WO2023087871A1 - 颗粒酶b靶向配合物、放射性药物及其制备方法和应用 - Google Patents

颗粒酶b靶向配合物、放射性药物及其制备方法和应用 Download PDF

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WO2023087871A1
WO2023087871A1 PCT/CN2022/118207 CN2022118207W WO2023087871A1 WO 2023087871 A1 WO2023087871 A1 WO 2023087871A1 CN 2022118207 W CN2022118207 W CN 2022118207W WO 2023087871 A1 WO2023087871 A1 WO 2023087871A1
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granzyme
complex
targeting
radionuclide
preparation
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French (fr)
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刘昭飞
杨兴
王琰璞
徐红闯
周昊毅
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北京大学
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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
    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/547—Chelates, e.g. Gd-DOTA or Zinc-amino acid chelates; Chelate-forming compounds, e.g. DOTA or ethylenediamine being covalently linked or complexed to the pharmacologically- or therapeutically-active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu

Definitions

  • the invention belongs to the field of nuclear medicine, and in particular relates to a granzyme B targeting complex and a preparation method thereof, a granzyme B targeting radiopharmaceutical and a preparation method thereof, and their application in nuclear medicine imaging diagnosis and treatment of related diseases such as tumors. Applications in therapeutic imaging monitoring.
  • Granzyme is a kind of serine protease. Human granzyme includes five kinds of A, B, H, K, and M, which exist in cytotoxic T lymphocytes (cytotoxic T lymphocytes, CTL) and natural killer cells (natural killer cells, NK). released cell granules. As one of the most important effector molecules of granzyme, granzyme B can enter cells, mediate the activation of downstream caspase signaling pathway, induce cellular DNA fragmentation, and thus lead to cell apoptosis. At the same time, granzyme B can also cut nuclear proteins, including NuMA and P and DNA-PKcs, etc., to promote and initiate nuclear apoptosis. Therefore, granzyme B is one of the important markers for the activation of CTL or NK cells and their killing effect.
  • A, B, H, K, and M which exist in cytotoxic T lymphocytes (cytotoxic T lymphocytes, CTL) and natural killer cells (natural killer cells, NK). released cell
  • tumor immunotherapy In the field of tumor treatment, immunotherapy is considered to be one of the most ideal ways to eliminate tumors and prevent tumor metastasis and recurrence.
  • tumor immunotherapy such as antibodies targeting immune checkpoints (such as CTLA-4, PD-1/PD-L1) and chimeric antigen receptor autologous T cell (CAR-T) therapy has achieved a series of major clinical breakthroughs .
  • immune checkpoints such as CTLA-4, PD-1/PD-L1
  • CAR-T chimeric antigen receptor autologous T cell
  • the current effective rate of tumor immunotherapy is low. Taking anti-PD-1 immune checkpoint inhibition as an example, its effective rate is often less than 30%. Therefore, it is very important to accurately predict the early efficacy of immunotherapy to effectively guide its precise treatment and improve the therapeutic efficacy.
  • 18 F-fluorodeoxyglucose 18 F-FDG
  • 18 F-FDG is currently the most widely used PET imaging drug in clinical practice.
  • 18 F-FDG is an analogue of glucose, its biological behavior is similar to that of glucose, and the occurrence and progress of the disease can be reflected by monitoring the uptake of glucose by the disease.
  • 18 F-FDG lacks tumor-specific, specific markers of active cells other than CTL or NK. It has obvious limitations in predicting and evaluating the efficacy of immunotherapy. Therefore, it is of great clinical significance to develop new nuclear medicine imaging drugs for monitoring the efficacy of tumor immunotherapy.
  • Granzyme B is a serine protease released by CTL and NK cells during the immune response, and its expression level is closely related to the immune response. Therefore, if nuclear medicine imaging drugs with good granzyme B target specificity, affinity and in vivo metabolic properties can be developed, especially PET and SPECT drugs labeled with 68 Ga, 18 F, 99m Tc, etc. It plays an important role in immunotherapy and efficacy monitoring, and has broad clinical application prospects.
  • granzyme B is also specifically highly expressed in other diseases, such as autoimmune diseases and immune-induced myocarditis.
  • diseases such as autoimmune diseases and immune-induced myocarditis.
  • the development of granzyme B target-specific nuclear medicine imaging drugs can also play an important role in the imaging diagnosis, treatment monitoring and curative effect judgment of such diseases.
  • the present invention provides a novel granzyme B targeting complex, which is formed by coupling a granzyme B targeting molecule with a bifunctional chelating agent.
  • Such complexes become radiopharmaceuticals for nuclear medicine imaging after being labeled with radionuclides.
  • Non-invasive and specific monitoring of granzyme B expression can be achieved through nuclear medicine PET or SPECT imaging, which is expected to be popularized and applied in the diagnosis and treatment monitoring of tumor immunotherapy and autoimmune diseases.
  • the granzyme B-specific radiopharmaceutical of the invention is simple to prepare and has good pharmacokinetic properties in vivo.
  • the first aspect of the present invention provides a granzyme B targeting complex, the structure of which is shown in the following formula (I):
  • R is any one of bifunctional chelating groups or derivatives thereof for radionuclide labeling.
  • the bifunctional chelating group is a group formed by a bifunctional chelating agent, preferably, the bifunctional chelating agent is DOTA, NOTA, HYNIC, MAG2, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, BAPEN, Df, DFO, TACN, NO2A, NOTAM, CB-DO2A, Cyclen, DO3A, DO3AP, MAS3, MAG3 or isonitrile.
  • the bifunctional chelating agent is DOTA, NOTA, HYNIC, MAG2, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, BAPEN, Df, DFO, TACN, NO2A, NOTAM, CB-DO2A, Cyclen, DO3A, DO3AP, MAS3, MAG3 or isonitrile.
  • the R is any one of the groups represented by formula (II), formula (III), formula (IV), formula (V), formula (VI) or their derivatives,
  • the second aspect of the present invention provides a granzyme B-targeted radiopharmaceutical obtained by labeling the complex with a radionuclide.
  • the radionuclide may be a diagnostic radionuclide or a therapeutic radionuclide.
  • the diagnostic radionuclide is preferably at least one of 68 Ga, 64 Cu, 18 F, 86 Y, 90 Y, 89 Zr, 111 In, 99m Tc, 11 C, 123 I, 125 I and 124 I.
  • the therapeutic radionuclides are preferably 177 Lu, 125 I, 131 I, 211 At, 111 In, 153 Sm, 186 Re, 188 Re, 67 Cu, 212 Pb, 225 Ac, 213 Bi, 212 Bi and 212 Pb at least one of the
  • the radionuclide is any one of 68 Ga, 64 Cu, 111 In, 18 F, 86 Y, and 99m Tc.
  • the third aspect of the present invention provides a method for preparing the above granzyme B targeting complex, comprising the following steps:
  • Reaction conditions (a) DCM solution of Fmoc-NHS and DIPEA; (b) DMF/DCM solution of 2-chlorotriphenyl chloride resin and DIPEA; (c) DMF solution of 20% piperidine, Fmoc-(2S, The DMF solution of 5S)-5-amino-1,2,4,5,6,7-hexahydroazepino[3,2,1-Hi]indole-4-one-2-carboxylic acid, HBTU, HOBt and DIPEA;( d) DMF solution of 20% piperidine, DMF solution of Fmoc-L-isoleucine, HBTU, HOBt and DIPEA; (e) DMF solution of 20% piperidine, Fmoc-(3-aminomethylphenyl) The DMF solution of acetic acid, HBTU, HOBt and EIPEA; (f) the DMF solution of 20% piperidine, the DMF solution of Fmoc-L-aspartic
  • the compounds used in the preparation of the granzyme B targeting complex of the present invention are commercially available or prepared by conventional organic synthesis methods.
  • the fourth aspect of the present invention provides a method for preparing a granzyme B-targeted radiopharmaceutical, comprising the following steps: dissolving the granzyme B-targeted complex in a radioactive labeling buffer, and then adding different radionuclides for reaction, after the reaction
  • the reaction solution was separated and purified by a Sep-Pak C18 chromatographic column to obtain the corresponding granzyme B-targeted radiopharmaceutical.
  • the preparation method includes The following steps:
  • Dissolve the DOTA coupling complex in an acidic buffer solution then add 68 GaCl 3 , 64 CuCl 2 , 111 InCl 3 or 86 YCl 3 , react at 37°C for 10-60 min, and then pass the reaction solution through Sep-Pak C18 Separation and purification by chromatographic column to obtain the corresponding 68 Ga, 64 Cu, 111 In or 86 Y labeled complexes.
  • the preparation method includes the following steps:
  • the preparation method includes the following steps:
  • the purified product is diluted with physiological saline and sterile filtered to obtain the injection of each complex.
  • the fifth aspect of the present invention provides the use of the granzyme B targeting complex, or the granzyme B targeting radiopharmaceutical in the preparation of nuclear medicine imaging reagents.
  • the nuclear medicine imaging reagent is used, for example, in tumor imaging diagnosis and immunotherapy monitoring.
  • the expression of granzyme B during tumor immunotherapy can be detected by imaging, so as to predict or monitor Efficacy of tumor immunotherapy; or other diseases that lead to overexpression of granzyme B, such as immune cardiomyopathy, side effects related to granzyme B caused by immunotherapy, etc.
  • the granzyme B targeting complex provided by the invention can be labeled with a radionuclide to prepare granzyme B targeting radiopharmaceuticals.
  • the provided granzyme B-targeted radiopharmaceutical is simple to prepare, and has better pharmacokinetic properties and in vivo metabolic stability than other granzyme B-targeted drugs. It can non-invasively and quantitatively monitor the expression level of granzyme B in vivo through nuclear medicine imaging.
  • Figure 1 is the mass spectrum of the DOTA-coupled granzyme B targeting complex.
  • Fig. 2 is a schematic diagram of the chemical structure of DOTA-coupled granzyme B targeting complex labeled with 68 Ga, 64 Cu, 111 In or 86 Y.
  • Figure 3 is the mass spectrum of the NOTA-coupled granzyme B targeting complex.
  • Fig. 4 is a schematic diagram of the chemical structure of 68 Ga or 64 Cu labeled NOTA-coupled granzyme B targeting complex.
  • Fig. 5 is a schematic diagram of the chemical structure of 18 F-labeled NOTA-coupled granzyme B targeting complex.
  • Figure 6 is the mass spectrum of HYNIC-coupled granzyme B targeting complex.
  • Fig. 7 is a schematic diagram of the chemical structure of 99m Tc-labeled HYNIC-coupled granzyme B targeting complex.
  • Fig. 8 shows the experimental results of binding specificity between 68 Ga-labeled DOTA complexes and coated granzyme B.
  • Figure 9 shows the in vitro stability results of 68 Ga-labeled DOTA complexes in PBS and FBS.
  • Figure 10 shows the metabolic stability results of 68 Ga-labeled DOTA complexes in mice.
  • Figure 11 shows the PET imaging of 68 Ga-labeled DOTA complex in tumor-bearing mice and its comparison with radiopharmaceutical imaging properties of similar structures.
  • Figure 12 shows the experimental results of the correlation between the uptake value of 68 Ga-labeled DOTA complex in MC38 tumor and the expression level of granzyme B in the tumor determined by in vitro Western blot.
  • Figure 13 is the PET imaging of 68 Ga-labeled DOTA complexes to monitor the results of tumor anti-PD-1 immunotherapy experiments.
  • Figure 14 shows the experimental results of PET imaging of 68 Ga-labeled DOTA complexes in predicting pseudo-progression of tumor immunotherapy.
  • Figure 15 shows the experimental results of PET imaging of 68 Ga-labeled NOTA complexes in tumor-bearing mice.
  • Figure 16 shows the experimental results of PET imaging of 18 F-labeled NOTA complexes in tumor-bearing mice.
  • Fig. 17 is the experimental result of SPECT imaging of 99m Tc-labeled HYNIC complex in tumor-bearing mice.
  • the granzyme B targeting complex (compound 7) was synthesized according to the following solid-phase synthesis route.
  • R is any one of bifunctional chelating groups or derivatives thereof for radionuclide labeling.
  • the bifunctional chelating group is a group formed by bifunctional chelating agents DOTA, NOTA, HYNIC, and MAG2.
  • Reaction conditions (a) DCM solution of Fmoc-NHS and DIPEA; (b) DMF/DCM solution of 2-chlorotriphenyl chloride resin and DIPEA; (c) DMF solution of 20% piperidine, Fmoc-(2S, 5S)-5-amino-1,2,4,5,6,7-hexahydroazepino[3,2,1-Hi]indole-4-one-2-carboxylic acid, HBTU, HOBt and DIPEA DMF solution; ( d) DMF solution of 20% piperidine, DMF solution of Fmoc-L-isoleucine, HBTU, HOBt and DIPEA; (e) DMF solution of 20% piperidine, Fmoc-(3-aminomethylphenyl) The DMF solution of acetic acid, HBTU, HOBt and EIPEA; (f) the DMF solution of 20% piperidine, the DMF solution of Fmoc-L-aspartic acid-1
  • Synthesis of Compound 4 The coupling of amino acids was carried out according to the standard Fmoc solid-phase synthesis method. Take a certain mass of resin 3 (0.25mmol) in a 10mL solid-phase synthesis tube, add 2mL dichloromethane (DCM) to swell, repeat three times, each time for 5min, and then wash three times with N,N-dimethylformamide (DMF) , 5 minutes each time. Use the DMF solution (v/v) containing 20% piperidine to remove the amino protecting group Fmoc, the specific operation is 2mL of 20% piperidine in DMF solution to react for 2min, 10min, 10min, then use 2mL DMF to wash 3-5 times, every 2 minutes each time.
  • DCM dichloromethane
  • DMF N,N-dimethylformamide
  • Synthesis of Compound 7 Take a certain mass of Compound 6 (5 ⁇ mol), dissolve it in 500 ⁇ L of DMSO, then add 10-fold molar ratio of bifunctional chelating agent-NHS or bifunctional chelating agent-p-SCN-Bn, and DIPEA (10 ⁇ mol). Mixed and reacted at room temperature for 2 hours, the crude product was purified by HPLC and freeze-dried.
  • the schematic diagram of its chemical structure is shown in Figure 4.
  • the schematic diagram of its chemical structure is shown in Figure 5.
  • Synthesis of HYNIC coupling complexes and labeling of 99m Tc radionuclides to prepare corresponding radiopharmaceuticals Specifically include the following steps:
  • Granzyme B was coated on the ELISA plate, and 7.4 KBq of 68 Ga-labeled DOTA complex was added to the coated plate. After reacting at room temperature for 0.5 h, the plate was washed, and the binding amount of 68 Ga-labeled DOTA complex in the plate was measured by a gamma counter. The results are shown in Figure 8. It can be seen that the binding of 68 Ga-labeled DOTA complex to granzyme B is significantly higher than that of the control group, which confirms the in vitro granzyme B binding specificity of 68 Ga-labeled DOTA complex.
  • mice were injected with 37MBq 68 Ga-labeled DOTA complex through the tail vein, and their serum and urine were collected 0.5 hours after the injection. After centrifugation, the supernatant was diluted with 50% acetonitrile aqueous solution. After filtering through a 0.22 ⁇ m filter membrane, its stability was analyzed by HPLC. The results are shown in Figure 10. It can be seen that the 68 Ga-labeled DOTA complex remains in the form of the original drug in both urine and serum, suggesting that it has excellent metabolic stability in vivo.
  • the 68 Ga-labeled DOTA complex of the present invention has the best tumor granzyme B uptake and low normal tissue (such as gastrointestinal tract) uptake compared with the other three comparative radiopharmaceuticals. It is suggested that the granzyme B targeting complex of the present invention has the most optimized internal medicine imaging properties in vivo.
  • PET imaging of 68 Ga-labeled DOTA complex quantifies the expression of granzyme B in tumors.
  • MC38 and 4T1 tumor cells were inoculated subcutaneously in the right axilla of C57 and BALB/c mice, respectively, to establish mouse models of pseudoprogression and true progression of immunotherapy.
  • 200 ⁇ g of anti-PD-1 antibody and anti-CTLA-4 antibody were injected intraperitoneally three times on day 0, day 3, and day 6, respectively.
  • tumor-bearing mice were injected with 7.4 MBq of newly prepared 68 Ga-labeled complex via tail vein.
  • small animal PET/CT imaging was performed after isoflurane anesthesia.
  • the tumor size changes in MC38 and 4T1 tumor-bearing mice were measured by vernier calipers. The results are shown in Figure 14.
  • the tumor uptake of 68 Ga-labeled complexes on day 6 was significantly higher than that on day 0.
  • the tumor uptake of the 68 Ga-labeled complex on day 6 was not significantly different from that on day 0. It is suggested that the 68 Ga-labeled complex can monitor the expression of granzyme B in the tumor during immunotherapy, and predict the true and false progress of tumor immunotherapy by reflecting the activation status of T cells.
  • 99m Tc-labeled HYNIC complex SPECT imaging tumor granzyme B. Take 18.5 MBq of newly prepared 99m Tc-labeled HYNIC complex and inject it into female C57 mice bearing MC38 tumors in the right axilla through the tail vein. After injection for 0.5 hours, perform small animal SPECT/CT imaging after isofluorane anesthesia . The results are shown in Figure 17. It can be seen that the 99m Tc-labeled HYNIC complex also has a good uptake value in MC38 tumors. It is suggested that granzyme B-targeting radiopharmaceuticals of the present invention can also perform granzyme B-targeting specific nuclear medicine imaging by replacing different bifunctional chelating agents with different radionuclide labels.

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Abstract

本发明属于核医学领域，涉及一种颗粒酶B靶向配合物、放射性药物及其制备方法和应用。所述颗粒酶B靶向配合物的结构如式(I)所示，其中，R为用于放射性核素标记的双功能螯合基团或其衍生物中的任意一种。本发明提供的颗粒酶B靶向配合物可经放射性核素标记制备成颗粒酶B靶向放射性药物。提供的颗粒酶B靶向放射性药物制备简单，与其他颗粒酶B靶向药物相比具有更佳的药代动力学特性和体内代谢稳定性。能够通过核医学显像无创监测体内颗粒酶B的表达水平。

Description

颗粒酶B靶向配合物、放射性药物及其制备方法和应用 技术领域
本发明属于核医学领域，具体地，涉及一种颗粒酶B靶向配合物及制备方法、一种颗粒酶B靶向放射性药物及制备方法，以及它们在肿瘤等相关疾病核医学显像诊断及治疗显像监测中的应用。
背景技术
颗粒酶是一种丝氨酸蛋白酶，人类颗粒酶包括A、B、H、K、M五种，存在于细胞毒性T淋巴细胞(cytotoxic T lymphocyte，CTL)和自然杀伤细胞(natural killer cell，NK)所释放的细胞颗粒中。颗粒酶B作为颗粒酶最主要的效应分子之一，可以进入细胞，介导下游caspase信号通路的激活，诱导细胞DNA的断裂，从而导致细胞凋亡。同时，颗粒酶B还可切割细胞核蛋白，包括NuMA和P以及DNA-PKcs等，促进和启动细胞核凋亡。因此，颗粒酶B是CTL或NK细胞激活并发挥细胞杀伤作用的重要标志物之一。
在肿瘤治疗领域，免疫治疗被认为是最为理想的消灭肿瘤和预防肿瘤转移和复发的方式之一。目前，肿瘤免疫治疗如针对免疫检查点(如CTLA-4、PD-1/PD-L1)的抗体以及嵌合抗原受体的自体T细胞(CAR-T)疗法取得了一系列重大的临床突破。然而，目前肿瘤免疫治疗的有效率低。以抗PD-1免疫检查点抑制为例，其有效率往往不足30％。因此，对免疫治疗进行早期疗效精确预测从而有效指导其精准治疗和提高治疗疗效至关重要。
以正电子发射计算机断层(positron emission tomography，PET)和单光子发射计算机断层(Single-photon emission computed tomography，SPECT)为代表的核医学分子影像为无创、动态、定量显像监测疾病治疗提供了有效的技术手段。 18F-氟代脱氧葡萄糖( 18F-FDG)是目前临床上应用最广泛的PET显像药物。 18F-FDG是葡萄糖的类似物，其生物学行为与葡萄糖相似，通过监测疾病对葡萄糖的摄取反映疾病的发生与进展。然而， 18F-FDG缺乏肿瘤特异性，非CTL或NK等活性细胞的特异性标志物。在用于免疫治疗疗效预测和评估方面具有明显的局限性。因此，研制针对肿瘤免疫治疗疗效监测的新型核医学显像药物具有重要的临床意义。
颗粒酶B是CTL和NK细胞在免疫应答过程中释放的丝氨酸蛋白酶，其表达量与免疫应答密切相关。因此，如果能够开发具有良好颗粒酶B靶点特异性、亲和力和体内代谢特性的核医学显像药物，特别是 68Ga、 18F、 99mTc等核素标记的PET和SPECT药物，将在肿瘤免疫治疗及疗效监测方面发挥重要的作用，具有广阔的临床应用前景。
另外，颗粒酶B在其他病变，如自身免疫病、免疫诱导的心肌炎等也呈现特异性高表达。研制颗粒酶B靶向特异性核医学显像药物同样能在这类疾病的显像诊断、治疗监测和疗效判断方面发挥重要的作用。
发明内容
基于上述背景，本发明提供一类新型颗粒酶B靶向的配合物，其由颗粒酶B靶向分子与双功能螯合剂偶联而成。该类配合物经放射性核素标记后成为核医学显像用放射性药物。通过核医学PET或SPECT显像可以实现对颗粒酶B表达量的无创特异性监测，有望在肿瘤免疫治疗、自身免疫病的诊断及治疗监测得以推广应用。本发明所述颗粒酶B特异性放射性药物制备简单、体内药代动力学特性佳。
本发明的第一方面提供一种颗粒酶B靶向配合物，其结构如下式(I)所示：
Figure PCTCN2022118207-appb-000001
其中，R为用于放射性核素标记的双功能螯合基团或其衍生物中的任意一种。
根据本发明，所述双功能螯合基团为双功能螯合剂形成的基团，优选地，所述双功能螯合剂为DOTA、NOTA、HYNIC、MAG2、NODA、NODAGA、DOTP、TETA、ATSM、PTSM、EDTA、EC、HBEDCC、DTPA、BAPEN、Df、DFO、TACN、NO2A、NOTAM、CB-DO2A、Cyclen、DO3A、DO3AP、MAS3、MAG3或异腈。
进一步优选地，所述R为式(II)、式(III)、式(IV)、式(V)、式(VI)所示基团或其衍生物中的任意一种，
Figure PCTCN2022118207-appb-000002
Figure PCTCN2022118207-appb-000003
本发明的第二方面提供一种颗粒酶B靶向放射性药物，由所述的配合物经放射性核素标记得到。
根据本发明，所述放射性核素可以为诊断用放射性核素，也可以为治疗用放射性核素。
所述诊断用放射性核素优选为 68Ga、 64Cu、 18F、 86Y、 90Y、 89Zr、 111In、 99mTc、 11C、 123I、 125I和 124I中的至少一种。
所述治疗用放射性核素优选为 177Lu、 125I、 131I、 211At、 111In、 153Sm、 186Re、 188Re、 67Cu、 212Pb、 225Ac、 213Bi、 212Bi和 212Pb中的至少一种。
根据本发明一些优选实施方式，所述放射性核素为 68Ga、 64Cu、 111In、 18F、 86Y、 99mTc中的任意一种。
本发明的第三方面提供上述颗粒酶B靶向配合物的制备方法，包括以下步骤：
a.根据如下固相合成路线合成颗粒酶B靶向前体；
Figure PCTCN2022118207-appb-000004
反应条件：(a)Fmoc-NHS和DIPEA的DCM溶液；(b)2-氯三苯基氯树脂和DIPEA的DMF/DCM溶液；(c)20％哌啶的DMF溶液，Fmoc-(2S,5S)-5-amino-1,2,4,5,6,7-hexahydroazepino[3,2,1-Hi]indole-4-one-2-carboxylic acid、HBTU、HOBt和DIPEA的DMF溶液；(d)20％哌啶的DMF溶液，Fmoc-L-异亮氨酸、HBTU、HOBt和DIPEA的DMF溶液；(e)20％哌啶的DMF溶液，Fmoc-(3-氨基甲基苯基)乙酸、HBTU、HOBt和EIPEA的DMF溶液；(f)20％哌啶的DMF溶液，Fmoc-L-天冬氨酸-1-叔丁酯、HBTU、HOBt和EIPEA的DMF溶液；(g)20％哌啶的DMF溶液，Fmoc-L-天冬氨酸-1-叔丁酯、HBTU、HOBt和EIPEA的DMF溶液；(h)20％哌啶的DMF溶液；(i)三氟乙酸，水，三异丙基硅烷；(j)DIPEA，DMSO溶液。
b.将双功能螯合剂偶联到所述颗粒酶B靶向前体上。
本发明颗粒酶B靶向配合物制备过程中使用的化合物可商购或通过常规有机合成方法制得。
以DOTA-NHS为例，采用如下合成路线将双功能螯合剂偶联到所述颗粒酶B靶向前体上：
Figure PCTCN2022118207-appb-000005
本发明的第四方面提供颗粒酶B靶向放射性药物的制备方法，包括以下步骤：将颗粒酶B靶向配合物溶于放射性标记缓冲液中，随后加入不同的放射性核素进行反应，反应后将反应液经Sep-Pak C18色谱柱分离纯化，得到相应的颗粒酶B靶向放射性药物。
根据本发明一种具体实施方式，当所述配合物为DOTA偶联配合物，所述放射性核素为 68Ga、 64Cu、 111In、 86Y中的任意一种时，所述制备方法包括以下步骤：
将所述DOTA偶联配合物溶于酸性缓冲溶液中，然后加入 68GaCl 3、 64CuCl 2、 111InCl 3或 86YCl 3，37℃反应10-60min，随后，将反应液经Sep-Pak C18色谱柱分离纯化，得到 相应的 68Ga、 64Cu、 111In或 86Y标记的配合物。
根据本发明一种具体实施方式，当所述配合物为NOTA偶联配合物，所述放射性核素为 68Ga、 64Cu、 18F中的任意一种时，所述制备方法包括以下步骤：
将所述NOTA偶联配合物溶于酸性缓冲溶液中，随后加入 68GaCl 3、 64CuCl 2，37℃反应10-30min，冷却后将反应液经Sep-Pak C18色谱柱分离纯化，得到相应的 68Ga或 64Cu标记的配合物；或者，
将 18F离子与AlCl 3混合于醋酸钠缓冲液，在室温下反应2-8min，随后，向混合液中加入所述NOTA偶联配合物并在加热105-115℃条件下反应10-20min，冷却后将反应液经Sep-Pak C18色谱柱分离纯化，得到相应的 18F标记的配合物。
根据本发明一种具体实施方式，当所述配合物为MAG2偶联配合物或HYNIC偶联配合物，所述放射性核素为 99mTc时，所述制备方法包括以下步骤：
将所述MAG2偶联配合物溶于醋酸铵和酒石酸缓冲液中，随后加入Na 99mTcO 4，混匀后加入新鲜制备的SnCl 2，随后加热到95-105℃反应40-80min，冷却后将反应液经Sep-Pak C18色谱柱分离纯化，得到相应的 99mTc标记的配合物；或者，
将所述HYNIC偶联配合物、TPPTS琥珀酸缓冲液及tricine琥珀酸缓冲液混合后加入Na 99mTcO 4，随后加热到95-105℃反应20-40min，冷却后将反应液经Sep-Pak C18色谱柱分离纯化，得到相应的 99mTc标记的配合物。
根据本发明，优选地，在分离纯化后，将纯化产物经生理盐水稀释后无菌过滤，可以得到各配合物的注射液。
本发明的第五方面提供所述颗粒酶B靶向配合物，或者所述颗粒酶B靶向放射性药物在制备核医学显像试剂中的用途。
所述核医学显像试剂例如用于肿瘤显像诊断、免疫治疗监测。具体地，例如显像检测肿瘤免疫治疗(如抗PD-1/PD-L1，抗CTLA-4，或CAR-T，CAR-NK抗肿瘤治疗)过程中颗粒酶B的表达，从而预测或监测肿瘤免疫治疗疗效；或者其他导致颗粒酶B过度表达的其他疾病，例如免疫性心肌病、免疫治疗导致的颗粒酶B相关的副反应等。
本发明的有益效果：
本发明提供的颗粒酶B靶向配合物可经放射性核素标记制备成颗粒酶B靶向放射性药物。提供的颗粒酶B靶向放射性药物制备简单，与其他颗粒酶B靶向药物相比具有更佳的药代动力学特性和体内代谢稳定性。能够通过核医学显像无创、定量监测体内颗粒酶B的表达水平。
本发明的其它特征和优点将在随后具体实施方式部分予以详细说明。
附图说明
通过结合附图对本发明示例性实施方式进行更详细的描述，本发明的上述以及其它目的、特征和优势将变得更加明显。
图1为DOTA偶联的颗粒酶B靶向配合物的质谱图。
图2为 68Ga、 64Cu、 111In或 86Y标记的DOTA偶联的颗粒酶B靶向配合物的化学结构示意图。
图3为NOTA偶联的颗粒酶B靶向配合物的质谱图。
图4为 68Ga或 64Cu标记的NOTA偶联的颗粒酶B靶向配合物的化学结构示意图。
图5为 18F标记的NOTA偶联的颗粒酶B靶向配合物的化学结构示意图。
图6为HYNIC偶联的颗粒酶B靶向配合物的质谱图。
图7为 99mTc标记的HYNIC偶联的颗粒酶B靶向配合物的化学结构示意图。
图8为 68Ga标记DOTA配合物与包被的颗粒酶B的结合特异性实验结果。
图9为 68Ga标记DOTA配合物在PBS和FBS中的体外稳定性结果。
图10为 68Ga标记DOTA配合物的小鼠体内的代谢稳定性结果。
图11为 68Ga标记DOTA配合物在荷瘤小鼠中的PET显像及其与类似结构对比放射性药物显像特性比较。
图12为 68Ga标记DOTA配合物在MC38肿瘤内的摄取值与离体Western blot测定的肿瘤内颗粒酶B的表达量相关性实验结果。
图13为 68Ga标记DOTA配合物的PET显像监测肿瘤抗PD-1免疫治疗实验结果。
图14为 68Ga标记DOTA配合物的PET显像预测肿瘤免疫治疗假性进展的实验结果。
图15为 68Ga标记NOTA配合物在荷瘤小鼠中的PET显像的实验结果。
图16为 18F标记NOTA配合物在荷瘤小鼠中的PET显像的实验结果。
图17为 99mTc标记HYNIC配合物在荷瘤小鼠中的SPECT显像的实验结果。
具体实施方式
下面将更详细地描述本发明的优选实施方式。虽然以下描述了本发明的优选实施方式，然而应该理解，可以以各种形式实现本发明而不应被这里阐述的实施方式所限制。
实施例1
颗粒酶B靶向配合物的合成。
根据如下固相合成路线合成颗粒酶B靶向配合物(化合物7)。其中，R为用于放射性核素标记的双功能螯合基团或其衍生物中的任意一种。所述双功能螯合基团为由双功能螯合剂DOTA、NOTA、HYNIC、MAG2形成的基团。
Figure PCTCN2022118207-appb-000006
具体合成步骤如下：
反应条件：(a)Fmoc-NHS和DIPEA的DCM溶液；(b)2-氯三苯基氯树脂和DIPEA的DMF/DCM溶液；(c)20％哌啶的DMF溶液，Fmoc-(2S,5S)-5-amino-1,2,4,5,6,7-hexahydroazepino[3,2,1-Hi]indole-4-one-2-carboxylic acid、HBTU、HOBt和DIPEA的DMF溶液；(d)20％哌啶的DMF溶液，Fmoc-L-异亮氨酸、HBTU、HOBt和DIPEA的DMF溶液；(e)20％哌啶的DMF溶液，Fmoc-(3-氨基甲基苯基)乙酸、HBTU、HOBt和EIPEA的DMF溶液；(f)20％哌啶的DMF溶液，Fmoc-L-天冬氨酸-1-叔丁酯、HBTU、HOBt和EIPEA的DMF溶液；(g)20％哌啶的DMF溶液，Fmoc-L-天冬氨酸-1-叔丁酯、HBTU、HOBt和EIPEA的DMF溶液；(h)20％哌啶的DMF溶液；(i)三氟乙酸，水，三异丙基硅烷；(j)DIPEA，DMSO溶液。
化合物2的合成：取1H-[1,2,3]噻唑-4-基甲基胺盐酸盐(1，200.00mg，2.04mmol)和9-芴甲基-N-琥珀酰亚胺基碳酸酯(825.77mg，2.45mmol)于50mL圆底烧瓶中，加入10mL DCM使之溶解，加入DIPEA(526.32mg,4.08mmol),室温电磁搅拌，反应4h后停止。减压蒸馏，往混合物中加入100mL DCM，水洗(50mL×2),收集有机相，经无水硫酸镁干燥后加压除去二氯甲烷，产物经硅胶柱纯化后得白色固体2。
树脂3的合成：将2-氯三苯基氯树脂(1.00g)于固相合成管中，加入2mL二氯甲烷(DCM)溶胀，重复三次，每次5min，随后用N,N-二甲基甲酰胺(DMF)洗涤三次， 每次5min。将化合物2(96.00mg，0.30mmol)溶于DCM和DMF的混合溶剂中，加入DIPEA(78mg，0.60mmol)，将以上混合物加入到固相合成管中，室温电磁搅拌，反应2h后停止；2mL二氯甲烷(DCM)洗涤，重复三次，每次5min；使用混合溶剂7mL(DCM:MeOH:DIPEA＝10mL:10mL:1mL)对树脂进行封闭，重复三次，每次5min；使用2mL二氯甲烷(DCM)洗涤，重复三次，每次5min，减压除去溶剂得到黄色树脂3。
化合物4的合成：氨基酸的偶连依照标准的Fmoc固相合成法进行。取一定质量的树脂3(0.25mmol)于10mL固相合成管，加入2mL二氯甲烷(DCM)溶胀，重复三次，每次5min，随后用N,N-二甲基甲酰胺(DMF)洗涤三次，每次5min。使用含20％哌啶的DMF溶液(v/v)脱去氨基保护基Fmoc，具体操作为2mL 20％哌啶的DMF溶液反应2min、10min、10min，随后使用2mL DMF洗涤3-5次，每次2min。相对于树脂(0.02mmol)，3倍化学量的Fmoc氨基酸在7.2倍化学量的DIPEA存在下，经3.6倍化学量的HBTU活化后加入到合成管中，电磁搅拌下反应1h。
化合物5的合成：依上述方法使用含20％哌啶的DMF溶液(v/v)脱去氨基保护基Fmoc。
化合物6的合成：化合物从树脂上解离和叔丁酯的脱去使用5mL三氟乙酸/三异丙基硅烷/水(95:2.5:2.5，v/v/v)搅拌2h完成，并用2mL三氟乙酸清洗树脂，收集所有滤液，经减压除去三氟乙酸后，粗产物经HPLC反向制备，冻干后得到。
化合物7的合成：取一定质量的化合物6(5μmol)，将其溶于500μL的DMSO中，然后加入10倍摩尔比的双功能螯合剂-NHS或双功能螯合剂-p-SCN-Bn，以及DIPEA(10μmol)。混匀后置室温反应2h，粗产物经HPLC纯化，冻干后得到。
实施例2
DOTA偶联配合物的合成及对其进行 68Ga、 64Cu、 111In、 86Y任意一种放射性核素的标记制备成相应的放射性药物。具体包括以下步骤：
取5μmol的化合物6，将其溶于500μL的DMSO中，然后加入50μmol的DOTA-NHS和10μmol的DIPEA。混匀后置室温反应2h，粗产物经HPLC纯化，冻干后得到DOTA偶联颗粒酶B靶向配合物。其质谱表征见图1。
将10nmol的DOTA偶联配合物溶于300μL的0.1M醋酸钠缓冲液(pH＝5.5)；随后加入185MBq的 68GaCl 3、 64CuCl 2、 111InCl 3或 86YCl 3，37℃反应30min，随后，将 反应液经Sep-Pak C18色谱柱分离纯化，纯化产物经生理盐水稀释后无菌过滤，即得到相应的 68Ga、 64Cu、 111In或 86Y标记的配合物注射液。其化学结构示意图见图2。
实施例3
NOTA偶联配合物的合成及对其进行 68Ga、 64Cu或 18F任意一种放射性核素的标记制备成相应的放射性药物。具体包括以下步骤：
取5μmol的化合物6，将其溶于500μL的DSMO中，然后加入50μmol的NOTA-NHS，以及10μmol的DIPEA。混匀后置室温反应2h，粗产物经HPLC纯化，冻干后得到NOTA偶联颗粒酶B靶向配合物。其质谱表征见图3。
68Ga或 64Cu放射性标记：将10nmol的NOTA偶联配合物溶于300μL的0.1M醋酸钠缓冲液(pH＝5.5)；随后加入185MBq的 68GaCl 3或 64CuCl 2，37℃反应15min，冷却后将反应液经Sep-Pak C18色谱柱分离纯化，纯化产物经生理盐水稀释后无菌过滤，即得到相应的 68Ga或 64Cu标记的配合物注射液。其化学结构示意图见图4。
18F放射性标记：将740MBq的 18F离子与24nmol AlCl 3混合于100μL的醋酸钠缓冲液(0.1M，pH＝4.0)，在室温下反应5min。随后，向混合液中加入40nmol的NOTA偶联配合物并在加热110℃条件下反应15min。冷却后将反应液经Sep-Pak C18色谱柱分离纯化，纯化产物经生理盐水稀释后无菌过滤，即得到相应的 18F标记的配合物注射液。其化学结构示意图见图5。
实施例4
HYNIC偶联配合物的合成及对其进行 99mTc放射性核素的标记制备成相应的放射性药物。具体包括以下步骤：
取5μmol的化合物6，将其溶于500μL的DMSO中，然后加入50μmol的HYNIC-NHS和10μmol的DIPEA。混匀后置室温反应2h，粗产物经HPLC纯化，冻干后得到HYNIC偶联颗粒酶B靶向配合物。其质谱表征见图6。
将10nmol的HYNIC偶联配合物、100μL的TPPTS(100μg/μL溶于25mM的琥珀酸缓冲液)及tricine(100mg/mL溶于25mM的琥珀酸缓冲液)混合后加入370MBq的Na 99mTcO 4，随后加热到100℃反应30min。冷却后将反应液经Sep-Pak C18色谱柱分离纯化，纯化产物经生理盐水稀释后无菌过滤，即得到相应的 99mTc标记的配合物注射液。其化学结构示意图见图7。
实施例5
68Ga标记DOTA配合物的体外颗粒酶B结合特异性。
将颗粒酶B包被于ELISA板中，将7.4KBq的 68Ga标记DOTA配合物加入到包被板中。室温反应0.5h后，洗板，通过伽马计数器测定板中 68Ga标记DOTA配合物的结合量。其结果示于图8，可见 68Ga标记DOTA配合物与颗粒酶B结合显著高于对照组，证实了 68Ga标记DOTA配合物的体外颗粒酶B结合特异性。
实施例6
68Ga标记DOTA配合物的体外稳定性。
取3.7MBq的 68Ga标记DOTA配合物溶于PBS或10％的胎牛血清(FBS)中，分别在室温孵育0h、0.5h、1h和2h后通过放射性薄层色谱检测其放射化学纯度(RCP)。其结果示于图9，可见 68Ga标记DOTA配合物在PBS和FBS均呈现良好的稳定性。
实施例7
68Ga标记DOTA配合物的体内代谢稳定性。
经尾静脉向BALB/c小鼠注射37MBq的 68Ga标记DOTA配合物，注射后0.5小时取其血清和尿液，离心后取上清用50％乙腈的水溶液稀释。经0.22μm滤膜滤过后用HPLC分析其稳定性。其结果示于图10，可见 68Ga标记DOTA配合物在尿液和血清中均保持原药形式，提示其具有优良的体内代谢稳定性。
实施例8
68Ga标记DOTA配合物在荷瘤小鼠中的PET显像及其与类似结构对比放射性药物显像特性比较。
为比较本发明所述的颗粒酶B靶向配合物的体内核医学显像优势，采用类似合成方法制备了3种结构类似的颗粒酶B靶向配合物。均经DOTA偶联后进行 68Ga标记得到相应的颗粒酶B靶向放射性化合物。取7.4MBq新制备的 68Ga标记配合物，经尾静脉注射到雌性荷MC38肿瘤的C57小鼠体内，注射0.5h后经异氟烷麻醉后进行小动物PET/CT显像。其结果示于图11，可见本发明的 68Ga标记DOTA配合物与其他3种对比放射性药物相比具有最佳的肿瘤颗粒酶B摄取及低的正常组织(如胃肠道)摄取。提示本发明所 述的颗粒酶B靶向配合物具有最优化的体内核医学显像特性。
实施例9
68Ga标记DOTA配合物的PET显像定量肿瘤颗粒酶B表达量。
取7.4MBq新制备的 68Ga标记配合物，经尾静脉注射到雌性右侧腋下荷MC38肿瘤的C57小鼠体内，注射0.5h后经异氟烷麻醉后进行小动物PET/CT显像。待显像结束后，将小鼠处死，取其肿瘤组织。将肿瘤组织研磨提取蛋白后，通过Western blot测定的肿瘤内颗粒酶B的表达量。结果示于图12，可见 68Ga标记DOTA配合物在MC38肿瘤内的摄取值与离体Western blot测定的肿瘤内颗粒酶B的表达量呈现良好的线性相关性。提示通过本发明所述颗粒酶B靶向放射性药物的核医学显像可以无创测定颗粒酶B的表达水平，并可动态监测其表达变化。
实施例10
68Ga标记DOTA配合物的PET显像监测肿瘤抗PD-1免疫治疗。右侧腋下荷MC38肿瘤的C57小鼠分别在第0天、第3天和第6天经腹腔注射200μg的抗PD-1抗体共计3次。在第9天，荷瘤小鼠经尾静脉注射7.4MBq新制备的 68Ga标记配合物。注射0.5h后经异氟烷麻醉后进行小动物PET/CT显像。通过游标卡尺测定MC38荷瘤小鼠肿瘤大小变化。其结果示于图13，可见 68Ga标记DOTA配合物在肿瘤摄取的高低可以很好地区分肿瘤是否对抗PD-1免疫治疗有响应。提示可通过本发明所述颗粒酶B靶向放射性药物的核医学显像无创监测颗粒酶B的表达水平来预测肿瘤免疫治疗的疗效。
实施例11
68Ga标记DOTA配合物的PET显像鉴定肿瘤免疫治疗的假性进展。
分别在C57和BALB/c小鼠右侧腋下皮下接种MC38和4T1肿瘤细胞，建立免疫治疗假性进展和真性进展小鼠模型。分别在第0天、第3天和第6天经腹腔注射200μg的抗PD-1抗体和抗CTLA-4抗体共计3次。在第12天，荷瘤小鼠经尾静脉注射7.4MBq新制备的 68Ga标记配合物。注射0.5h后经异氟烷麻醉后进行小动物PET/CT显像。通过游标卡尺测定MC38和4T1荷瘤小鼠肿瘤大小变化。其结果示于图14，可见在MC38假性进展小鼠肿瘤模型中， 68Ga标记配合物在第6天的肿瘤摄取显著高于第0天。而在4T1真性进展小鼠肿瘤模型中， 68Ga标记配合物在第6天的肿瘤摄取与第0天无明显差 别。提示 68Ga标记配合物可以监测免疫治疗过程中的颗粒酶B在肿瘤内表达，通过反应T细胞激活状态预测肿瘤免疫治疗的真、假性进展。
实施例12
68Ga标记NOTA配合物的PET显像肿瘤颗粒酶B。
取7.4MBq新制备的 68Ga标记的NOTA配合物，经尾静脉注射到右侧腋下雌性荷MC38肿瘤的C57小鼠体内，注射0.5h后经异氟烷麻醉后进行小动物PET/CT显像。结果示于图15，可见 68Ga标记NOTA配合物同样在MC38肿瘤具有较好的摄取值。提示本发明所述颗粒酶B靶向放射性药物更换不同的双功能螯合剂不影响其颗粒酶B靶向特性。
实施例13
取7.4MBq新制备的 18F标记的NOTA配合物，经尾静脉注射到雌性右侧腋下荷MC38肿瘤的C57小鼠体内，注射0.5h后经异氟烷麻醉后进行小动物PET/CT显像。结果示于图16，可见 18F标记NOTA配合物同样在MC38肿瘤具有较好的摄取值。提示本发明所述颗粒酶B靶向放射性药物更换不同的双功能螯合剂及更换不同的放射性核素不影响其颗粒酶B靶向特性。
实施例14
99mTc标记HYNIC配合物的SPECT显像肿瘤颗粒酶B。取18.5MBq新制备的 99mTc标记HYNIC配合物，经尾静脉注射到右侧腋下雌性荷MC38肿瘤的C57小鼠体内，注射0.5h后经异氟烷麻醉后进行小动物SPECT/CT显像。结果示于图17，可见 99mTc标记HYNIC配合物同样在MC38肿瘤具有较好的摄取值。提示本发明所述颗粒酶B靶向放射性药物更换不同的双功能螯合剂进行不同的放射性核素标记同样可以进行颗粒酶B靶向特异性核医学显像。
以上已经描述了本发明的各实施例，上述说明是示例性的，并非穷尽性的，并且也不限于所披露的各实施例。在不偏离所说明的各实施例的范围和精神的情况下，对于本技术领域的普通技术人员来说许多修改和变更都是显而易见的。

Claims (10)

  1. 一种颗粒酶B靶向配合物，其结构如下式(I)所示：
    Figure PCTCN2022118207-appb-100001
    其中，R为用于放射性核素标记的双功能螯合基团或其衍生物中的任意一种。
  2. 根据权利要求1所述的颗粒酶B靶向配合物，其特征在于，所述双功能螯合基团为双功能螯合剂形成的基团，所述双功能螯合剂为DOTA、NOTA、HYNIC、MAG2、NODA、NODAGA、DOTP、TETA、ATSM、PTSM、EDTA、EC、HBEDCC、DTPA、BAPEN、Df、DFO、TACN、NO2A、NOTAM、CB-DO2A、Cyclen、DO3A、DO3AP、MAS3、MAG3或异腈。
  3. 根据权利要求2所述的颗粒酶B靶向配合物，其特征在于，所述R为式(II)、式(III)、式(IV)、式(V)、式(VI)所示基团或其衍生物中的任意一种，
    Figure PCTCN2022118207-appb-100002
    Figure PCTCN2022118207-appb-100003
  4. 一种颗粒酶B靶向放射性药物，由权利要求1-3中任意一项所述的配合物经放射性核素标记得到；所述放射性核素为诊断用放射性核素或治疗用放射性核素；其中，
    所述诊断用放射性核素优选为 68Ga、 64Cu、 18F、 86Y、 90Y、 89Zr、 111In、 99mTc、 11C、 123I、 125I和 124I中的至少一种；
    所述治疗用放射性核素优选为 177Lu、 125I、 131I、 211At、 111In、 153Sm、 186Re、 188Re、 67Cu、 212Pb、 225Ac、 213Bi、 212Bi和 212Pb中的至少一种。
  5. 权利要求1-3中任意一项所述的颗粒酶B靶向配合物的制备方法，包括以下步骤：
    a.根据如下固相合成路线合成颗粒酶B靶向前体；
    Figure PCTCN2022118207-appb-100004
    b.将双功能螯合剂偶联到所述颗粒酶B靶向前体上。
  6. 权利要求4所述的颗粒酶B靶向放射性药物的制备方法，包括以下步骤：将颗粒酶B靶向配合物溶于放射性标记缓冲液中，随后加入不同的放射性核素进行反应，反应后将反应液经Sep-Pak C18色谱柱分离纯化，得到相应的颗粒酶B靶向放射性药物。
  7. 根据权利要求6所述的颗粒酶B靶向放射性药物的制备方法，其特征在于，所述配合物为DOTA偶联配合物，所述放射性核素为 68Ga、 64Cu、 111In、 86Y中的任意一种，所述制备方法包括以下步骤：
    将所述DOTA偶联配合物溶于酸性缓冲溶液中，然后加入 68GaCl 3、 64CuCl 2、 111InCl 3或 86YCl 3，37℃反应10-60min，随后，将反应液经Sep-Pak C18色谱柱分离纯化，得到相应的 68Ga、 64Cu、 111In或 86Y标记的配合物。
  8. 根据权利要求6所述的颗粒酶B靶向放射性药物的制备方法，其特征在于，所述配合物为NOTA偶联配合物，所述放射性核素为 68Ga、 64Cu、 18F中的任意一种，所述制备方法包括以下步骤：
    将所述NOTA偶联配合物溶于酸性缓冲溶液中，随后加入 68GaCl 3、 64CuCl 2，37℃反应10-30min，冷却后将反应液经Sep-Pak C18色谱柱分离纯化，得到相应的 68Ga或 64Cu标记的配合物；或者，
    将 18F离子与AlCl 3混合于醋酸钠缓冲液，在室温下反应2-8min，随后，向混合液中加入所述NOTA偶联配合物并在加热105-115℃条件下反应10-20min，冷却后将反应液经Sep-Pak C18色谱柱分离纯化，得到相应的 18F标记的配合物。
  9. 根据权利要求6所述的颗粒酶B靶向放射性药物的制备方法，其特征在于，所述配合物为MAG2偶联配合物或HYNIC偶联配合物，所述放射性核素为 99mTc，所述制备方法包括以下步骤：
    将所述MAG2偶联配合物溶于醋酸铵和酒石酸缓冲液中，随后加入Na 99mTcO 4，混匀后加入新鲜制备的SnCl 2，随后加热到95-105℃反应40-80min，冷却后将反应液经Sep-Pak C18色谱柱分离纯化，得到相应的 99mTc标记的配合物；或者，
    将所述HYNIC偶联配合物、TPPTS琥珀酸缓冲液及tricine琥珀酸缓冲液混合后加入Na 99mTcO 4，随后加热到95-105℃反应20-40min，冷却后将反应液经Sep-Pak C18色谱柱分离纯化，得到相应的 99mTc标记的配合物。
  10. 权利要求1-3中任意一项所述的颗粒酶B靶向配合物，或者权利要求4所述的颗粒酶B靶向放射性药物在制备核医学显像试剂中的用途；优选地，所述核医学显像试剂用于肿瘤显像诊断、免疫治疗监测。
PCT/CN2022/118207 2021-11-18 2022-09-09 颗粒酶b靶向配合物、放射性药物及其制备方法和应用 WO2023087871A1 (zh)

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