYNIC-PEG 4-(Aoc-rk) 2与HER2有较高的亲和力，并且是特异性结合。
99mTc-HYNIC-PEG 4-(Aoc-rk) 2在荷瘤鼠中SPECT/CT显像结果如图4所示：在SKBR3乳腺癌肿瘤模型中， 99mTc-HYNIC-PEG 4-(Aoc-rk) 2的肿瘤摄取清晰可见，明显高于单体，且全身背景干净，可用于早期肿瘤诊断。多肽在肿瘤部位滞留时间较长，注射后四小时肿瘤依然清晰可见，可以延迟显像，更有利于肿瘤的诊断，显像结果如图6所示。阻断实验结果如图5所示，肿瘤摄取明显降低，说明多肽与HER2位点的特异性结合。在SKBR3乳腺癌模型Herceptin阻断实验组中，肿瘤摄取基本没有变化，说明探针与多肽的结合位点不同，可以用于Herceptin治疗病人的实时疗效监测。 99mTc-HYNIC-PEG 4-(Aoc-rk) 2在HER2高表达肿瘤模型(SKBR3人乳腺癌肿瘤)，HER2中度表达肿瘤模型(HT29人结肠癌肿瘤)，HER2低表达肿瘤模型(BxPC3人胰腺癌肿瘤)中SPECT/CT显像图如图7所示，结果显示 99mTc-HYNIC-PEG 4-(Aoc-rk) 2的摄取与HER2表达水平线性相关，能用于检测肿瘤HER2的表达情况。
99mTc-HYNIC-PEG 4-(Aoc-rk) 2在荷瘤鼠中生物分布：将NOD SCID鼠荷SKBR3乳腺癌肿瘤，每组4只。各组小鼠分别经尾静脉注射不同的 99mTc标记多肽，与注射后30分钟、60分钟处死，取血及主要脏器，称重并测量放射 性计数，经衰变校正后计算每克组织百分注射剂量率(％ID/g)。实验结果如图8显示，30分钟肿瘤摄取为3.7％ID/g，除肾脏外，血液及其他脏器摄取较低。
99mTc-HYNIC-PEG 4-(Aoc-rk) 2肿瘤摄取与HER2表达量的相关性：选择拥有不同HER2表达量的不同肿瘤类型的细胞，包括人乳腺癌细胞系SKBR3、MCF7、MDA-MB-468，人结直肠癌细胞系HT29，人胰腺癌细胞系BxPC3，分别进行了细胞流式实验，对细胞的HER2表达量进行定量分析；之后用相应细胞的肿瘤模型进行SPECT/CT成像，并定量分析肿瘤的摄取；最后，对HER2表达量与肿瘤摄取的相关性进行研究。
细胞流式：将上述细胞用0.25％的EDTA/胰酶消化下来后封闭15min，用PBS离心洗净后分为实验组和对照组，实验组加入PE直标的抗人HER2抗体(1:100稀释)，对照组用等体积PBS重悬，4℃避光孵育0.5h，用冷PBS洗3次，使用流式细胞仪进行分析，通过流式曲线计算出每种细胞上结合抗体的平均荧光强度(MFI)。
SPECT/CT显像：选取肿瘤大小相近的不同肿瘤模型，经尾静脉注射1mCi 99mTc-HYNIC-PEG 4-(Aoc-rk) 2，于注射后0.5h进行SPECT/CT成像，显像后对SPECT图像进行重建，与CT图像融合得到小鼠3D成像图，用InVivoScope软件扣取肿瘤部位进行定量分析。
HER2表达量与肿瘤摄取的相关性分析：用prism 7.0软件作图分析HER2表达量(MFI)和肿瘤摄取(％ID/g)相对应的线性关系，计算R 2值。
实验结果如图14所示，肿瘤摄取与HER2表达量呈正相关(R 2＝0.977)，说明探针 99mTc-HYNIC-PEG 4-(Aoc-rk) 2在肿瘤的摄取可以准确的反应出HER2表达的高低。
99mTc-HYNIC-PEG 4-(Aoc-rk) 2在乳腺癌患者中的SPECT/CT显像：经北京协和医院伦理审查委员会的批准和知情同意，招募20名经乳房钼靶或超声检查怀疑患有乳腺癌的女性患者，于手术前1周分别进行SPECT/CT和PET/CT成像。患者静脉注射11.1MBq(0.3mCi)/千克体重的 99mTc-HYNIC-PEG 4-(Aoc-rk) 2，30min后进行SPECT/CT扫描；静脉注射5.6MBq(0.15mCi)/千克体重的 18F-FDG，60min后进行PET/CT扫描。其中有15名患者被病理诊断为乳腺癌，其中6例患者出现同侧淋巴结转移。临床试验结果如图15所示，在15例患者中， 99mTc-HYNIC-PEG 4-(Aoc-rk) 2较准确的显像出14例患者的肿瘤(93％)，在6例出现淋巴结转移的患者中，有5例显像出转移灶(83％)，另一例患者仅有轻微的淋巴结转移。乳腺癌对 99mTc-HYNIC-PEG 4-(Aoc-rk) 2的摄取强度(SUVmax)与HER2的免疫组化结果呈显著的相关性(R 2＝0.863,P＝0.001)。临床试验过程中未发现与放射药物相关的不良反应。
实施例2：
本实施例以 99mTc-HYNIC-PEG 4-(PEG 4-rk) 2多肽放射性药物及其制备方法为例。
99mTc-HYNIC-PEG 4-(PEG 4-rk) 2中，rk多肽单体为D型氨基酸线性多肽rnwelrlk，rk多肽二聚体是将连接剂PEG4与rk多肽单体连接，再将两个连接有PEG 4的rk多肽单体二聚化而成的rk多肽二聚体，放射性核素 99mTc通过一个双功能螯合剂HYNIC标记所述rk多肽二聚体，rk多肽二聚体与双功能螯合剂之间还连接有药代动力学修饰分子PEG 4，所述rk多肽放射性药物为 99mTc-HYNIC-PEG 4-(PEG 4-rk) 2，所述rk多肽放射性药物为无色透明液体针剂。
99mTc-HYNIC-PEG 4-(PEG 4-rk) 2制备方法如下：
HYNIC-PEG 4-COOH的制备：将Fmoc保护的PEG 4-COOH溶于DMF，加入哌啶使终浓度为20％，室温反应20分钟后，加入10ml 4℃乙醚使PEG 4-COOH沉淀，4000rpm 4℃离心5分钟，弃掉上清，沉淀用4℃乙醚洗涤3次，旋蒸除去残留的乙醚，获得产物为NH 2-PEG 4-COOH；将HYNIC-NHS和NH 2-PEG 4-COOH溶于DMF，加入DIEA调节pH值至8.5-9.0，室温搅拌过夜，粗产品经YMC-Pack ODS-A半制备柱HPLC分离纯化。HPLC方法为使用Agilent 1260HPLC系统配备YMC-Pack ODS-A半制备柱(250×10mm,I.D.S-5μm，12nm)，梯度淋洗30分钟，流速4mL/min,其中流动A相为去离子水(含0.05％TFA)，流动B相为乙腈(含0.05％TFA)。淋洗梯度设定为起始时90％A和10％B，25分钟时50％A和50％B，30分钟时90％A和10％B。收集目标物的馏分，合并收集液并冻干，获得产物经MALDI-TOF-MS质谱分析m/z＝568.60([M+H] +)，确认为预期产物HYNIC-PEG 4-COOH。
HYNIC-PEG 4-OSu的制备：将HYNIC-PEG 4-COOH溶于DMF，加入NHS和EDC·HCl，室温搅拌7小时，向反应液中加入体积分数50％ACN的水溶液并过滤，滤液经YMC-Pack ODS-A半制备柱HPLC分离纯化。HPLC方法为使用Agilent 1260HPLC系统配备YMC-Pack ODS-A半制备柱(250×10mm,I.D.S-5μm，12nm)，梯度淋洗30分钟，流速4mL/min,其中流动A相为去离子水(含0.05％TFA)，流动B相为乙腈(含0.05％TFA)。淋洗梯度设定为起始时90％A和10％B，25分钟时50％A和50％B，30分钟时90％A和10％B。收集目标物的馏分，合并收集液并冻干，获得产物经MALDI-TOF-MS质谱分析m/z＝665.67([M+H] +)，确认为预期产物HYNIC-PEG 4-OSu。
(PEG 4-rk-Dde) 2-Glu的制备：将PEG 4-rk-Dde和OSu 2-Glu-Boc溶于DMF，加入DIEA调节pH值至8.5-9.0，室温搅拌过夜，粗产品经YMC-Pack ODS-A半制备柱HPLC分离纯化。HPLC方法为使用Agilent 1260HPLC系统配备YMC-Pack ODS-A半制备柱(250×10mm,I.D.S-5μm，12nm)，梯度淋洗30分钟，流速4mL/min,其中流动A相为去离子水(含0.05％TFA)，流动B相为乙腈(含0.05％TFA)。淋洗梯度设定为起始时90％A和10％B，25分钟时50％A和50％B，30分钟时90％A和10％B。收集目标物的馏分，合并收集液并冻干，获得产物经MALDI-TOF-MS质谱分析m/z＝3262.85([M+H]+)，确认为预期产物(PEG 4-rk-Dde) 2-Glu-Boc；将冻干产物(PEG 4-rk-Dde) 2-Glu-Boc溶于1ml TFA，室温反应5min，反应液用氮气吹干，获得产物经MALDI-TOF-MS质谱分析m/z＝3162.73([M+H] +)，确认为预期产物(PEG 4-rk-Dde) 2-Glu。
HYNIC-PEG 4-(PEG 4-rk) 2的制备：将(PEG 4-rk-Dde) 2-Glu和HYNIC-PEG 4-OSu溶于DMF，加入DIEA调节pH值至8.5-9.0，室温搅拌过夜，粗产品经YMC-Pack ODS-A半制备柱HPLC分离纯化，HPLC方法为使用Agilent 1260HPLC系统配备YMC-Pack ODS-A半制备柱(250×10mm,I.D.S-5μm，12nm)，梯度淋洗30分钟，流速4mL/min,其中流动A相为去离子水(含0.05％TFA)，流动B相为乙腈(含0.05％TFA)。淋洗梯度设定为起始时90％A和10％B，25分钟时50％A和50％B，30分钟时90％A和 10％B。收集目标物的馏分，合并收集液并冻干，获得产物经MALDI-TOF-MS质谱分析m/z＝3713.32([M+H] +)，确认为预期产物HYNIC-PEG 4-(PEG 4-rk-Dde) 2；将HYNIC-PEG 4-(PEG 4-rk-Dde) 2溶于体积分数2％水合肼的DMF溶液，室温反应30分钟，粗产品经YMC-Pack ODS-A半制备柱HPLC分离纯化，HPLC方法为使用Agilent 1260HPLC系统配备YMC-Pack ODS-A半制备柱(250×10mm,I.D.S-5μm，12nm)，梯度淋洗30分钟，流速4mL/min,其中流动A相为去离子水(含0.05％TFA)，流动B相为乙腈(含0.05％TFA)。淋洗梯度设定为起始时90％A和10％B，25分钟时50％A和50％B，30分钟时90％A和10％B。收集目标物的馏分，合并收集液并冻干，获得产物经MALDI-TOF-MS质谱分析m/z＝3384.91([M+H] +)，确认为预期产物HYNIC-PEG 4-(PEG 4-rk) 2。
99mTc-HYNIC-PEG 4-(PEG4-rk) 2的制备：配制含三苯基膦三磺酸钠(TPPTS)5.0mg，三羟甲基甘氨酸(tricine)6.5mg，琥珀酸二钠38.5mg，琥珀酸12.7mg和50μg的HYNIC-PEG 4-(PEG 4-rk) 2的混合液500μL于10mL西林瓶中，将混合液冻干。在冻干粉末中加入1.0-1.5mL的Na 99mTcO 4溶液(10～35mCi)，100℃水浴加热西林瓶反应20～25分钟，待反应结束后室温冷却10分钟，制成rk多肽放射性药物。
对rk多肽放射性药物取样进行放射性HPLC分析。HPLC方法为使用Agilent 1260HPLC系统配备YMC-Pack ODS-A分析柱(250×4.6mm,I.D.S-5μm，12nm)，梯度淋洗20分钟，流速1mL/min,其中流动A相为去离子水(含0.05％TFA)，流动B相为乙腈(含0.05％TFA)。淋洗梯度设定为起始时90％A和10％B，20分钟时30％A和70％B。 99mTc-HYNIC-PEG 4-(PEG 4-rk) 2的标记率>95％，经Sep-Pak C 18柱纯化后放射化学纯度>98％。
99mTc-HYNIC-PEG 4-(PEG 4-rk) 2在荷瘤鼠中SPECT/CT显像如图9所示：在SKBR3乳腺癌肿瘤模型中， 99mTc-HYNIC-PEG 4-(PEG 4-rk) 2的肿瘤摄取清晰可见，明显高于单体，且全身背景干净。在阻断实验组中，肿瘤摄取明显降低，说明多肽与HER2位点的特异性结合。
对比例1：
公开号为CN 109045313A的中国专利公开一种基于refvffly多肽序列(D型氨基酸线性多肽的序列为：Arg-Glu-Phe-Val-Phe-Phe-Leu-Tyr，简称ref多肽)的化合物 99mTc-HYNIC-PKM-ref(PKM＝PEGn，n＝1-24)，也是一种靶向HER2阳性肿瘤的多肽放射性药物。本发明在该化合物的基础上进行了改进。
本发明所选用的多肽水溶性优于refvffly多肽。改进后的化合物的靶向特异性优于 99mTc-HYNIC-PKM-ref。详见图10。图10A是同一模型显像对比和定量分析结果。图10B是从图10A定量分析出来的结果。
如图10所示，本发明申请的显像剂 99mTc-HYNIC-PEG 4-(Aoc-rk) 2(附图中简写成 99mTc-HPArk2)不管是相对于CN 109045313A中的 99mTc-HYNIC-PEG 4-ref(简称 99mTc-HP 4-ref)或者 99mTc-HYNIC-PEG 24-ref(简称 99mTc-HP 24-ref)显像剂，最终在HER2阳性的乳腺癌模型中的显像效果都有明显差异。由于本发明最终是要用于乳腺癌的原位显像，那么肿瘤摄取和在胸腔的肝脏摄取本底最为关键， 99mTc-HPArk2的肿瘤摄取比 99mTc-HP 24-ref更高，与 99mTc-HP 4-ref无明显差异。而 99mTc-HPArk2的肝脏摄取最低，比 99mTc-HP 4-ref和 99mTc-HP 24-ref都低。因此， 99mTc-HPArk2的肿瘤与肝脏的比值最高。这对于在胸腔显像原位的乳腺癌肿瘤尤其关键，因此本发明的 99mTc-HPArk2的显像剂在显像乳腺癌原位肿瘤方面更有优势。
本发明与公开号为CN 109045313 A的专利文件的放射性药物具体体内药代动力学分布的具体差异如图11所示。图11的肿瘤摄取和对比度跟图10的显像是对应的数据。图11是定量数据，图10是直观影像数据。图11B图是图11A计算的结果。就把肿瘤和每个脏器的摄取值做了对比分析。由图11可见，本发明的放射性药物 99mTc-HPArk2与 99mTc-HP 4-ref和 99mTc-HP 24-ref相比，在肿瘤的摄取相对更高，有助于提高肿瘤显像的灵敏度，更小的肿瘤都能清晰显像，这点在图10中也可见， 99mTc-HPArk2显像了更小的肿瘤，显像效果仍然有保证。 99mTc-HPArk2在血液，心脏，肝脏，脾，肠，胃，骨等组织中的摄取均相对更低，肿瘤与正常组织血、心脏、肝脏、脾、肠、胃的比值相对更高。这样在显像的对比度上有更好的效果，尤其是在胸腔里的心脏和肝脏的摄取相对更低，有利于乳腺癌的原位肿瘤清晰显像。
如图12所示，为了验证本发明药物的效果，进行了该放射性药物对HER2 特异性的检测。不管在蛋白层面还是在细胞层面都具有良好的特异性。
由于EGFR和HER2是同一家族的蛋白，结构相似，一般靶向分子很难对其有较好的区分，这样在检测肿瘤时，难免会造成有假阳性和假阴性。与公开号为CN 109045313 A的专利文本的药物进行了在同一模型的对比，公开号为CN 109045313 A的专利文本所公布的放射性药物在显像HER2阴性EGFR阳性的MDA-MB-468乳腺癌模型时，呈假阳性结果，区分效果不如本发明的药物。本发明药物显示阴性，区分度更高，不会因为EGFR表达得到假阳性结果。可见本发明放射性药物在区分HER2和EGFR高表达的肿瘤时有明显的优势，减少了假阳性结果的产生，具有明显的优势。

Claims (10)

  1. 一种靶向HER2的rk多肽放射性药物，其特征在于：包括rk多肽二聚体和放射性核素，所述放射性核素通过螯合剂标记所述rk多肽二聚体，所述rk多肽二聚体是将PKM与rk多肽单体相连，再将两个连接有PKM的rk多肽单体二聚化而成的多肽二聚体；所述rk多肽单体为D型氨基酸线性8元多肽，其序列为：Arg-Asn-Trp-Glu-Leu-Arg-Leu-Lys；所述PKM表示药代动力学修饰分子。
  2. 根据权利要求1所述的靶向HER2的rk多肽放射性药物，其特征在于：所述rk多肽二聚体与所述螯合剂之间还连接有PKM。
  3. 根据权利要求1所述的靶向HER2的rk多肽放射性药物，其特征在于：PKM是聚乙二醇或者8-氨基辛酸。
  4. 根据权利要求1所述的靶向HER2的rk多肽放射性药物，其特征在于：所述放射性核素为 99mTc， 68Ga， 64Cu， 111In， 90Y和 177Lu中任意一种。
  5. 根据权利要求1所述的靶向HER2的rk多肽放射性药物，其特征在于：螯合剂为HYNIC，NOTA，DOTA和DTPA中任意一种。
  6. 根据权利要求1所述的靶向HER2的rk多肽放射性药物，其特征在于：所述rk多肽放射性药物为无色透明液体针剂。
  7. 根据权利要求1所述的靶向HER2的rk多肽放射性药物，其特征在于：所述放射性核素为 99mTc，所述rk多肽二聚体与所述螯合剂之间连接有PKM，PKM是聚乙二醇或者为8-氨基辛酸，螯合剂为HYNIC，所述rk多肽放射性药物表示为 99mTc-HYNIC-PKM-(PKM-rk) 2。
  8. 根据权利要求7所述的靶向HER2的rk多肽放射性药物，其特征在于：rk多肽二聚体与螯合剂之间连接的PKM是聚合度为4的聚乙二醇，与rk多肽单体相连的PKM是聚合度为4的聚乙二醇或者8-氨基辛酸，所述rk多肽放射性药物表示为 99mTc-HYNIC-PEG 4-(Aoc-rk) 2及 99mTc-HYNIC-PEG 4-(PEG 4-rk) 2。
  9. 一种靶向HER2的rk多肽放射性药物的制备方法，其特征在于：
    所述方法包括以下步骤：
    a、HYNIC-PKM-COOH的制备
    将Fmoc保护的PKM-COOH溶于终浓度体积分数20％哌啶的DMF溶液，室温反应15～30分钟后，加入乙醚使PKM沉淀，离心，弃掉上清，沉淀用乙 醚洗涤，除去残留的乙醚，获得预期产物NH 2-PKM-COOH；将HYNIC-NHS和NH 2-PKM-COOH溶于DMF，加入DIEA调节pH值至8.5-9.0，室温搅拌过夜，粗产品经YMC-Pack ODS-A半制备柱HPLC分离纯化，收集目标物的馏分，合并收集液并冻干，获得产物HYNIC-PKM-COOH；
    b、HYNIC-PKM-OSu的制备
    将HYNIC-PKM-COOH溶于DMF，加入NHS和EDC·HCl，室温搅拌5～10小时，向反应液中加入体积分数50％ACN的水溶液并过滤，滤液经YMC-Pack ODS-A半制备柱HPLC分离纯化，收集目标物的馏分，合并收集液并冻干，获得预期产物HYNIC-PKM-OSu；
    c、(PKM-rk-Dde) 2-Glu的制备
    将PKM-rk-Dde和OSu 2-Glu-Boc溶于DMF，加入DIEA调节pH值至8.5-9.0，室温搅拌过夜，粗产品经YMC-Pack ODS-A半制备柱HPLC分离纯化，收集目标物的馏分，合并收集液并冻干，获得预期产(PKM-rk-Dde) 2-Glu-Boc；将冻干产物(PKM-rk-Dde) 2-Glu-Boc溶于1ml TFA，室温反应5min，反应液用氮气吹干，获得预期产物(PKM-rk-Dde) 2-Glu；其中以rk表示rk多肽单体；
    d、HYNIC-PKM-(PKM-rk) 2的制备
    将(PKM-rk-Dde) 2-Glu和HYNIC-PKM-OSu溶于DMF，加入DIEA调节pH值至8.5-9.0，室温搅拌过夜，粗产品经YMC-Pack ODS-A半制备柱HPLC分离纯化，收集目标物的馏分，合并收集液并冻干，获得预期产物HYNIC-PKM-(PKM-rk-Dde) 2；将HYNIC-PKM-(PKM-rk-Dde) 2溶于体积分数2％水合肼的DMF溶液，室温反应30分钟，粗产品经YMC-Pack ODS-A半制备柱HPLC分离纯化，收集目标物的馏分，合并收集液并冻干，获得预期产物HYNIC-PKM-(PKM-rk) 2；
    e、 99mTc-HYNIC-PKM-(PKM-rk) 2的制备
    配制含三苯基膦三磺酸钠、三羟甲基甘氨酸、琥珀酸二钠、琥珀酸和HYNIC-PKM-(PKM-rk) 2的混合液，混合液中上述各物质的质量比为：4～6:6～7:38～39:12～13:0.04，将混合液冻干。在冻干粉末中加入1ml Na 99mTcO 4溶液，100℃水浴加热反应20～25分钟，待反应结束后室温冷却，制成 99mTc-HYNIC-PKM-(PKM-rk) 2，即为所述rk多肽放射性药物。
  10. 根据权利要求9所述的rk多肽放射性药物的制备方法，其特征在于：所述HPLC为使用Agilent 1260 HPLC系统配备YMC-Pack ODS-A半制备柱或分析柱，梯度淋洗30分钟，其中流动A相为去离子水，含0.05％TFA，流动B相为乙腈，含0.05％TFA；步骤a：配备半制备柱，流速4mL/min，淋洗梯度设定为起始时90％A和10％B，25分钟时50％A和50％B，30分钟时90％A和10％B；步骤b：配备分析柱，流速1mL/min，淋洗梯度为初始时90％A和10％B，20分钟时30％A和70％B。
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