Heterodimer radionuclide coupling medicine as well as preparation and application thereof

Abstract

The invention relates to a GRPR and integrin alpha V beta 3 heterodimer radionuclide coupled drug with a novel structure, which has higher specificity on two receptors of integrin alpha V beta 3 and GRPR, shows stronger tumor uptake ability and longer tumor retention time compared with a monomer receptor, has good safety in clinical application, and can be used for preparing a novel drug for treating tumor tumors. Compared with the prior art, the method provided by the invention not only has higher tumor uptake rate and tumor-to-non-target ratio, but also has great potential in distinguishing primary tumor parts and metastases, provides more accurate information for tumor diagnosis and staging, monitors metastasis conditions in the treatment process, and has great clinical application prospects.

Description

Heterodimer radionuclide conjugated drug, preparation and application thereof Technical Field

[0001] The present invention relates to the field of radionuclide conjugated drugs, in particular to a heterodimer radionuclide conjugated drug containing GRPR. The present invention also relates to the preparation and application of the radionuclide conjugated drug. Background Art

[0002] Radionuclide drug conjugates (RDCs) are an emerging class of precision-targeted tumor drugs. These drugs are structurally composed of a targeting ligand (e.g., an antibody, small molecule, or peptide), a linker, a chelator, and a tracer (radionuclide). The ligand mediates specific targeting, and the linker and chelator work together to guide the radionuclide to a precise location, thereby concentrating the radiation produced by the radionuclide to a localized tissue, thereby achieving diagnostic and therapeutic goals. Therefore, the specificity and targeting properties of the ligand, the type of tracer, and the type of linker and chelator used to connect them are all key factors influencing the properties of the final RDC. Furthermore, due to the heterogeneity of tumors and the complex tumor microenvironment, receptor imaging based on a single target still has certain clinical limitations and cannot meet the diverse characteristics of tumors. Consequently, heterodimeric RDCs have emerged. Heterodimeric radionuclide-conjugated drugs are radionuclide-conjugated drugs that can simultaneously target two antigens or two different epitopes on a single antigen. Compared to conventional radionuclide-conjugated drugs, heterodimeric radionuclide-conjugated drugs have an additional specific antigen-binding site, resulting in enhanced specificity and more accurate tumor cell targeting, expanding the detection range, thereby reducing false-negative rates and improving diagnostic sensitivity. However, because heterodimeric radionuclide-conjugated drugs involve the coupling and synergistic effects of multiple ligands, R&D and production challenges in ligand selection, linkage methods, quality, and stability have severely hindered drug development and widespread clinical application.

[0003] The gastrin-releasing peptide receptor (GRPR) is a member of the bombesin G protein-coupled receptor family. It is expressed in tissues such as the central nervous system, gastrointestinal tract, pancreas, and adrenal cortex, regulating a variety of physiological functions. Furthermore, GRPR is overexpressed in a variety of malignant tumors, including prostate cancer, breast cancer, gastrointestinal stromal tumors, pancreatic cancer, gastric cancer, cervical cancer, small cell lung cancer, and glioblastoma. GRPR overexpression in malignant tissues has prompted the development of GRPR-targeted radiopharmaceuticals to better treat or diagnose cancers with high GRPR expression.

[0004] The first generation of bombesin analogue radionuclide conjugated drugs are GRPR agonists derived from the C-terminal fragment of the amphibian tetradecapeptide bombesin (BBN, Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2). GRPR agonists have internalization properties after binding to the receptor, which allows them to remain in the target cells for a long time. Therefore, it has been considered a prerequisite for high in vivo absorption rate for many years. To date, several BBN-based GRPR agonist radionuclide conjugated drugs (such as 68 Ga-AMBA, 68 Ga-BBN, 68 Ga-BBN-RGD, etc.) have been introduced into clinical practice [Reference 1: Cagnolini, A., Chen, J., Ramos, K., Marie Skedzielewski, T., Lantry, LE, Nunn, AD,…Linder, KE (2010). Automated synthesis, characterization and biological evaluation of [ 68 Ga]Ga-AMBA,and the synthesis and characterization of natGa-AMBA and[ 67 Ga]Ga-AMBA.Applied Radiation and Isotopes, 68(12),2285–2292.; Literature 2; Zhang J, Li D, Lang L, Zhu Z, Wang L, Wu P, Niu G, Li F, Chen X. 68Ga-NOTA-Aca-BBN(7-14)PET / CT in Healthy Volunteers and Glioma Patients.J Nucl Med.2016Jan;57(1):9-14.; Document 3: Zhang J, Niu G, Lang L, Li F, Fan X, Yan X, Yao S, Yan W, Huo L, Chen L, Li Z, Zhu Z, Chen X.Clinical Translation of a Dual Integrin αvβ3-and Gastrin-Releasing Peptide Receptor-Targeting PET Radiotracer, 68 Ga-BBN-RGD. J Nucl Med. 2017 Feb; 58(2): 228-234.], used for cancer diagnosis and radioligand therapy. However, since agonist-induced GRPR activation can have adverse effects on the gastrointestinal system (such as release of gastrointestinal peptide hormones, stimulation of exocrine gland secretion, smooth muscle contraction, etc.) [Reference 4: Maina, T., Nock, BA, Kulkarni, H., Singh, A., & Baum, RP (2017). Theranostic Prospects of Gastrin-Releasing Peptide Receptor–Radioantagonists in Oncology. PET Clinics, 12(3), 297–309.], although they can be internalized in cancer cells, they have not been further developed.

[0005] Compared with GRPR agonists, GRPR antagonists have more favorable pharmacokinetics. Although studies have shown that GRPR antagonists do not activate receptors after binding, they can successfully target GRPR-expressing cancer lesions and remain fully retained therein in animal models and humans, while being rapidly cleared from physiological organs. Therefore, research on GRPR antagonist radionuclide-conjugated drugs has gradually increased, such as RM2, NeoBOMB1, etc. However, in patients and preclinical animal models, it was found that these GRPR antagonist radionuclide-conjugated drugs accumulate in large quantities in normal organs (especially the pancreas). This high uptake not only affects the detection of lesions, but also limits the maximum tolerated dose of targeted radiotherapy applications [Reference 5: Kurth J., Krause BJ, SM, Bergner C., Hakenberg OW, Heuschkel M. First-in-human dosimetry of gastrin-releasing peptide receptor antagonist[ 177 Lu] Lu-RM2: A radiopharmaceutical for the treatment of metastatic castration-resistant prostate cancer. Eur.J.Nucl.Med.Mol.Imaging.2020; 47:123–135.; Document 6: Nock BA, Kaloudi A., Lymperis E., Giarika A., Kulkarni HR, Klette I., Singh A., Krenning EP, de Jong M., Maina T. Theranostic perspectives in prostate cancer with the gastrin-releasing peptide receptor antagonist NeoBOMB1: Preclinical and first clinical results. J. Nucl. Med. 2017; 58:75–80.]. RM26 is a high-affinity GRPR antagonist discovered by modifying the peptide backbone of a bombesin analog (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2). A clinical study confirmed that 68 Ga-RM26 showed high specific uptake and high tumor-to-background ratio in tumors. 68 Ga-RM26 showed good tolerability and was more effective than GRPR agonists. 68 Ga-BBN, 68 Ga-RM26 can detect more primary lesions and lymph node metastases, and the tracer accumulation is also higher. 68 Ga-BBN is high, which indicates 68 Ga-RM26 is expected to be superior to GRPR agonists as an imaging marker for evaluating GRPR expression [Reference 7: Zhang J, Niu G, Fan X, Lang L, Hou G, Chen L, Wu H, Zhu Z, Li F, Chen X. PET Using a GRPR Antagonist 68Ga-RM26 in Healthy Volunteers and Prostate Cancer Patients. J Nucl Med. 2018 Jun; 59(6): 922-928.] However, some clinical studies have found that RM26-based radiotherapy has high pancreatic and bladder toxicity, which to some extent limits the clinical application of GRPR antagonists.

[0006] Integrin α V β3 is a cell adhesion receptor that is very important for the formation, survival and maturation of new blood vessels during angiogenesis. Since its expression level is related to the invasiveness of tumors, integrin α V β3 has become an important biological target for molecular imaging probes in early tumor diagnosis. Synthetic peptides derived from the arginine-glycine-aspartic acid (RGD) sequence bind to integrin α V β3 has high affinity and binding specificity and has been developed for integrin α V Imaging of β3-positive cancers, but although RGD performs well in primary tumor detection, it has not shown sufficient sensitivity for lymph node staging [Reference 8: Beer AJ, Niemeyer M, Carlsen J, Sarbia M, J, Watzlowik P, et al. Patterns of alphavbeta3 expression in primary and metastatic human breast cancer as shown by 18F-Galacto-RGD PET. J Nucl Med. 2008; 49:255-9.].

[0007] Therefore, there is still a huge unmet clinical need in the accurate diagnosis, staging and treatment of cancer. Summary of the Invention

[0008] To solve the above problems, the present invention has developed a novel structure of GRPR and integrin α V The β3 heterodimer radionuclide-coupled drug provided by the present invention has enhanced tumor uptake and significantly improved tumor-to-non-target ratio compared to the monomer, has significant advantages in cancer diagnosis, and has great potential for clinical transformation.

[0009] Specifically, the present invention provides a heterodimeric radionuclide-conjugated drug, which is a compound represented by formula (I) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt:

[0010] in:

[0011] The A and B are targeting groups;

[0012] The L1 and L2 are connecting groups;

[0013] Said R is a metal chelator containing at least one radionuclide; and

[0014] The targeting group A targets integrin α v β3;

[0015] The targeting group B is a GRPR antagonist.

[0016] Furthermore, the GRPR antagonist is selected from:

[0017] Furthermore, the structure of the targeting group A is:

[0018] Furthermore, the structure of the linking group L1 is:

[0019] Furthermore, the structure of the linking group L2 is:

[0020] In some preferred embodiments, when L1 is When L2 is

[0021] In other preferred embodiments, when L1 is When L2 is

[0022] In other preferred embodiments, when L1 is When L2 is

[0023] In other preferred embodiments, when L1 is When L2 is

[0024] In other preferred embodiments, when L1 is When L2 is

[0025] In other preferred embodiments, when L1 is When L2 is

[0026] In other preferred embodiments, when L1 is When L2 is

[0027] In other preferred embodiments, when L1 is The L2 is

[0028] In other preferred embodiments, when L1 is The L2 is

[0029] In some more preferred embodiments, the Selected from: It can be understood that the remaining structures not given can be given without any doubt with reference to the above-mentioned connection method.

[0030] In some more preferred embodiments, the heterodimeric radionuclide-drug conjugate structure is selected from the following (wherein the wavy line represents the covalent attachment site), wherein R is a metal chelator comprising at least one radionuclide:

[0031] In other preferred embodiments, the heterodimer radionuclide-drug conjugate structure is selected from the following: Wherein R is a metal chelator containing at least one radioactive nuclide.

[0032] Furthermore, the metal chelator is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), DOTAGA-TETRA (t-Bu edter), and NODAGA, and their structures are shown below (in the state without radionuclide attached), wherein the wavy line marks the site where the metal chelator and the linker L1 are covalently attached:

[0033] In some preferred embodiments, the metal chelator is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA) and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

[0034] To further illustrate the heterodimeric radionuclide-conjugated drug provided by the present invention, in some specific embodiments, the structure of the heterodimeric radionuclide-conjugated drug described in the present invention can be exemplified by the following structure, wherein M is any optional radionuclide (it is understood that other structures not shown can also be obtained without doubt according to the following exemplary structure):

[0035] Furthermore, the radionuclide is selected from C-11, N-13, O-15, F-18, Na-24, P-32, P-33, K-42, Sc-43, Sc-44, Sc-47, Cr-51, Mn-51, Fe-52, Mn-52, Mn-52m, Co-55, Co-57, Fe-59, Co-60, Cu-62, Cu-64, Cu-67, Ga-67 , Ga-68, As-72, Br-75, Se-75, Br-76, As-77, Rb-82m, Sr-83, Y-86, Y-88, Zr-89, Sr-89, ln-9 0. Y-90, Tc-94, Mo-99, Tc-99m, Pd-103, Rh-105, Ru-106, Pd-109, In-110, In-111, Ag-111, I- 120, I-123, I-124, I-125, I-131, Xe-133, Cs-137, Pr-142, Pr-143, Tb-149, Tb-151, Gd-152 , Gd-153, Sm-153, Gd-154, Gd-155, Gd-156, Gd-157, Gd-158, Tb-161, Dy-165, Dy-166, Ho-166 , Er-169, Yb-169, Lu-177, Yb-177, Re-186, Re-188, Re-189, Ir-192, Ir-194, Au-198, Au-199, At-211, Pb-211, Bi-212, Pb-212, Bi-213, Ra-223, Ac-225, Fm-255, Th-226, Th-227.

[0036] To further illustrate the heterodimer radionuclide conjugated drug provided by the present invention, in some exemplary embodiments, the radionuclide is 68 Ga, the heterodimer radionuclide conjugated drug structure is selected from the following:

[0037] In order to further illustrate the heterodimer radionuclide conjugated drug provided by the present invention, in other exemplary embodiments, the radionuclide is 18 F, the heterodimer radionuclide-conjugated drug structure is selected from the following:

[0038] In order to further illustrate the heterodimer radionuclide conjugated drug provided by the present invention, in other exemplary embodiments, the radionuclide is 177 Lu, the heterodimer radionuclide-coupled drug structure is selected from the following:

[0039] In other specific embodiments, the metal chelator and radionuclide may also have other options. Other structures not provided can all be obtained without doubt with reference to the above-mentioned connection mode. It is understood that the present invention is intended to provide a heterodimer radionuclide conjugated drug, and those skilled in the art can select suitable chelators and nuclides according to clinical needs, so the selection of chelators and nuclides should not be regarded as a limitation of the present invention.

[0040] The present invention also provides a heterodimeric compound that can be labeled with a radionuclide, characterized in that the heterodimeric compound that can be labeled with a radionuclide is a compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt:

[0041] in:

[0042] The A and B are targeting groups;

[0043] The L1 and L2 are connecting groups;

[0044] The R' is a metal chelating agent; and

[0045] The targeting group A targets integrin α v β3;

[0046] The targeting group B is a GRPR antagonist.

[0047] Furthermore, the GRPR antagonist is selected from:

[0048] Furthermore, the structure of the targeting group A is:

[0049] Furthermore, the structure of the linking group L1 is:

[0050] Furthermore, the structure of the linking group L2 is:

[0051] In some preferred embodiments, when L1 is When L2 is

[0052] In other preferred embodiments, when L1 is When L2 is

[0053] In other preferred embodiments, when L1 is When L2 is

[0054] In other preferred embodiments, when L1 is When L2 is

[0055] In other preferred embodiments, when L1 is When L2 is

[0056] In other preferred embodiments, when L1 is When L2 is

[0057] In other preferred embodiments, when L1 is When L2 is

[0058] In other preferred embodiments, when L1 is The L2 is

[0059] In other preferred embodiments, when L1 is The L2 is

[0060] In some more preferred embodiments, the Selected from: It is understandable that other structures not shown can also be obtained without any doubt by referring to the above connection method.

[0061] In some more preferred embodiments, the structure of the heterodimeric compound that can be labeled with a radionuclide is selected from the following (wherein the wavy line represents a covalent attachment site), wherein R' is a metal chelator:

[0062] In a further preferred embodiment, the structure of the heterodimer compound that can be labeled with radionuclides is selected from the following:

[0063] Wherein R' is a metal chelating agent.

[0064] Furthermore, the metal chelator is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), DOTAGA-TETRA (t-Bu edter), and NODAGA, and their structures are shown below (in the state without radionuclide attached), wherein the wavy line marks the site where the metal chelator and the linker L1 are covalently attached:

[0065] In some preferred embodiments, the metal chelator is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA) and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA).

[0066] To further illustrate the heterodimeric compound that can be labeled with a radionuclide provided by the present invention, in some specific embodiments, the heterodimeric compound that can be labeled with a radionuclide according to the present invention can be exemplified by the following structure (other structures not shown can also be obtained according to the following exemplary structure without any doubt):

[0067] In other specific embodiments, the metal chelator may also have other options. Other structures not given can be obtained without doubt with reference to the above-mentioned connection mode. It is understood that the present invention is intended to provide a heterodimeric compound that can be radiolabeled. Those skilled in the art can select a suitable chelating agent according to clinical needs, so the selection of the chelating agent should not be regarded as a limitation of the present invention.

[0068] The present invention also provides a heterodimer compound, wherein the heterodimer compound is a compound represented by formula (III) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt: A-L'1-L2-B (III)

[0069] in:

[0070] The A and B are targeting groups;

[0071] The L'1 and L2 are linking groups; and

[0072] The targeting group A targets integrin α v β3;

[0073] The targeting group B is a GRPR antagonist.

[0074] Furthermore, the GRPR antagonist is selected from:

[0075] Furthermore, the structure of the targeting group A is:

[0076] Furthermore, the structure of the linking group L'1 is:

[0077] Furthermore, the structure of the linking group L2 is:

[0078] In some preferred embodiments, when L'1 is When L2 is

[0079] In other preferred embodiments, when L'1 is When L2 is

[0080] In other preferred embodiments, when L'1 is When L2 is

[0081] In other preferred embodiments, when L'1 is When L2 is

[0082] In other preferred embodiments, when L'1 is When L2 is

[0083] In other preferred embodiments, when L'1 is When L2 is

[0084] In other preferred embodiments, when L'1 is When L2 is

[0085] In other preferred embodiments, when L'1 is

[0086] In other preferred embodiments, when L'1 is The L2 is

[0087] In some more preferred embodiments, the Selected from: It is understood that other structures not shown can also be obtained without any doubt by referring to the above coupling methods.

[0088] In some more preferred embodiments, the heterodimeric compound structure is selected from the following (wherein the wavy line represents the covalent attachment site):

[0089] In a further preferred embodiment, the structure of the heterodimer compound is selected from the following:

[0090] The present invention also provides a pharmaceutical composition comprising any of the heterodimeric radionuclide-conjugated drugs described above, or any of the heterodimeric compounds described above that can be labeled with a radionuclide, or any of the heterodimeric compounds described above.

[0091] Furthermore, the pharmaceutical composition described above further comprises a pharmaceutically acceptable carrier, excipient, diluent and / or additive.

[0092] The present invention also provides any of the heterodimer radionuclide conjugated drugs described above, or any of the heterodimer compounds that can be labeled with radionuclides described above, or any of the heterodimer compounds described above, or any of the pharmaceutical compositions described above for use in the preparation of a drug for binding / inhibiting GRPR and / or binding / inhibiting α v Application of β3 drugs.

[0093] The present invention also provides the use of any of the above-mentioned heterodimeric radionuclide-conjugated drugs, or any of the above-mentioned heterodimeric compounds that can be labeled with radionuclides, or any of the above-mentioned heterodimeric compounds, or any of the above-mentioned pharmaceutical compositions in the preparation of molecular imaging agents.

[0094] Furthermore, in the above application, the heterodimer radionuclide-conjugated drug, or the heterodimer compound that can be labeled with a radionuclide, or the heterodimer compound, or the pharmaceutical composition is an effective amount of a radiopharmaceutical.

[0095] Furthermore, the above-mentioned developer is used to v β3-positive tumors were imaged.

[0096] Furthermore, the imaging agent described above is used to image GRPR-overexpressing tumors.

[0097] Furthermore, the above-mentioned developer is used to v Tumors correlating β3 positivity and GRPR overexpression were imaged.

[0098] Furthermore, the above-mentioned tumors include but are not limited to prostate cancer, breast cancer, colon cancer, lung cancer, head and neck cancer, kidney cancer, pancreatic cancer, brain tumor, liver cancer, and ovarian cancer.

[0099] The present invention also provides the use of any of the above-mentioned heterodimeric radionuclide-conjugated drugs, or any of the above-mentioned heterodimeric compounds that can be labeled with radionuclides, or any of the above-mentioned heterodimeric compounds, or any of the above-mentioned pharmaceutical compositions in the preparation of drugs for treating or preventing tumors.

[0100] Furthermore, in the above application, the heterodimer radionuclide-conjugated drug, or the heterodimer compound that can be labeled with a radionuclide, or the heterodimer compound, or the pharmaceutical composition is a therapeutically effective amount.

[0101] Furthermore, the above-mentioned tumor is α v Tumors associated with β3 positive overexpression.

[0102] Furthermore, the above-mentioned tumor is a GRPR overexpression-related tumor.

[0103] Furthermore, the above-mentioned tumor is α v β3-positive and GRPR-overexpression-related tumors.

[0104] Furthermore, the tumor includes but is not limited to prostate cancer, breast cancer, colon cancer, lung cancer, head and neck cancer, kidney cancer, pancreatic cancer, brain tumor, liver cancer, and ovarian cancer.

[0105] The present invention also provides a kit, which contains a predetermined amount of any of the above-mentioned heterodimer compounds that can be labeled with radionuclides, and a nuclide for radioactively labeling the heterodimer compound that can be labeled with radionuclides.

[0106] The present invention also provides another kit, which contains a predetermined amount of any one of the above-mentioned heterodimers and a metal chelator-nuclide complex for radiolabeling the heterodimer.

[0107] The present invention also provides any of the above heterodimeric radionuclide-conjugated drugs, or any of the above heterodimeric compounds that can be labeled with radionuclides, or a method for preparing any of the above heterodimeric compounds.

[0108] The present invention also provides any of the above heterodimeric radionuclide-conjugated drugs, or any of the above heterodimeric compounds that can be labeled with radionuclides, or a preparation route for any of the above heterodimeric compounds.

[0109] The heterodimer radionuclide conjugated drug provided by the present invention is effective for integrin α V The two receptors, β3 and GRPR, have high specificity and show stronger tumor uptake ability and longer tumor retention time compared with monomeric receptors. The heterodimeric radionuclide-conjugated drugs provided by the present invention are safely tolerated by all test patients in clinical cancer diagnosis, without any adverse reactions after injection, and their effective whole-body dose is far lower than the dose limit required by the FDA, and have good safety in clinical applications. PET imaging of the heterodimeric radionuclide-conjugated drugs provided by the present invention can detect more metastatic lymph nodes, brain metastases and bone metastases, and can provide more information for the progression and aggressiveness of tumors in the diagnosis of primary malignant tumors and metastases, and provide molecular details for the design of treatment plans. The heterodimeric radionuclide-conjugated drugs provided by the present invention can make up for the shortcomings of PSMA-based radioactive tracers in detecting PSMA-negative or low-expressing prostate cancer, can play an important role in early diagnosis, and provide guidance for potential targeted radiotherapy using tracers labeled with α and β particles. In summary, the heterodimeric radionuclide-conjugated drug provided by the present invention not only has a higher tumor uptake rate and tumor-to-nontarget ratio, but also has great potential in distinguishing primary tumor sites from metastatic lesions. This will provide more precise information for tumor diagnosis and staging, and may also enable monitoring of metastasis during treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1 68 HPLC spectrum of Ga-NOTA-II-1, wherein Figure A is 68 HPLC spectrum of Ga-NOTA-II-1 purity; Figure B, Figure C, Figure D are 68 Stability HPLC profiles of Ga-NOTA-II-1 in phosphate-buffered saline (PBS) at 0.5, 1, and 2 h after incubation.

[0111] Figure 2 68Cellular uptake of Ga-NOTA-II-1 and blocking assay of unlabeled precursors 1, 2, and 3 on PC3 tumor cells.

[0112] Figure 3 68 Saturation binding experiment of Ga-NOTA-II-1 on PC3 tumor cells.

[0113] Figure 4 shows 68 Cellular uptake of Ga-DOTA-II-4 and blocking assay of unlabeled precursors 1, 4, and 5 on PC3 tumor cells.

[0114] Figure 5 68 Saturation binding experiment of Ga-DOTA-II-4 on PC3 tumor cells.

[0115] Figure 6 shows the injection 68 Ga-NOTA-II-1、 68 Ga-labeled precursor 1, 68 Representative whole-body PET images after Ga-labeled precursor 2, where panel A is 68 PET images of Ga-NOTA-II-1 at 0.5, 1, and 2 hours of static scanning for 5 minutes in PC3 tumor-bearing mice; Figure C 68 PET images of Ga-labeled precursor 1 in PC3 tumor-bearing mice at 0.5, 2 hours, and 5 minutes of static scanning; Figure E is 68 PET images of Ga-labeled precursor 2 in PC3 tumor-bearing mice at 0.5 and 2 hours of static scanning for 5 minutes; Figures B, D, and F are 68 Ga-NOTA-II-1、 68 Ga-labeled precursor 1, 68 Tissue uptake values ​​and tumor / non-target (T / NT) ratios of Ga-labeled precursor 2 at different time points.

[0116] Figure 7 68 The temporal trend of Ga-NOTA-II-1 tissue uptake in PC3 tumor-bearing mice, including heart (A), muscle (B), tumor (C), blood (D), liver (E), and kidney (F).

[0117] Figure 8 shows the injection 68 Ga-DOTA-II-4, 68 Ga-labeled precursor 1, 68 Representative whole-body PET images after Ga-labeled precursor 4, where panel A is 68 PET images of Ga-DOTA-II-4 in PC3 tumor-bearing mice at 0.5, 1, and 2 hours; Figure B is 68Tissue uptake values ​​and tumor / non-target (T / NT) ratios of Ga-DOTA-II-4; Figures C and D are 68 Ga-labeled precursor 1 and 68 PET images of Ga-labeled pro-4 at 0.5 h and 2 h post-inoculation in PC3 tumor mice.

[0118] Figure 9 68 The temporal trend of Ga-DOTA-II-4 tissue uptake in PC3 tumor-bearing mice, including heart (A), muscle (B), tumor (C), blood (D), liver (E), and kidney (F).

[0119] Figure 10 68 Tumor uptake of Ga-DOTA-compound 16 in the PC3 tumor mouse model.

[0120] Figure 11 68 Tumor uptake of Ga-DOTA-compound 17 in the PC3 tumor mouse model.

[0121] Figure 12 68 Tumor uptake of Ga-DOTA-compound 18 in the PC3 tumor mouse model.

[0122] Figure 13 68 Average 2-h time-activity curves (TACs) of Ga-NOTA-II-1 in normal organs and tumors over time.

[0123] Figure 14 shows 68 PET / CT maximum intensity projection (MIP) images of Ga-NOTA-II-1 over time in patient 1.

[0124] Figure 15 68 Ga-NOTA-II-1 and 18 F-FDG PET / CT comparison diagram, where Figure A is 68 PET / CT MIP images of Ga-NOTA-II-1 in patients with recurrent breast cancer, Figure E 18 F-FDG PET / CT MIP images in patients with recurrent breast cancer; Figures B, F, I, and K are 68 Axial PET and PET / CT images of Ga-NOTA-II-1; Figures C, G, J, and L are 18 Figure D is the patient's axial CT scan image; Figure H is the patient's axial MRI scan image. 18 Compared with F-FDG, 68Ga-NOTA-II-1PET / CT showed more metastatic lymph nodes (circle mark and 1# arrow), bone metastasis (2# arrow), and brain metastasis (3# arrow), and the imaging effect was better. 18 F-FDG is clearer.

[0125] Figure 16 68 Ga-NOTA-II-1 and 18 F-FDG PET / CT images of axillary lymph node metastasis in patient No. 7, where Figure A is 68 PET / CT MIP spectrum of Ga-NOTA-II-1; Figure BD is 68 Axial image of Ga-NOTA-II-1; Figure E is 18 F-FDG PET / CT MIP atlas; Figures FH are 18 Axial image of F-FDG.

[0126] Figure 17 shows the PET / CT MIP and axial images of patient No. 10, where Figures A to D are 68 PET / CT MIP and axial images of Ga-NOTA-II-1; Figures E-H are 18 F-FDG PET / CT MIP and axial images.

[0127] Figure 18 68 Biodistribution profile of Ga-DOTA-II-4 in normal organs and tumors.

[0128] Figure 19 shows representative images of patient No. 8, where A and E represent 68 PET / CT fusion image, CT and PET images of Ga-labeled precursor 1, C and G represent 68 PET / CT fusion image, CT and PET images of Ga-DOTA-II-4, B is HE staining of the lesion, D is GRPR receptor staining of the lesion, F is integrin α V β3 receptor staining. DETAILED DESCRIPTION

[0129] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as they may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0130] Preferably, the terms used herein are defined in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Klb1, Hb, eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0131] Unless the context requires otherwise, throughout the specification and the claims that follow, the word "comprise" and its variations such as "comprising" and "containing" will be understood to implicitly include the stated integers or steps, or groups of integers or steps, but not to exclude any other integers or steps, or groups of integers or steps. In the following paragraphs, the same aspects of the invention will be defined in more detail. Each aspect so defined can be combined with any other aspect or aspects unless there is a clear indication to the contrary. In particular, any feature that is optional, preferred or advantageous can be combined with any other feature or features that are optional, preferred or advantageous.

[0132] Throughout this specification, some documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's instructions, operating instructions, etc.), whether above or below, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to such disclosures as prior inventions. Certain documents cited herein are identified as "incorporated by reference." In the event that a definition or teaching in such an incorporated reference conflicts with a definition or teaching described in this specification, the text of this specification shall prevail.

[0133] The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. Example 1 Preparation of Compound (II-1-NOTA)

[0134] The preparation route and method of compound (II-1-NOTA) are as follows:

[0135] Synthesis of compound 3: Compound 1 (60 mg, 0.1 mmol) and compound 2 (56 mg, 0.1 mmol) were dissolved in DMF (5 ml), and N, N-diisopropylethylamine (65 mg, 0.5 mmol) was added. The mixture was reacted at room temperature for 6 h. DMF was removed under high vacuum, and the residue was redissolved in DCM-TFA (volume ratio of 2:1, 10 ml). The reaction was monitored by high performance liquid chromatography, and the reaction was completed within 4 h. After the reaction, the reaction mixture was concentrated in vacuo, and the residue was redissolved in DMF (10 ml). NOTA-NHS (42 mg, 0.1 mmol) and N, N-diisopropylethylamine (130 mg, 1 mmol) were added. The reaction was monitored by high performance liquid chromatography, and the reaction was completed within 5 h. After the reaction was complete, piperidine (0.5 mm L) was gradually added, DMF was removed under high vacuum, and then DCM (4 mL) and TFA (4 mL) were added. The reaction was monitored by high performance liquid chromatography and was completed within 3 h. Mobile phase A (deionized water) and mobile phase B (acetonitrile) were acidified to pH = 3 with trifluoroacetic acid. The gradient elution process was as follows: 10% mobile phase B, 0-3 minutes; 10-90% mobile phase B, 3-14 minutes; 90% mobile phase B, 14-16 minutes; 90-10% mobile phase B, 16-18 minutes; 10% mobile phase B, 18-20 minutes. The product was concentrated in vacuo and purified by Pre-HPLC to obtain 33 mg of compound 3.

[0136] Synthesis of Compound 5: Weigh 10g of MBHA resin and add it to a 1L solid-phase synthesizer. Then add 300ml of dichloromethane and agitate with N2. Swell the resin for half an hour, then filter. Mix 80ml of DMF and 20ml of piperidine and add them to the solid-phase synthesizer. Agitate with N2. Separately, dissolve 10.61g of Fmoc-Leu-OH, 5.70g of HBTU, 0.16g of HOBT, and 2.63g of DIPEA in a 500ml beaker in 100ml of DMF. Stir and activate at room temperature. After a half-hour reaction, filter and wash with 2 x 150ml of dichloromethane, 150ml of methanol, and 2 x 150ml of DMF, then filter. Add the activated solution to the solid-phase synthesizer and agitate with N2 for 1 hour. A small amount of the resin was aspirated for color development, and the resin appeared colorless. Filter with suction and wash with 2 x 150 ml of dichloromethane, 150 ml of methanol, and 2 x 150 ml of DMF, sequentially. Continue solid-phase synthesis to connect Fmoc-Sta-OH, Fmoc-His(Boc)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln-OH, and Fmoc-D-Phe-OH. After amino acid connection, mix 80 ml of DMF and 20 ml of piperidine and add to the solid-phase synthesizer. Incite the reaction with N2. After half an hour of reaction, filter with suction and wash with 2 x 150 ml of dichloromethane, 150 ml of methanol, and 2 x 150 ml of DMF, and filter with suction. Add 100 ml of 95% trifluoroacetic acid solution to cut the resin. After 2 hours of reaction, filter and collect the liquid. The liquid was added dropwise to methyl tert-butyl ether, stirred and crystallized, and the solid was collected by filtration to obtain Compound 5.

[0137] Synthesis of Compound (II-1-NOTA): Compound 5 (65 mg, 0.05 mmol) was dissolved in DMF (10 ml), and N,N'-dicyclohexylcarbodiimide (12 mg, 0.06 mmol) and N-hydroxysuccinimide (7 mg, 0.06 mmol) were added. The reaction mixture was stirred at room temperature for 12 hours. Compound 3 (50 mg, 0.05 mmol) and N,N-diisopropylethylamine (13 mg, 0.1 mmol) were then added. The reaction was monitored by HPLC and completed within 6 hours. Mobile phase A (deionized water) and mobile phase B (acetonitrile) were acidified to pH 3 with trifluoroacetic acid. The gradient elution process was as follows: 10% mobile phase B, 0-3 minutes; 10-90% mobile phase B, 3-14 minutes; 90% mobile phase B, 14-16 minutes; 90-10% mobile phase B, 16-18 minutes; 10% mobile phase B, 18-20 minutes. The product was purified by Pre-HPLC to obtain 30 mg of compound (II-1-NOTA). Example 2 Preparation of compound (II-4-DOTA)

[0138] The preparation route and method of compound (II-4-DOTA) are as follows:

[0139] Synthesis of Compound 8: Compound 6 (62 mg, 0.1 mmol) and Compound 7 (56 mg, 0.1 mmol) were dissolved in DMF (5 ml), and N,N-diisopropylethylamine (65 mg, 0.5 mmol) was added. The reaction mixture was stirred at room temperature for 6 hours. The DMF was removed under high vacuum, and the residue was redissolved in DCM-TFA (2:1 volume ratio, 10 mL). After reacting for 4 hours, the mixture was concentrated in vacuo and purified by Pre-HPLC to yield 39 mg of Compound 6.

[0140] Synthesis of compound 9: Compound 4 (111 mg, 0.1 mmol) was dissolved in DMF (10 mL), and 2,5-dioxopyrrolidin-1-yl 3-(2-(3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-3-oxopropoxy)ethoxy)propanoate (43 mg, 0.1 mmol) and N,N-diisopropylethylamine (65 mg, 0.5 mmol) were added. The reaction mixture was stirred at room temperature for 6 hours. The DMF was removed under high vacuum, and the mixture was purified by Pre-HPLC to obtain 90 mg of compound 9.

[0141] Synthesis of compound (II-4-DOTA): Compound 8 (14 mg, 0.02 mmol) and compound 9 (28 mg, 0.02 mmol) were dissolved in 5 mL of DMF. The reaction was shaken at room temperature overnight. DOTA-NHS (15 mg, 0.025 mmol) and N,N-diisopropylethylamine (13 mg, 0.1 mmol) were then added. Mobile phase A (deionized water) and mobile phase B (acetonitrile) were acidified to pH 3 with trifluoroacetic acid. Gradient elution was performed as follows: 10% mobile phase B, 0-3 minutes; 10-90% mobile phase B, 3-14 minutes; 90% mobile phase B, 14-16 minutes; 90-10% mobile phase B, 16-18 minutes; 10% mobile phase B, 18-20 minutes. After purification by Pre-HPLC, 17 mg of compound (II-4-DOTA) was obtained.

[0142] Example 3 Preparation of Compound (II-1-DOTA), Compound (II-2-NOTA), Compound (II-2-DOTA), Compound (II-3-NOTA), Compound (II-3-DOTA), and Compound (II-4-NOTA)

[0143] Compound (II-1-DOTA), compound (II-2-NOTA), compound (II-2-DOTA), compound (II-3-NOTA), compound (II-3-DOTA), and compound (II-4-NOTA) were prepared with reference to the preparation routes and methods of Examples 1 and 2. Those skilled in the art can prepare the compounds by replacing the corresponding raw materials based on the basis / inspiration of Examples 1-2, and the specific preparation methods are not described in detail here.

[0144] (1) Preparation route of compound (II-1-DOTA)

[0145] (2) Preparation route of compound (II-2-NOTA)

[0146] (3) Preparation route of compound (II-2-DOTA)

[0147] (4) Preparation route of compound (II-3-NOTA)

[0148] (5) Preparation route of compound (II-3-DOTA)

[0149] (6) Preparation route of compound (II-4-NOTA) Example 4 Other Preparation Examples

[0150] The following compounds were prepared with reference to the preparation routes and methods of Examples 1, 2 and 3. Those skilled in the art can prepare the compounds by replacing the corresponding raw materials based on the basis / inspiration of Examples 1-2, and the specific routes and preparation methods are not described in detail. Example 5 Preparation of Compound (III-1), Compound (III-2), Compound (III-3), Compound (III-4) and Other Heterodimer Compounds

[0151] Compound (III-1), compound (III-2), compound (III-3), compound (III-4) and other heterodimeric compounds were prepared with reference to the preparation routes and / or methods of Examples 1-3. Those skilled in the art can replace and / or eliminate the corresponding raw materials based on / inspired by Examples 1-3 for preparation, and the specific preparation methods are not described in detail here.

[0152] (1) Preparation route of compound (III-1)

[0153] (2) Preparation route of compound (III-2)

[0154] (3) Preparation route of compound (III-3)

[0155] (4) Preparation route of compound (III-4) Other heterodimeric compounds can also be prepared by referring to the preparation routes and / or methods of Examples 1-3. Those skilled in the art can replace and / or eliminate the corresponding raw materials based on / inspired by Examples 1-3 for preparation, and the specific preparation methods are not described in detail. Example 6 Radiolabeling

[0156] In this example, a general method is used to radiolabel heterodimeric compounds provided by the present invention that can be labeled with radionuclides (such as compound (II-1-NOTA), compound (II-1-DOTA), compound (II-2-NOTA), compound (II-2-DOTA), compound (II-3-NOTA), compound (II-3-DOTA), compound (II-4-NOTA), compound (II-4-DOTA), compound II-9-NOTA, compound II-9-DOTA, compound II-10-NOTA, compound II-10-DOTA, compound II-17-NOTA, compound II-17-DOTA, compound II-18-NOTA, compound II-18-DOTA):68 GaCl3 was eluted with 0.1M HCl, 68 GaCl3 is made from 68 Ge / 68 Ga was generated by a Ga generator (ITG, Berlin, Germany) and mixed with 0.25 M NaOAc buffer, and the pH value was adjusted to 4.0. 50 μg of the heterodimer compound to be labeled with radionuclide was reacted with the above mixed buffer at 95°C for 15 min to obtain radionuclide 68 Ga-labeled heterodimer radionuclide conjugate drug, denoted as: 68 Ga-NOTA-II-1、 68 Ga-DOTA-II-1, 68 Ga-NOTA-II-2, 68 Ga-DOTA-II-2, 68 Ga-NOTA-II-3、 68 Ga-DOTA-II-3, 68 Ga-NOTA-II-4、 68 Ga-DOTA-II-4, 68 Ga-NOTA-II-9, 68 Ga-DOTA-II-9, 68 Ga-NOTA-II-10、 68 Ga-DOTA-II-10, 668 Ga-NOTA-II-17、 68 Ga-DOTA-II-17, 68 Ga-NOTA-II-18、 68 Ga-DOTA-II-18. The radiochemical purity of the radioactive nuclide marker was greater than 95% as verified by TLC (Mini-Scan instant thin layer chromatography scanner, Bioscan, USA) and HPLC (high performance liquid chromatography, Thermo Scientific, USA). 177 Lu labeling The heterodimer compound provided by the present invention can be labeled with radioactive nuclides, and its labeling method is the same as 68 The labeling method of Ga is similar. 68 The GaCl3 solution was replaced by 177 Lu hydrochloric acid aqueous solution, namely obtain 177 Lu heterodimer radionuclide conjugated drug: 177 LU-DOTA-II-1, 177 LU-DOTA-II-2, 177 LU-DOTA-II-3, 177 LU-DOTA-II-4, 1177LU-DOTA-II-9, 177 LU-DOTA-II-10, 177 LU-DOTA-II-17, 177 LU-DOTA-II-18.

[0157] Also refer to the patents CN102123739B and / or CN102066974B 18 The F-Al labeling method is used to label heterodimers that can be labeled with radionuclides. 18 F mark, get 18 Heterodimer radionuclide conjugated drug of F: 18 F-NOTA-II-1, 18 F-NOTA-II-2, 18 F-NOTA-II-3, 18 F-NOTA-II-4, 18 F-NOTA-II-9, 18 F-NOTA-II-10, 18 F-NOTA-II-17, 18 F-NOTA-II-18.

[0158] Other general labeling methods can also be used to label the heterodimer compound provided by the present invention that can be labeled with radioactive nuclides for radiolabeling. Optional radionuclides include C-11, N-13, O-15, Na-24, P-32, P-33, K-42, Sc-43, Sc-44, Sc-47, Cr-51, Mn-51, Fe-52, Mn-52, Mn-52m, Co-55, Co-57, Fe-59, C o-60, Cu-62, Cu-64, Cu-67, Ga-67, As-72, Br-75, Se-75, Br-76, As-77, Rb-82m, Sr-83, Y-86, Y- 88. Zr-89, Sr-89, ln-90, Y-90, Tc-94, Mo-99, Tc-99m, Pd-103, Rh-105, Ru-106, Pd-109, In-110 , In-111, Ag-111, I-120, I-123, I-124, I-125, I-131, Xe-133, Cs-137, Pr-142, Pr-143, Tb-149 , Tb-151, Gd-152, Gd-153, Sm-153, Gd-154, Gd-155, Gd-156, Gd-157, Gd-158, Tb-161, Dy-165, D Any one of y-166, Ho-166, Er-169, Yb-169, Yb-177, Re-186, Re-188, Re-189, Ir-192, Ir-194, Au-198, Au-199, At-211, Pb-211, Bi-212, Pb-212, Bi-213, Ra-223, Ac-225, Fm-255, Th-226, Th-227, etc.

[0159] After radiolabeling, HPLC results showed that all radiolabeled compounds had high radiochemical purity and in vitro stability. 68 Ga-labeled heterodimeric radionuclide-conjugated drug 68 The HPLC spectrum of Ga-NOTA-II-1 is shown in Figure 1.

[0160] Example 7 Preparation of control compound

[0161] Referring to the preparation routes and methods and labeling methods provided in Examples 1-3 and Example 5, DOTA-compound 16, DOTA-compound 17, and DOTA-compound 18 were prepared, and the three compounds were 68 Ga labeling, 68 Ga-DOTA-compound 16, 68 Ga-DOTA-compound 17,68 Ga-DOTA-Compound 18:

[0162] Example 8 Cellular Uptake and Blockade

[0163] a) 68 Cellular uptake and blocking experiments of Ga-NOTA-II-1

[0164] PC3 tumor cells (purchased from American Type Culture Collection, ATCC, Maryland, USA) were cultured in a 37°C, 5% CO2 incubator in RPMI 1640 medium containing 10% fetal bovine serum (FBS). 2×10 cells were seeded into each well of a 24-well plate. 5 cells until they reach 80% confluence, and 37 kBq 68 Ga-NOTA-II-1 was added to 0.5 mL of RPMI 1640 medium and incubated at 37°C for 10, 30, 60, 90, and 120 minutes. The blocking group used excess unlabeled precursor 1 (i.e., compound 4), precursor 2 (i.e., compound 1), and precursor 3 (i.e., compound III-1) as inhibitors of the labeled radiotracer.

[0165] The results are shown in Figure 2. The results show that on PC3 tumor cells, 68 Ga-NOTA-II-1 showed rapid specific uptake within 10 minutes (1.05±0.28%), and the uptake gradually increased over time (1.42±0.50%, 1.67±0.62% and 2.43±0.91% after 30, 60 and 90 minutes, respectively), reaching a peak at 90 minutes. 68 The uptake of Ga-NOTA-II-1 could be blocked by unlabeled precursors 1, 2, and 3, which also verified the specific uptake of the radiolabeled precursor.

[0166] In this example, PC3 tumor cells were used to 68 Ga-NOTA-II-1 was subjected to a saturation binding test to verify its binding specificity and to calculate the equilibrium dissociation constant (Kd) and maximum specific binding (Bmax) by specific binding. The saturation binding test was performed in triplicate by increasing the amount of Ga-NOTA-II-1 in the absence or presence of precursor 3 (i.e., compound III-1) (10 μM). 68 The concentration of Ga-NOTA-II-1 was used to observe its saturation binding with PC3 cells. The experimental results are shown in Figure 3. 68The receptor binding affinity of Ga-NOTA-II-1 was Kd = 40.45 ± 10.54 nM, Bmax = 11.12 ± 1.03 (×10 4 CPM). This indicates that: 68 Ga-NOTA-II-1 has high binding affinity and specificity to PC3 tumor cells.

[0167] b) 68 Cellular uptake and blocking experiments of Ga-DOTA-II-4

[0168] PC3 tumor cell line was purchased from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). PC3 cells were plated at 2×10 5 The number of cells / well was inoculated into a 24-well plate overnight. Then 37 kBq of 68 Ga-DOTA-II-4 was added to adherent PC3 tumor cells and incubated at 37°C for 10, 30, 60, 90, and 120 minutes. The culture medium was then removed, the cells were rinsed with cold PBS, and lysed with 1M NaOH. In the blocking experiment, excess unlabeled precursor 1 (i.e., compound 4), precursor 4 (i.e., compound 6), and precursor 5 (i.e., compound III-4) were used as inhibitors. 68 Ga-DOTA-II-4 was added to the cells at the same time, the cell lysate was collected, and the radioactivity was measured using a γ counter.

[0169] From 10 minutes to 120 minutes, 68 The uptake of Ga-DOTA-II-4 into PC3 cells gradually increased. As shown in Figure 4, 68 The cellular uptake value of Ga-DOTA-II-4 was the highest at 120 minutes. The addition of unlabeled precursors 1, 4, and 5 could block the cellular uptake to some extent, which demonstrated its targeting specificity.

[0170] 68 The saturation binding curve of Ga-DOTA-II-4 in PC3 tumor cells is shown in Figure 5. After calculation, 68 The receptor binding affinity of Ga-DOTA-II-4 is Kd=50.89±18.36nM. Bmax=2.17±0.30(×10 4 CPM). This indicates that: 68 Ga-DOTA-II-4 has high binding affinity and specificity to PC3 tumor cells and 68 Ga-NOTA-II-1 has comparable affinity.

[0171] The above method is used to treat the heterodimer compound that can be labeled with radionuclides provided by the present invention. 68 Ga radiolabeled (e.g. 68 Ga-DOTA-II-1, 68 Ga-NOTA-II-2, 68 Ga-DOTA-II-2, 68 Ga-NOTA-II-3、 68 Ga-DOTA-II-3, 68 Ga-NOTA-II-4、 68 Ga-NOTA-II-9, 68 Ga-DOTA-II-9, 68 Ga-NOTA-II-10、 68 Ga-DOTA-II-10, 68 Ga-NOTA-II-17、 68 Ga-DOTA-II-17, 68 Ga-NOTA-II-18、 68 The results of the cell uptake and blocking experiments showed that the heterodimer radionuclide conjugated drugs provided by the present invention have good targeting specificity and high binding affinity.

[0172] a) 68 Animal tumor uptake experiment and biodistribution study of Ga-NOTA-II-1

[0173] 4-6 week old male BALB / c nude mice were injected with 5×10 6 PC3 tumor cells (100 μL PBS) were added to establish the PC3 tumor model. When the tumor volume reached approximately 200-300 mm 3 PET imaging of mice was performed at 4 ℃ and 8 ℃. 68 Ga-NOTA-II-1 was imaged at 0.5, 1, and 2 h post-injection (pi). PET images were reconstructed after attenuation correction and analyzed by drawing regions of interest (ROIs).

[0174] Figure 6 shows the injection 68 Ga-NOTA-II-1、 68 Ga-labeled precursor 1 (chelating group is NOTA), 68 Representative whole-body PET images of Ga-labeled precursor 2 (chelating group is NOTA). Results show: 68 Ga-NOTA-II-1 was rapidly absorbed within 0.5 hours (2.88±0.38% ID / g).68 The uptake of Ga-NOTA-II-1 was lower than that in tumors, except for the kidney (2.24±0.29% ID / g at 2 h pi) (see Figure 6B). 68 PET imaging of Ga-labeled precursor 1 is shown in Figure 6C , which shows low tumor uptake (1.09 ± 0.21% ID / g), but significantly higher uptake in the liver (9.17 ± 0.72% ID / g) and kidney (6.73 ± 0.26% ID / g) after 2 hours (see Figure 6D ). 68 The tumor uptake signal of Ga-labeled precursor 2 was low (see Figure 6E). PET imaging clearly showed 68 More importantly, compared with the monomer (i.e. 68 Ga-labeled precursor 1, 68 Compared with Ga-labeled precursor 2), 68 Ga-NOTA-II-1 showed a substantial increase in the ratio of tumor to normal tissue.

[0175] In the biodistribution study, 1.48 MBq was injected into BALB / c nude mice bearing PC3 tumors. 68 Ga-NOTA-II-1 was added and sacrificed at different time points (30, 60, 90, and 120 minutes; n = 4). The heart, muscle, tumor, blood, liver, and kidney were dissected and wet weighed. The radioactivity in these organs was then measured using a gamma counter and the results were calculated as a percentage of the injected dose per gram (%ID / g).

[0176] 68 The biodistribution results of Ga-NOTA-II-1 in PC3 tumor mice are shown in Figure 7. After 120 minutes, the uptake in the heart, blood, and muscle rapidly decreased to almost baseline levels, while the tumor uptake remained at more than half of the initial uptake (from 2.91±0.44%ID / g to 1.75±0.17%ID / g). In addition, high absorption rate in the kidney was also observed in the biodistribution study. These results confirm that 68 Ga-NOTA-II-1 exhibited good tumor retention in PC3 tumors and demonstrated that the radiotracer could be cleared by the kidneys.

[0177] b) 68 Animal tumor uptake experiment and biodistribution study of Ga-DOTA-II-4

[0178] 5×10 6 PC3 tumor cells were used to establish the tumor model. When the tumor volume reached about 200-300 mm 3MicroPET imaging and biodistribution studies were performed on tumor-bearing mice.

[0179] PC3 tumor mice were intravenously injected with approximately 7.4 MBq of 68 Static PET imaging was performed using Ga-DOTA-II-4. At 0.5, 1, and 2 h post-injection (pi), mice with tumors were anesthetized and placed in the imaging chamber for image acquisition. For comparison, mice with PC3 tumors were injected with 68 Ga-labeled precursor 1 and 68 Ga-labeled precursor 4 was used, and PET imaging was performed at 0.5 and 2 h after injection.

[0180] Figure 8 shows the injection 68 Ga-DOTA-II-4, 68 Ga-labeled precursor 1 (chelating group is DOTA) and 68 Representative whole-body PET scan images of Ga-labeled precursor 4 (chelating group is DOTA). 68 The uptake of Ga-DOTA-II-4 was significant (see Figure 8A), with tumor uptake values ​​of 1.62 ± 0.08, 1.48 ± 0.08, and 1.38 ± 0.11% ID / g after 0.5, 1, and 2 hours, respectively. 68 The uptake of Ga-DOTA-II-4 was lower than that of tumor (see Figure 8B). 68 PET imaging of Ga-labeled precursor 1 is shown in Figure 8C. Tumor uptake is low, but liver and kidney uptake is high. After 2 hours, the tumor signal is almost invisible. PET imaging clearly shows that 68 Increased tumor uptake of Ga-DOTA-II-4.

[0181] To further evaluate 68 The pharmacokinetic properties of Ga-DOTA-II-4 were studied, and the biodistribution of PC3 tumor mice was studied. In the biodistribution study, PC3 tumor mice were randomly divided into 4 groups (n=4 / group), and each group was injected with approximately 740 kBq 68 Ga-DOTA-II-4 was removed and collected from relevant tissues and organs at 0.5, 1, and 2 hours after injection, weighed, and the activity was measured using a gamma counter. The biodistribution results were calculated as the percentage of injected dose per gram of tissue (%ID / g). The results are shown in Figure 9: From 10 minutes (1.62±0.23%ID / g) to 120 minutes (0.54±0.09%ID / g), the tumor 68The uptake of Ga-DOTA-II-4 gradually decreased, with muscle uptake decreasing more rapidly (from 1.23±0.47%ID / g at 10 minutes to 0.22±0.07%ID / g at 120 minutes). This suggests that: 68 Ga-DOTA-II-4 has a high tumor / muscle ratio and low background signal, and has a rapid in vivo clearance rate.

[0182] c) 68 Ga-DOTA-compound 16, 68 Ga-DOTA-compound 17 and 68 Animal tumor uptake experiment of Ga-DOTA-compound 18

[0183] Follow the above method 68 Ga-DOTA-compound 16, 68 Ga-DOTA-compound 17 and 68 Animal tumor uptake of Ga-DOTA-compound 18.

[0184] 4-6 week old male BALB / c nude mice were injected with 5×10 6 PC3 tumor cells (100 μL PBS) were added to establish the PC3 tumor model. When the tumor volume reached approximately 200-300 mm 3 PET imaging of mice was performed at 4 ℃ and 8 ℃. 68 Ga-DOTA-compound 16, 68 Ga-DOTA-compound 17 and 68 Ga-DOTA-Compound 18, imaged at 0.5, 1, and 2 hours post-injection (pi).

[0185] Figures 10, 11, and 12 show the injection 68 Ga-DOTA-compound 16, 68 Ga-DOTA-compound 17 and 68 Representative whole-body PET images after Ga-DOTA-compound 18. Results show: 68 Ga-DOTA-compound 16, 68 Ga-DOTA-compound 17 and 68 PET imaging of Ga-DOTA-compound 18 showed high background signals and low tumor uptake values, and the probe was quickly cleared from the tumor site, as shown in Figure 2. 68There was no obvious radioactive uptake of Ga-DOTA-compound 18 at the tumor site 1 hour and 2 hours after injection, while the radioactive signals in the kidney, intestine and bladder were still very high, indicating that the probes 68Ga-DOTA-compound 16, 68Ga-DOTA-compound 17 and 68Ga-DOTA-compound 18 had low tumor / kidney and tumor / normal tissue ratios and were not suitable for clinical application.

[0186] The above method is used to treat the heterodimer compound that can be labeled with radionuclides provided by the present invention. 68 Ga radiolabeled (e.g. 68 Ga-DOTA-II-1, 68 Ga-NOTA-II-2, 68 Ga-DOTA-II-2, 68 Ga-NOTA-II-3、 68 Ga-DOTA-II-3, 68 Ga-NOTA-II-4、 68 Ga-NOTA-II-9, 68 Ga-DOTA-II-9, 68 Ga-NOTA-II-10、 68 Ga-DOTA-II-10, 68 Ga-NOTA-II-17、 68 Ga-DOTA-II-17, 68 Ga-NOTA-II-18、 68 The results of animal tumor uptake experiments and biodistribution studies using Ga-DOTA-II-18) showed that the heterodimer radionuclide conjugated drugs provided by the present invention have good tumor retention in PC3 tumors and can be cleared by the kidneys, showing good clinical application prospects. 68 Real-world clinical study of Ga-NOTA-II-1

[0187] Inclusion criteria

[0188] (1) Aged 18 and above;

[0189] (2) ultrasound or mammography showed breast lesions BI-RADS ≥ 4 in the past 3 months;

[0190] (3) biopsy or surgical histopathological examination will be performed;

[0191] (4) Be able to understand and sign the informed consent form;

[0192] (5) Follow-up can be performed within the prescribed follow-up period, and the predicted survival period is more than 6 months.

[0193]

Exclusion criteria

[0194] (1) Patients with severe hepatic and renal insufficiency;

[0195] (2) Patients with claustrophobia;

[0196] (3) Pregnant or breastfeeding women.

[0197] Research subjects

[0198] A total of 11 patients with suspected breast tumors (mean age (±SD), 49±7 years; age range, 43-63 years) were included in this study. The patient information is shown in Table 1. None of these patients received any treatment before PET examination. Table 1 Patient information

[0199] Research plan and methods

[0200] (1) Experimental group 1: 6 patients underwent whole-body PET scan

[0201] Six patients who underwent whole-body PET scans were 68 Ga-NOTA-II-1 radiation dose measurement. Before PET scanning, the patient was intravenously injected with 68 Ga-NOTA-II-1 (119 ± 20 MBq) was administered, followed by low-dose CT scans (120 keV, 200 mA), and continuous whole-body dynamic PET scans were acquired in 10 beds in three-dimensional mode. Acquisition durations were: 30 seconds per bed 5 minutes after injection; 1 minute per bed 10, 25, 35, and 50 minutes after injection; and 2 minutes per bed 70 and 100 minutes after injection, using a 192 × 192 matrix with a 5.0 × 5.0 full width at half maximum (FWHM). PET data were reconstructed using ordered subset expectation maximization (OSEM) and corrected for randomness, dead time, scatter, and attenuation using the CT data.

[0202] Radiation dose estimation used Hybrid-Dosimetry software (Hermes Medical Solutions, Sweden) to determine organ delineation for each organ and the accumulated activity at each imaging time point. Time-activity curve fitting and subsequent dose calculation were performed using OLINDA / EXM, version 2.2.0. Red bone marrow dose measurements were performed using image-based three-dimensional volumetric analysis of the L2–L4 vertebrae, assuming that the L2–L4 vertebrae comprise 6.7% of the total bone marrow volume. A ROI for the organ of interest was delineated from the examination with the most accurate organ delineation and then replicated for all other time points to calculate the time-activity curve. Using an adult female as a model, the administration time and the corrected dose were input, and a biexponential curve-fitting parameter was used to obtain the best curve-fit value for the residence time of the activity in the source organ. The absorbed dose for all organs, including the whole-body effective dose, was generated in units of mSv / MBq and rem / mCi. Time-activity plots for each organ were generated using GraphPad Prism software (version 9.0).

[0203] All data are expressed as mean ± SD. Comparisons were made using the Wilcoxon signed-rank test. 68 SUV and tumor-to-background ratio of primary lesions detected by Ga-NOTA-II-1 and 18F-FDG. P values ​​< 0.05 were considered statistically significant.

[0204] (2) Experimental group 2: 5 patients underwent scanning from the top of the skull to the root of the femur

[0205] The imaging process and data analysis of the five patients were the same as clinical routine, and the scanning range was from the top of the skull to the root of the femur.

[0206] (3) Control group: 18 F-FDG PET scan

[0207] All 11 patients underwent a second 18 F-FDG PET scan for comparison. PET / CT scan after intravenous injection of 270-419 MBq 18 1 hour after F-FDG.

[0208] Adverse event monitoring

[0209] Monitor and record vital signs (blood pressure, temperature, and heart rate) and clinical symptoms within 4 hours after injection. Monitor the patient's basic vital signs within one week after the examination and record any drug-related adverse reactions.

[0210] Research Results

[0211] (1) All patients tolerated the examination well. Vital parameters remained stable, and no obvious adverse reactions related to the tracer injection were observed.

[0212] (2) 68 The biological distribution of Ga-NOTA-II-1 in normal organs and tumors is shown in FIG13 , and the maximum intensity projection (MIP) over time is shown in FIG14 . 68 The physiological and biological distribution of Ga-NOTA-II-1 involves the pancreas, kidney, liver, cardiac contents, spleen, uterus, and bladder. The results showed that the kidney was the main excretion organ, which was due to the strong radioactive signal in the bladder. The pancreas also had a high accumulation of radioactive tracer, with an average SUV of 11.72±4.81 at 35 minutes after injection. The uterus, liver, and spleen had lower absorption rates, with an SUV of 11.72±4.81. mean The values ​​were (2.46±0.52), (1.58±0.42) and (1.55±0.52) respectively. The background activity of brain, lung and muscle was very low, and SUV mean The values ​​were 0.12±0.12, 0.36±0.08 and 0.42±0.10, respectively.

[0213] (3) Table 2 lists the organ status of the six patients. 68 The estimated absorbed dose of Ga-NOTA-II-1 is highest in the bladder wall (5.06E-01±1.34E-01mSv / MBq) due to the high accumulation of radioactivity in the bladder, followed by the pancreas (8.31E-02±2.99E-02mSv / MBq), uterus (8.15E-02±4.80E-02mSv / MBq) (for females), and kidney (3.28E-02±5.76E-03mSv / MBq). 68 The whole-body absorbed dose and effective dose of Ga-NOTA-II-1 were 9.93E-03±1.27E-03 and 2.91E-02±5.71E-03 mSv / MBq, respectively. Table 2 Organs 68 Estimated absorbed dose of Ga-NOTA-II-1 (mSv / MBq)

[0214] (4) 11 patients (100%) passed 68 Ga-NOTA-II-1 and 18 F-FDG PET / CT detected 12 positive primary lesions. Eight patients underwent surgery, four of whom had lymph node metastases. Three patients received neoadjuvant therapy (NACT), two of whom underwent surgery after NACT. Patient 1 underwent surgery for invasive ductal carcinoma of the left breast in 2016 but did not receive standard postoperative treatment and recently experienced a recurrence.68 Ga-NOTA-II-1 PET / CT showed that the patient had bilateral breast cancer metastasis, multiple lymph node metastasis, brain metastasis and bone metastasis. Compared with 18F-FDG, 68 The SUVmeanT / B ratio of Ga-NOTA-II-1 to right temporal lobe metastases was higher (50.5 vs. 1.2). 18 Compared with F-FDG, 68 Ga-NOTA-II-1 PET / CT detected more metastatic lymph nodes and clearly showed brain and bone metastases in patients with advanced recurrent breast cancer (Figure 15).

[0215] (5) Patient No. 7 passed 68 Ga-NOTA-II-1PET / CT was used to evaluate the metastasis of the axillary lymph nodes, which was 0.5 cm long. 68 Ga-NOTA-II-1 PET / CT showed positive uptake, SUV max is 3.3, but 18 F-FDG PET / CT showed negative uptake ( Figure 16 ).

[0216] (6) Patient No. 10 passed 68 Ga-NOTA-II-1PET / CT showed right invasive ductal carcinoma. 68 SUV in Ga-NOTA-II-1 max As high as 7.5, while 18 SUV in F-FDG max The test was done during the secretory phase of the menstrual cycle, and normal breast tissue 68 Ga-NOTA-II-1 has moderate physiological uptake (Figure 17). 68 Ga-NOTA-II-1 and 18 Average SUV of F-FDG max There was no significant difference (8.9±3.2 vs. 10.3±6.9; p=0.459).

[0217]

in conclusion

[0218] (1) In previous clinical studies, RM26-based radiotherapy has high pancreatic and bladder toxicity; while RGD has good performance in primary tumor detection, it has not shown sufficient sensitivity in lymph node staging [Reference 8: Beer AJ, Niemeyer M, Carlsen J, Sarbia M, J, Watzlowik P, et al. Patterns of alphavbeta3 expression in primary and metastatic human breast cancer as shown by 18F-Galacto-RGD PET. J Nucl Med. 2008; 49: 255-9. 】. The present invention provides 68 Ga-NOTA-II-1 to integrin α V The specificity of the two receptors, β3 and GRPR, has been confirmed by blocking experiments with RGD or RM26. 68 Ga-NOTA-II-1 has more chances to localize in tumors because the number of two receptors is the highest than that of a single receptor. 68 Ga-NOTA-II-1 exhibited stronger PC3 tumor uptake and longer tumor retention compared to the monomeric receptor.

[0219] (2) The present invention provides 68 In clinical cancer diagnosis, Ga-NOTA-II-1 was safely tolerated by all patients tested, and no adverse reactions occurred after injection. 68 The average injected activity of Ga-NOTA-II-1 was 119±20MBq, and the effective whole-body dose was 2.91E-02±5.71E-03mSv / MBq, which is far below the dose limit required by the FDA [Reference 9: Mittra ES, Goris ML, Iagaru AH, Kardan A, Burton L, Berganos R, et al. Pilot pharmacokinetic and dosimetric studies of 18F-FPPRGD2:a PET radiopharmaceutical agent for imagingαvβ3integrin levels. Radiology. 2011; 260: 182-91.], which shows that the present invention provides 68 Ga-NOTA-II-1 has good safety in clinical applications.

[0220] (3) Metastasis is one of the hallmarks of cancer and is considered to be the key cause of cancer treatment failure and death [Reference 10: Fares J, Fares MY, Khachfe HH, Salhab HA, Fares Y. Molecular principles of metastasis: a hallmark of cancer revisited. Signal Transduct Target Ther. 2020; 5: 28.], so a comprehensive and accurate diagnosis of metastasis beyond the primary site is of great significance. In clinical studies, 18 Compared with F-FDG, 68 PET imaging of Ga-NOTA-II-1 can detect more metastatic lymph nodes, brain metastases and bone metastases. There is a close connection between angiogenesis and tumor metastasis. 68 Ga-NOTA-II-1 can provide more information on tumor progression and aggressiveness in the diagnosis of primary malignant tumors and metastatic tumors, and provide molecular details for the design of treatment plans. 68 Ga-NOTA-II-1 can make up for the shortcomings of PSMA-based radiotracers in detecting PSMA-negative or low-expressing prostate cancer, which is well documented in the early stages of cancer [Reference 11: Adnan A, Basu S. PSMA Receptor-Based PET-CT: The Basics and Current Status in Clinical and Research Applications. Diagnostics (Basel). 2023; 13: 158.]. This shows that the present invention provides 68 Ga-NOTA-II-1 could play an important role in early diagnosis and provide guidance for potential targeted radiotherapy using tracers labeled with alpha and beta particles.

[0221] In summary, the present invention provides 68 Ga-NOTA-II-1 not only has a higher tumor uptake rate and tumor-to-nontarget ratio, but also has great potential in distinguishing primary tumor sites from metastatic lesions. This will provide more precise information for tumor diagnosis and staging, and may also monitor metastasis during treatment.

[0222] Example 11 68 Ga-DOTA-II-4 Real World Clinical Study

[0223] Research subjects

[0224] A total of 9 patients (7 males, 2 females; mean age (±SD), 61.7±15.5 years; age range, 34-86 years; mean weight (±SD), 70.1±8.8 kg; weight range, 60-85 kg, see Table 3 for specific patient information) were recruited in this study, including 4 patients with suspected prostate tumors and 5 patients with suspected brain tumors. Among the 4 patients with suspected prostate cancer, 1 underwent surgical treatment, and 3 chose non-surgical treatment or clinical observation. Among the 5 patients with suspected brain tumors, 4 patients underwent surgical resection after PET examination, and 1 patient refused surgery. Table 3 Patient information

[0225]

Exclusion criteria

[0226] (1) Patients with severe hepatic and renal insufficiency;

[0227] (2) Patients with claustrophobia;

[0228] (3) Pregnant or breastfeeding women.

[0229] Research plan and methods

[0230] Experimental Group 1: 6 patients underwent whole-body PET scan

[0231] Six patients (patient numbers 1-6) who underwent whole-body PET scans were analyzed. 68 Ga-DOTA-II-4 radiation dose measurement. Low-dose CT scan (120keV; 100mAs; 1.3mm spacing; 2.5mm slice thickness). Before PET scan, the patient was intravenously injected 68 Ga-DOTA-II-4 (155 ± 6.5 MBq). Continuous whole-body dynamic PET scans were acquired in 10 beds in three-dimensional mode. Acquisition times were: 4 minutes after injection, 30 seconds per bed; 10, 25, 35, and 50 minutes after injection, 1 minute per bed; and 70 and 100 minutes after injection, 2 minutes per bed.

[0232] Radiation dosimetry was estimated using Hybrid-Dosimetry software (Hermes Medical Solutions, Sweden). Organ parcellation and accumulated activity for each imaging time point were determined for each organ. Time-activity curve fitting and subsequent dose calculations were performed using OLINDA / EXM, version 2.2.0. Red bone marrow dosimetry was measured using image-based three-dimensional volumetric analysis of the L2–L4 vertebrae, which were considered to comprise 6.7% of the total bone marrow volume. A ROI for the organ of interest was delineated based on the examination results and replicated for all other time points to calculate the time-activity curve. An adult male or female phantom was entered, along with the administration time and corrected dose. A biexponential curve-fitting parameter was used to determine the best curve fit for the residence time of activity in the source organ. The absorbed dose for all organs, including the whole-body effective dose, was generated in units of mSv / MBq and rem / mCi. Time-activity plots for each organ were generated using GraphPad Prism software (version 9.0).

[0233] All data are expressed as mean ± SD. Calculations were performed using SPSS (IBM Corp., Armonk, NY, USA, version 22). P values ​​less than 0.05 were considered statistically significant.

[0234] Experimental Group 2: 3 patients underwent head PET scan

[0235] Three patients with suspected brain tumors (patient numbers 7-9) received intravenous injections of 117-128 MBq of 68 A low-dose CT scan (120 kV, 35 mA, 3 mm slice, 512 × 512 matrix, 70 cm field of view) was performed, followed by an intravenous injection of 117–128 MBq of Ga-DOTA-II-4. 68 Ga-DOTA-II-4 After 45 ± 13.5 minutes, a 10-minute PET acquisition covering the patient's entire head was performed.

[0236] PET data were reconstructed using ordered subset expectation maximization (OSEM) and corrected for randomness, dead time, scatter, and attenuation using CT data.

[0237] Control group: 68 Ga-labeled precursor 1

[0238] Four patients (patient numbers 6-9) used it within 1 week. 68 A second scan of Ga-labeled precursor 1 was performed for comparison. Each patient received 110-156.5 MBq intravenously. 68Ga-labeled precursor 1. Imaging procedures and data analysis 68 Ga-DOTA-II-4 PET / CT was the same as for MRI. If the patient had an MRI scan, MRI was performed according to standard clinical procedures.

[0239] Adverse event monitoring

[0240] Monitor and record vital signs (blood pressure, body temperature, and heart rate) and clinical symptoms within 2 hours after injection. Observe the patient's basic life indicators within one week after the examination and record any drug-related side effects.

[0241] Research Results

[0242] All patients tolerated the examination well. Vital parameters remained stable, and no significant adverse reactions related to the tracer injection were observed.

[0243] 68 The biodistribution of Ga-DOTA-II-4 in normal organs and tumors is shown in Figure 18, which shows the maximum intensity projection (MIP) over time. As can be seen from the MIP, the kidney is the main excretion organ, as the bladder shows strong radioactivity. The pancreas also has high accumulation, with an average SUV of 6.93 ± 1.32 at 35 minutes after injection. The liver and spleen show moderate uptake, with an average SUV mean The background activity of brain, lung and muscle was low, with an average SUV of 1.33±0.47 and 1.43±0.55 respectively. mean They are 0.13±0.08, 0.44±0.17 and 0.44±0.17 respectively.

[0244] Table 4 shows the organ status of patients No. 1 to No. 6. 68 Estimated absorbed dose of Ga-DOTA-II-4. Due to the high accumulation of radioactivity in the bladder, the organ with the highest absorbed dose is the bladder wall (4.30E-01±8.08E-02mSv / MBq), followed by the pancreas (5.54E-02±1.95E-02mSv / MBq), prostate (5.15E-02±3.43E-02mSv / MBq) (males), and kidney (3.16E-02±9.98E-03mSv / MBq). The whole-body absorbed dose and effective dose of 68Ga-RM26-RGD are 8.71E-03±1.56E-03 and 2.43E-02±4.64E-03mSv / MBq, respectively. Table 4 Organs 68 Estimated absorbed dose of Ga-DOTA-II-4 (mSv / MBq)

[0245] 68 Ga-DOTA-II-4 and 68 Comparison of tumor uptake of Ga-labeled precursor 1 in one prostate cancer patient (patient number 5) and five brain tumor patients (patient numbers 1, 6-9) 68 Ga-DOTA-II-4PET / CT detected 7 positive lesions, of which 6 lesions (3 lesions were glioblastoma (grade IV), 1 lesion was glioma (grade I), 1 lesion was meningioma (grade I), and 1 lesion was prostate cancer) were confirmed by surgery. 68 Ga-DOTA-II-4 and 68 Ga-labeled precursor 1 PET / CT examination. 68 Average SUV of Ga-DOTA-II-4 max Higher than 68 Ga-labeled precursor 1, but in 5 brain lesions, 68 Ga-DOTA-II-4 and 68 Average SUV of Ga-labeled precursor 1 max and average SUV mean There was no significant difference (1.96±0.98 vs. 1.41±0.48, p=0.138; 1.25±0.70 vs. 0.84±0.26, p=0.138). Figure 19 is a representative image of patient No. 8, 68 The uptake of the two lesions shown by Ga-DOTA-II-4 was significantly higher than 68 Ga-labeled precursor 1 (SUVmax: 3.33 vs. 1.82, 2.58 vs. 1.46; T / B are 30.3 vs. 22.8 and 23.5 vs. 18.25, respectively). A and E represent 68 PET / CT fusion image, CT and PET images of Ga-labeled precursor 1, C and G represent 68 PET / CT fusion image, CT and PET images of Ga-DOTA-II-4. B is HE staining of the lesion, D is GRPR receptor staining of the lesion, and F is integrin α V β3 receptor staining.

[0246]

in conclusion

[0247] In summary, the present invention provides 68 Ga-DOTA-II-4 also has a higher tumor uptake rate and tumor to non-target ratio, and has great potential in distinguishing primary tumor sites from metastatic lesions. This will provide more accurate information for tumor diagnosis and staging, and may also monitor metastasis during treatment. Example 12 Real-world clinical studies of other heterodimeric radionuclide-conjugated drugs

[0248] This study aims to evaluate the efficacy of other heterodimeric radionuclide conjugated drugs provided by the present invention (such as 68 Ga-DOTA-II-1, 68 Ga-NOTA-II-2, 68 Ga-DOTA-II-2, 68 Ga-NOTA-II-3、 68 Ga-DOTA-II-3, 68 Ga-NOTA-II-4、 68 Ga-NOTA-II-9, 68 Ga-DOTA-II-9, 68 Ga-NOTA-II-10、 68 Ga-NOTA-II-17、 68 Ga-DOTA-II-17, 68 Ga-NOTA-II-18、 68 Ga-DOTA-II-18) in real-world patient tolerance and PET / CT imaging, tumor uptake, etc.

[0249] Results showed that all patients tolerated the examination well. Vital parameters remained stable, and no significant adverse reactions related to the tracer injection were observed. Furthermore, the heterodimeric radionuclide-conjugated drug provided by the present invention not only has a higher tumor uptake rate and tumor-to-nontarget ratio, but also has great potential in distinguishing primary tumor sites from metastatic lesions. This will provide more precise information for tumor diagnosis and staging, and may also enable monitoring of metastasis during treatment.

[0250] In summary, the heterodimer radionuclide conjugated drug provided by the present invention is effective for integrin α VThe two receptors, β3 and GRPR, have high specificity and show stronger tumor uptake ability and longer tumor retention time compared with monomeric receptors. The heterodimeric radionuclide-conjugated drugs provided by the present invention are safely tolerated by all test patients in clinical cancer diagnosis, without any adverse reactions after injection, and their effective whole-body dose is far lower than the dose limit required by the FDA, and have good safety in clinical applications. PET imaging of the heterodimeric radionuclide-conjugated drugs provided by the present invention can detect more metastatic lymph nodes, brain metastases and bone metastases, and can provide more information for the progression and aggressiveness of tumors in the diagnosis of primary malignant tumors and metastases, and provide molecular details for the design of treatment plans. The heterodimeric radionuclide-conjugated drugs provided by the present invention can make up for the shortcomings of PSMA-based radioactive tracers in detecting PSMA-negative or low-expressing prostate cancer, can play an important role in early diagnosis, and provide guidance for potential targeted radiotherapy using tracers labeled with α and β particles. In summary, the heterodimeric radionuclide-conjugated drug provided by the present invention not only has a higher tumor uptake rate and tumor-to-nontarget ratio, but also has great potential in distinguishing primary tumor sites from metastatic lesions. This will provide more precise information for tumor diagnosis and staging, and may also enable monitoring of metastasis during treatment.

[0251] The above description is merely a preferred embodiment, which is intended to be illustrative and non-limiting of the combinations of features necessary to implement the present invention. The titles provided are not intended to limit the various embodiments of the present invention. Terms such as "comprising," "including," and "including" are not intended to be limiting. In addition, unless otherwise indicated, the absence of a numeral modifier includes the plural form, and "or" and "or" mean "and / or." Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0252] All disclosures and patents mentioned in this application are incorporated herein by reference. Without departing from the scope and spirit of the present invention, multiple modifications and variants of the described method and composition of the present invention will be apparent to those skilled in the art. Although the present invention has been described by specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, those multiple variants of the described pattern that are apparent to those skilled in the relevant art are intended to be included in the scope of the appended claims.

Claims

1. A heterodimer radionuclide conjugated drug, characterized in that: The heterodimer radionuclide conjugated drug is a compound as shown in formula (I) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a pharmaceutically acceptable salt thereof: in: The A and B are targeting groups; The L1 and L2 are connecting groups; The R is a metal chelator containing at least one radionuclide; and The targeting group A targets integrin α v β3; The targeting group B is a GRPR antagonist. Preferably, the structure of the GRPR antagonist is selected from:

2. The heterodimer radionuclide conjugated drug according to claim 1, characterized in that: The structure of the targeting group A is:

3. The heterodimer radionuclide conjugated drug according to claim 1, characterized in that: The structure of the linking group L1 is:

4. The heterodimer radionuclide conjugated drug according to claim 1, characterized in that: The structure of the linking group L2 is:

5. The heterodimer radionuclide conjugated drug according to any one of claims 1 to 4, characterized in that: The metal chelating agent is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), DOTAGA-TETRA (t-Bu edter), and NODAGA.

6. The heterodimer radionuclide conjugated drug according to any one of claims 1 to 5, characterized in that: The radioactive nuclide is selected from C-11, N-13, O-15, F-18, Na-24, P-32, P-33, K-42, Sc-43, Sc-44, Sc-47, Cr-51, Mn-51, Fe-52, Mn-52, Mn-52m, Co-55, Co-57, Fe-59, Co-60, Cu-62, Cu-64, Cu-67, Ga-67, Ga- 68. As-72, Br-75, Se-75, Br-76, As-77, Rb-82m, Sr-83, Y-86, Y-88, Zr-89, Sr-89, ln-90, Y- 90. Tc-94, Mo-99, Tc-99m, Pd-103, Rh-105, Ru-106, Pd-109, In-110, In-111, Ag-111, I-120 , I-123, I-124, I-125, I-131, Xe-133, Cs-137, Pr-142, Pr-143, Tb-149, Tb-151, Gd-152, G d-153, Sm-153, Gd-154, Gd-155, Gd-156, Gd-157, Gd-158, Tb-161, Dy-165, Dy-166, Ho-166, Any one of Er-169, Yb-169, Lu-177, Yb-177, Re-186, Re-188, Re-189, Ir-192, Ir-194, Au-198, Au-199, At-211, Pb-211, Bi-212, Pb-212, Bi-213, Ra-223, Ac-225, Fm-255, Th-226, Th-227.

7. A heterodimeric compound that can be labeled with a radionuclide, characterized in that: The heterodimer compound that can be labeled with a radionuclide is a compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a pharmaceutically acceptable salt thereof: in: The A and B are targeting groups; The L1 and L2 are connecting groups; The R' is a metal chelator; and The targeting group A targets integrin α v β3; The targeting group B is a GRPR antagonist. Preferably, the structure of the GRPR antagonist is selected from:

8. The heterodimeric compound that can be labeled with a radionuclide according to claim 7, characterized in that: The structure of the targeting group A is:

9. The heterodimeric compound that can be labeled with a radionuclide according to claim 7, characterized in that: The structure of the linking group L1 is:

10. The heterodimeric compound that can be labeled with a radionuclide according to claim 7, characterized in that: The structure of the linking group L2 is:

11. The heterodimer radionuclide conjugated drug according to any one of claims 7 to 10, characterized in that: The metal chelating agent is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), DOTAGA-TETRA (t-Bu edter), and NODAGA.

12. A heterodimer compound, characterized in that The heterodimer compound is a compound represented by formula (III) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a pharmaceutically acceptable salt thereof: A-L'1-L2-B (III) in: The A and B are targeting groups; The L'1 and L2 are connecting groups; and The targeting group A targets integrin α v β3; The structure of the targeting group B is a GRPR antagonist. Preferably, the structure of the GRPR antagonist is selected from:

13. The heterodimeric compound according to claim 12, characterized in that The structure of the targeting group A is:

14. The heterodimeric compound according to claim 12, characterized in that The structure of the linking group L'1 is:

15. The heterodimeric compound according to claim 12, characterized in that The structure of the linking group L2 is:

16. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the heterodimer radionuclide-coupled drug according to any one of claims 1 to 6, the heterodimer compound that can be labeled with a radionuclide according to any one of claims 7 to 11, or the heterodimer compound according to any one of claims 12 to 15.

17. The pharmaceutical composition according to claim 16, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, diluent and / or additive.

18. The heterodimer radionuclide conjugated drug according to any one of claims 1 to 6, the heterodimer compound labeled with a radionuclide according to any one of claims 7 to 11, the heterodimer compound according to any one of claims 12 to 15, or the pharmaceutical composition according to any one of claims 16 to 17 in the preparation of a drug for binding to / inhibiting GRPR and / or binding to / inhibiting α v Application of β3 drugs.

19. Use of the heterodimer radionuclide-conjugated drug according to any one of claims 1 to 6, the heterodimer compound labeled with a radionuclide according to any one of claims 7 to 11, the heterodimer compound according to any one of claims 12 to 15, or the pharmaceutical composition according to any one of claims 16 to 17 in the preparation of a molecular imaging agent.

20. The use according to claim 19, characterized in that The imaging agent is used to v Tumors associated with β3 positivity and / or GRPR overexpression were imaged.

21. Use of the heterodimer radionuclide-conjugated drug according to any one of claims 1 to 6, the heterodimer compound labeled with a radionuclide according to any one of claims 7 to 11, the heterodimer compound according to any one of claims 12 to 15, or the pharmaceutical composition according to any one of claims 16 to 17 in the preparation of a drug for treating or preventing tumors, wherein the tumor α v β3 positivity and / or GRPR overexpression.

22. The use according to claim 20 or claim 21, characterized in that The tumors include, but are not limited to, prostate cancer, breast cancer, colon cancer, lung cancer, head and neck cancer, kidney cancer, pancreatic cancer, brain tumors, liver cancer, and ovarian cancer.

23. A kit, characterized in that The kit contains a predetermined amount of the heterodimer compound that can be labeled with a radionuclide according to any one of claims 7 to 11, and a nuclide used for radioactively labeling the heterodimer compound that can be labeled with a radionuclide.

24. A kit, characterized in that The kit contains a predetermined amount of the heterodimer according to any one of claims 12 to 15, and a metal chelator-nuclide complex for radioactively labeling the heterodimer.

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