Double-targeting multi-mode diagnosis and treatment integrated probe as well as preparation method and application thereof

Abstract

The invention provides a dual-targeting multi-mode diagnosis and treatment integrated probe as well as a preparation method and application thereof. The probe comprises a marking tracing part, a target point binding ligand and a cyanine fluorescent dye part, the probe can be rapidly combined with target tissues after reaching a body, the target tissues with high target point expression can be effectively imaged in a bimodal mode and effectively distinguished, and the probe can effectively perform surgical navigation on the target tissues with high target point expression, so that the target tissues with high target point expression can be obtained. The probe is a diagnosis and treatment integrated probe with a good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biological probes, and particularly to a dual-targeted multimodal diagnosis and treatment integrated probe, a preparation method thereof, and an application thereof. Background Art

[0002] Prostate cancer is one of the common malignant tumors, and its incidence rate ranks second among male malignant tumors globally. According to the data of the National Cancer Center, prostate cancer has become the most frequently occurring tumor in the male urinary system in China since 2008. The incidence rate reached 9.8 / 100,000 in 2014, ranking sixth among male malignant tumors; the mortality rate reached 4.22 / 100,000, ranking ninth among male malignant tumors. In recent years, the incidence rate of prostate cancer in China has shown an increasing trend year by year, especially among elderly men. There are huge differences in the staging composition of prostate cancer patients between China and Western developed countries. Among the newly diagnosed cases in China, only 30% are clinically localized patients at the time of diagnosis, and the rest are patients with local advanced or widespread metastases. These patients cannot receive local radical treatment and have a poor prognosis. Early, comprehensive, non-invasive, and dynamic detection of the primary focus and metastatic foci of prostate cancer is of great significance for guiding accurate clinical staging, judging prognosis, selecting treatment plans, and judging treatment efficacy.

[0003] Currently, the most commonly used imaging method for prostate cancer diagnosis is MRI, which has the characteristics of high sensitivity, relatively low specificity, and high false positive rate, and is relatively limited in the detection of distant metastases and biochemical recurrence. Recently, prostate-specific membrane antigen (PSMA)-targeted PET imaging has received increasing attention and has good application value for prostate diagnosis and treatment. The 2021 Chinese CSCO guidelines first recommend (Class IA, Grade I recommendation) PSMA PET / CT for distant staging of prostate cancer. In a meta-analysis, 68 The sensitivity and specificity of 68Ga-PSMA PET / CT for preoperative regional lymph node metastases in intermediate- and high-risk prostate cancer are 65% and 95% respectively, showing a relatively high level.

[0004] PSMA is a type II transmembrane protein composed of 19 intracellular, 24 transmembrane, and 707 extracellular amino acids. The external groups of PSMA can be linked to various ligands with different functions, while the internal groups contain functional factors that initiate endocytosis. When the PSMA ligand or specific antibody for imaging binds to the external group of PSMA, the internalization group will initiate the endocytosis of cells, and the PSMA ligand or antibody will be internalized into the cytoplasm or retained in lysosomes, so as to achieve the accumulation of radioactive imaging agents inside cells; while the unbound ligands, due to their small size, are rapidly cleared by the body, thus forming a high contrast between tumors and the background. PSMA is significantly highly expressed in prostate cancer cells, 100 - 1000 times that of normal cells, and is even more highly expressed in advanced prostate cancer and castration-resistant cancer cells, and increases with the elevation of tumor stage and grade. Although PSMA is not specifically expressed in prostate cancer, PSMA expression can also be observed in the neovessels of some normal organs including the kidney, salivary gland, lacrimal gland, small intestine, etc. and some tumors such as renal cell carcinoma, but the PSMA expression level in these tissues is lower than that in prostate cancer. Based on these biological characteristics above, PSMA has become a biological target with great development potential.

[0005] The properties of different PSMA radioligands are slightly different. Among them, the inhibitors of PSMA are mainly divided into 3 categories: phosphorous-based, thiol-based, and urea-based. The most widely used PSMA PET imaging agent is the urea-based small molecule, such as PSMA-11, 68 Ga-PSMA-617, 68 Ga-PSMA-I&T, 18 F-DCFBC, 18 F-DCFPyL, and 18 F-PSMA-1007, etc. A number of studies have shown that compared with other PET imaging or traditional imaging, PSMA PET imaging has significant advantages, including higher specificity, sensitivity, and positive predictive value. 68 Ga-labeled PSMA ligands, as the first PSMA PET imaging agents applied to prostate cancer, have shown good application prospects in multiple aspects such as tumor localization, local staging, BCR, metastasis detection, and guiding treatment. [2] 。

[0006] Among these ligands, PSMA-11 is the most widely used clinically. 68 Ga-PSMA-11 was approved by the FDA for marketing in December 2020 for patients with suspected metastatic prostate cancer who are intended for initial radical treatment and patients with suspected recurrent prostate cancer with elevated PSA levels. Another product of the same kind, LOCAMETZ ® ( 68Gozetotide was approved by the FDA for marketing in March 2022 for patients with suspected metastatic prostate cancer undergoing initial radical treatment, patients with suspected recurrent prostate cancer with elevated PSA levels, and screening for metastatic prostate cancer patients suitable for 177 targeted therapy with Lu-PSMA-617.

[0007] However, all such probes are radionuclide diagnostic or diagnostic and therapeutic probes. First, such radionuclide probes can only provide a diagnostic function and cannot provide guidance during surgery; second, there is a lack of research and development of more diagnostic and therapeutic probes. Currently, only 177 a radiopharmaceutical such as Lu-PSMA-617 has obtained FDA approval for the treatment of prostate cancer. Therefore, there is a need to develop more specific therapeutic probes; third, there is a lack of development of PSMA-targeted multimodal molecular probes that integrate diagnosis and treatment and can be used for intraoperative navigation. Therefore, there is an urgent need to develop a PSMA-targeted probe with strong specificity, high tumor uptake, long retention time, and the ability to perform intraoperative navigation for integrated diagnosis, surgery, and treatment of prostate cancer patients. Summary of the Invention

[0008] The object of the present invention is to provide a dual-targeted multimodal integrated diagnosis and treatment probe, its preparation method and application. The probe of the present invention includes a labeled tracer part, a target-binding ligand, and a cyanine fluorescent dye part. After the probe reaches the body, it can quickly bind to the target tissue, can effectively perform dual-modal imaging of the target tissue with high expression of the target and effectively distinguish it, and the probe can effectively perform surgical navigation for the target tissue with high expression of the target. It is a very promising integrated diagnosis and treatment probe in clinical practice.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] In the first aspect, the present invention provides a dual-targeted multimodal integrated diagnosis and treatment probe, the probe includes the following parts: a labeled tracer part, a target-binding ligand, and a cyanine fluorescent dye part, wherein the labeled tracer part is a group labeled with a radioactive isotope or a group capable of chelating a radioactive isotope, the cyanine fluorescent dye is a double-claw structure group with heterocycles at both ends and a bridging chain in the middle, the labeled tracer part is connected to the bridging chain of the cyanine fluorescent dye molecular skeleton, and a target-binding ligand is connected to each of the heterocycles at both ends of the cyanine fluorescent dye molecule to form a double-ligand structure.

[0011] Preferably, the group labeled with a radioactive isotope is selected from 18 RSECA-TFP labeled with 18FAlF, 18 N-propargyl-N,N-dimethylammonium methyl boron trifluoride labeled with 18F, 18 OTS group labeled with 18F,18 a quaternary ammonium salt group labeled with F, 124 / 125 / 131 a phenol group labeled with I, 89 any one or more of a DFO group labeled with Zr.

[0012] Preferably, the group capable of chelating a radioactive isotope is selected from those capable of 68 / 69 Ga or 177 any one or more of DOTA, NOTA, NODA, NODAGA, DOTP, TETA, NOTAM, DO3AP chelating groups labeled with Lu.

[0013] Preferably, the cyanine fluorescent dye is a cyanine fluorescent dye with near-infrared fluorescence.

[0014] Preferably, the target-binding ligand is selected from any one or more bindable ligands selected from a ligand targeting PSMA for prostate cancer, an FAPI ligand targeting the tumor-targeting fibroblast target FAP, a ligand targeting the somatostatin receptor SSTR for neuroendocrine tumors, an RGD ligand targeting the tumor-targeting integrin αvβ3, and a ligand targeting GPC-3 for liver cancer or hepatocellular carcinoma.

[0015] More preferably, the labeled tracer moiety is DOTA, the target-binding ligand is a PSMA ligand, and the cyanine fluorescent dye is IR-780.

[0016] Furthermore, the probe has the following molecular structure:

[0017] .

[0018] In a second aspect, the present invention provides a method for preparing the dual-targeting multimodal diagnosis and treatment integrated probe as described above. The method includes: first reacting the target-binding ligand with the cyanine fluorescent dye to connect it to the heterocycles at both ends of the dye molecule, and then reacting the labeled tracer moiety with the cyanine fluorescent dye to connect it to the bridging chain of the dye molecule, or first reacting the labeled tracer moiety with the cyanine fluorescent dye to connect it to the bridging chain of the dye molecule, and then reacting the target-binding ligand with the cyanine fluorescent dye to connect it to the heterocycles at both ends of the dye molecule, to form a dual-targeting multimodal diagnosis and treatment integrated probe having dual target-binding ligands and simultaneously having radioactive tracer and fluorescent tracer capabilities.

[0019] Preferably, the reaction is a click chemical reaction, in which an alkynyl group and an azide group are introduced into the reaction sites of the target-binding ligand and the cyanine fluorescent dye respectively. After the reaction, an alkynyl group and an azide group are introduced into the reaction sites of the labeling tracer part and the cyanine fluorescent dye respectively and then the reaction continues, or an alkynyl group and an azide group are introduced into the reaction sites of the labeling tracer part and the cyanine fluorescent dye respectively. After the reaction, an alkynyl group and an azide group are introduced into the reaction sites of the target-binding ligand and the cyanine fluorescent dye respectively and then the reaction continues.

[0020] Preferably, the cyanine fluorescent dye used is IR-780, the labeling tracer part is DOTA, and the target-binding ligand is PSMA. The specific reaction is as follows:

[0021] (a) Perform a click chemical reaction on the compound IR-780-Alkyne and the compound N3-PSMA to introduce a dual PSMA targeting group;

[0022] (b) After step (a) is completed, the product is directly subjected to a substitution reaction without purification to introduce an azide group;

[0023] (c) The reaction product of step (b) is directly subjected to a click chemical reaction with the compound PG without purification to introduce an alkynylamine short chain to obtain the compound IR-780-C-4;

[0024] (d) Perform a condensation reaction on the compound IR-780-C-4 and DOTA-NHS to introduce a labeling group to obtain a labeling precursor DOTA-IR-780-C-4.

[0025] Further, the method further includes the step of radioisotope labeling the obtained labeling precursor.

[0026] In a third aspect, the present invention provides the use of the dual-targeted multimodal diagnosis and treatment integrated probe as described above or the dual-targeted multimodal diagnosis and treatment integrated probe prepared by the method as described above in the imaging of target tissues.

[0027] Preferably, the tissue imaging method is PET or SPECT.

[0028] Further, the target tissue is a tumor tissue.

[0029] In a fourth aspect, the present invention also provides the use of the dual-targeted multimodal diagnosis and treatment integrated probe as described above or the dual-targeted multimodal diagnosis and treatment integrated probe prepared by the method as described above in guiding fluorescence surgical navigation.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention combines receptor-mediated binding and retention effects with a multimodal strategy to design and synthesize a dual-targeted, long-retention multimodal imaging probe. By binding to the target, the probe can effectively stay at tissues with high target expression, thus avoiding rapid clearance; through radionuclide labeling, the probe can generate strong radioactive signals at the target tissue, which can be acquired by the instrument and corrected by the computer to obtain images, thereby realizing more accurate and higher signal-to-background ratio imaging detection and analysis of the target tissue; then through fluorescence imaging, the long retention and high signal-to-background ratio of the probe at the target tissue mediate surgical navigation to resect the lesion. In the examples, the 68 Ga]DOTA-IR-780-C-4 probe synthesized according to the method of the present invention was used in prostate cancer patients for clinical PET imaging, and it was found that the probe has good specificity and sensitivity, high signal-to-background ratio, and is beneficial to the diagnosis of prostate cancer. The 69 Ga]DOTA-IR-780-C-4 probe was used for rapid detection of prostate cancer tumors and sections in patients, to measure the PSMA expression and malignancy degree of prostate cancer tumors, realizing the dual combination of imaging and detection, for the purpose of rapid diagnosis of prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the principle mechanism of the dual-targeted multimodal diagnosis and treatment integrated probe of the present invention;

[0032] Figure 2 For the probe 68 Ga]DOTA-IR-780-C-4 for 68 Ga radioactive labeling synthesis and HPLC analysis of its stability in PBS and serum, and 69 UV and fluorescence spectra of

[0033] Figure 3 For the probe 68 Ga]DOTA-IR-780-C-4 binding affinity study;

[0034] Figure 4 For the probe 69 / 68 Ga]DOTA-IR-780-C-4 specificity study in 22RV1 and PC-3 cells (cell uptake and fluorescence imaging);

[0035] Figure 5 For the probe 68 Ga]DOTA-IR-780-C-4 and 68 Ga]PSMA-617 PET imaging and quantitative analysis in different tumor-bearing mice (Lncap, 22RV1, PC-3), indicating that the probe68 Ga]DOTA-IR-780-C-4 has better targeting specificity, higher tumor uptake, and stronger retention, compared with the clinical drug 68 Ga]PSMA-617, it has higher tumor uptake and tumor / muscle uptake ratio, and has good clinical transformation value;

[0036] Figure 6 as the probe 68 Ga]DOTA-IR-780-C-4 and 68 Ga]PSMA-617 in the biodistribution in tumor-bearing mice at 3 h;

[0037] Figure 7 for 69 Ga]DOTA-IR-780-C-4 in fluorescence imaging and quantitative analysis in different tumor-bearing mice (Lncap, 22RV1, PC-3), indicating that it has good targeting specificity to detect tumors with different PSMA expressions, and has the advantage of fluorescence surgical navigation for tumors with high PSMA expression;

[0038] Figure 8 as the probe 69 Ga]DOTA-IR-780-C-4 in fluorescence imaging in different tumor-bearing mice (Lncap, 22RV1, PC-3) after 24 h, fluorescence imaging, HE staining and immunohistochemical analysis of tumor sections;

[0039] Figure 9 for the injected probe 69 Ga]DOTA-IR-780-C-4 fluorescence surgical navigation map of Lncap tumor-bearing mice 24 hours later, this probe can indeed guide fluorescence surgical navigation and guide the resection of tumors with high PSMA expression;

[0040] Figure 10 for the fluorescence biodistribution of tumors and various tissues and organs in tumor-bearing mice after surgical navigation;

[0041] Figure 11 as the probe 69 Ga]DOTA-IR-780-C-4 fluorescence distribution in sections of the kidney;

[0042] Figure 12 as the probe 177 Lu]DOTA-IR-780-C-4 for 177 Lu radiolabeled synthesis and HPLC analysis of its stability in PBS and serum;

[0043] Figure 13 as the probe 177Lu]DOTA-IR-780-C-4 and 177 SPECT imaging and quantitative analysis of 177 Lu]PSMA-617 in Lncap tumor-bearing mice showed that the probe

[0044] Figure 14 Lu]DOTA-IR-780-C-4 had better targeting specificity and long retention at the tumor site; 177 For the biodistribution of 177 Lu]DOTA-IR-780-C-4 and 177 Lu]PSMA-617 in Lncap tumor-bearing mice at 84 h, 177 the Lncap tumor uptake in the Specific Embodiments

[0045] The present invention developed a dual-targeted multimodal diagnosis and treatment integrated probe. The probe of the present invention includes a labeled tracer part, a target-binding ligand, and a cyanine fluorescent dye part. After reaching the body, the probe can quickly bind to the target tissue, can effectively perform bimodal imaging on the target tissue with high expression of the target, and can effectively distinguish, and the probe can effectively perform surgical navigation on the target tissue with high expression of the target, and is a very promising diagnosis and treatment integrated probe in clinical practice.

[0046] Figure 1 The schematic diagram of the principle mechanism of the dual-targeted multimodal diagnosis and treatment integrated probe of the present invention is shown. For the probe of the present invention, its dual-targeting enhances the binding force, and the radionuclide labeling is used for accurate diagnosis, while the near-infrared fluorescence backbone is conducive to surgical navigation. Taking prostate cancer tumor tissue and the highly expressed PSMA therein as an example of the target tissue and target, the present invention provides a dual-PSMA-targeted multimodal molecular probe and its preparation method, so as to realize more accurate and higher signal-to-background ratio PET and SPECT imaging detection and analysis of prostate cancer with high PSMA expression, and perform intraoperative navigation to remove the lesion.

[0047] In the following examples, the present invention designed and synthesized a dual-PSMA-targeted, long-retention multimodal imaging probe 68 / 69 Ga / 177 Lu]DOTA-IR-780-C-4. By binding to PSMA, 68 / 69 Ga / 177 Lu]DOTA-IR-780-C-4 can effectively stay at the tumor with high PSMA expression, thus avoiding rapid clearance; through the radionuclide68 Ga and 177 Lu, the probe can generate a strong radioactive signal at the tumor site, which can be acquired by the instrument, and the computer corrects it to obtain an image, so as to realize more accurate and higher signal-to-background ratio imaging detection and analysis of prostate cancer; then through fluorescence imaging, the long retention and high signal-to-background ratio of the probe at the tumor site mediate the surgical navigation to resect the lesion. Using 68 Ga]DOTA-IR-780-C-4 in prostate cancer patients for clinical PET imaging, it is concluded that the probe has good specificity and sensitivity, with a high signal-to-background ratio, which is beneficial to the diagnosis of prostate cancer. Using 69 Ga]DOTA-IR-780-C-4 for the rapid detection of prostate cancer tumors and slices in patients, measuring the PSMA expression and malignancy degree of prostate cancer tumors, realizing the dual combination of imaging and detection, so as to achieve the purpose of rapid diagnosis of prostate cancer.

[0048] In the exemplary embodiment, the technical solution adopted by the present invention is as follows:

[0049] A dual-PSMA-targeted multimodal molecular probe, which is composed of the following parts bonded together:

[0050] A short-chain compound PG connecting the fluorescent backbone and the DOTA-labeled group:

[0051] ;

[0052] N3-PSMA that specifically binds to the PSMA-targeted ligand:

[0053] ;

[0054] Having 68 / 69 Ga and 177 Lu chelation labeling function ligand DOTA-NHS:

[0055]

[0056] DOTA-NHS;

[0057] And cyanine fluorescent dyes with near-infrared fluorescence (IR-780-Alkyne or IR780-SO3 - ):

[0058] .

[0059] Specifically, the probe has the following molecular structure:

[0060] .

[0061] Furthermore, the present invention also provides a method for preparing the above-mentioned dual-PSMA multimodal molecular probe, which includes the following steps:

[0062] (a) Perform a click chemical reaction on the compound IR-780-Alkyne and the compound N3-PSMA to introduce a PSMA targeting group;

[0063] (b) After step (a) is completed, the product is directly subjected to a substitution reaction without purification to introduce an azide (N3) group;

[0064] (c) The reaction product of step (b) is directly subjected to a click chemical reaction with a PG short chain without purification to obtain the compound IR-780-C-4;

[0065] (d) Condense the compound IR-780-C-4 to introduce a labeling group DOTA-NHS to obtain a labeling precursor DOTA-IR-780-C-4;

[0066] The reaction formulas of the above steps are as follows:

[0067] .

[0068] Among them, in step (a), the click chemical reaction is to dissolve the compound IR-780-Alkyne, the compound N3-PSMA, anhydrous copper sulfate (CuSO4), sodium ascorbate, and tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) in a solvent and carry out the reaction; among them, the molar ratio of the compound IR-780-Alkyne, the compound N3-PSMA, sodium ascorbate, anhydrous copper sulfate, and tris(3-hydroxypropyltriazolylmethyl)amine is 1:2.5, 1:1, 1:2.5, 1:1, the reaction temperature is 25 °C, and the reaction is carried out for 30 - 45 min under stirring, preferably 30 min. The solvent includes but is not limited to dimethyl sulfoxide, preferably dimethyl sulfoxide, and a certain amount of H2O is added, (V DMSO :V H2O = 10:1).

[0069] In step (b), sodium azide (NaN3) is dissolved in a solvent and added to the reaction product of step (a) for a substitution reaction; among them, the molar ratio of the compound IR-780-Alkyne to sodium azide (NaN3) is 1:2, preferably 1:2; the reaction temperature is 25 °C, and the reaction is carried out for 10 - 15 min under stirring, preferably 15 min. The solvent includes but is not limited to secondary water, preferably secondary water (double-distilled water).

[0070] In step (c), compound PG is dissolved in a solvent and added to the reaction product of step (b) for reaction; wherein, the molar ratio of compound IR-7810-Alkyne to compound PG is 1:1.2, preferably 1:1.2; the reaction temperature is 25 °C, preferably room temperature, and the reaction is carried out for 30 - 45 min, preferably 40 min under stirring to obtain a reaction solution containing compound IR-780-C-4, which is purified and freeze-dried to obtain compound IR-780-C-4; the solvent includes but is not limited to dimethyl sulfoxide, preferably dimethyl sulfoxide.

[0071] In step (d), compound IR-780-C-4, DOTA-NHS and N,N-diisopropylethylamine are dissolved in a solvent for reaction; wherein, the molar ratio of compound IR-780-C-4, DOTA-NHS and N,N-diisopropylethylamine is 1:1.2:3, the reaction temperature is 25 °C, and the reaction is carried out for 2 - 3 h under stirring; the reaction solvent includes but is not limited to DMF, preferably DMF. After the reaction, the reaction solution containing the compound is purified and freeze-dried to obtain the labeling precursor DOTA-IR-780-C-4.

[0072] The structure of the said DOTA-NHS is:

[0073]

[0074] Furthermore, the present invention also claims the use of the above-mentioned dual-PSMA multimodal small molecule probe for detecting the binding to PSMA in vitro.

[0075] Furthermore, the present invention also claims the use of the above-mentioned dual-PSMA multimodal small molecule probe for PET and SPECT imaging detection of different prostate cancer tumors.

[0076] Furthermore, the present invention also claims the use of the above-mentioned dual-PSMA multimodal small molecule probe for fluorescence surgical navigation of prostate cancer tumors.

[0077] Furthermore, the present invention also claims the use of the above-mentioned dual-PSMA multimodal small molecule probe for clinical PET imaging of prostate cancer patients.

[0078] Furthermore, the present invention also claims the use of the above-mentioned dual-PSMA multimodal small molecule probe for rapid detection of prostate cancer clinical tumors.

[0079] The probe DOTA-IR-780-C-4 of the present invention contains 68 / 69 Ga / 177The DOTA group radiolabeled with Lu, the N3-PSMA group with PSMA targeting, and the IR-780-Alkyne group (808 nm) with near-infrared fluorescence group. When intravenously injected 68 / 69 Ga / 177 Lu]DOTA-IR-780-C-4, 68 / 69 Ga / 177 Lu]DOTA-IR-780-C-4 can quickly reach the tumors with high PSMA expression and bind to PSMA. Due to the dual-PSMA structure of this probe, the probe binds strongly to PSMA, has a long residence time at the tumor site, and the uptake at the tumor site shows a trend of continuous increase and slow decrease, and the signal-to-background ratio (compared with muscle tissue, heart, liver, etc.) continuously increases. The probe is mainly metabolized by the kidneys in non-target tissues (such as the heart and liver with relatively fast metabolism). First, through 68 Ga]DOTA-IR-780-C-4 in the PET imaging of prostate cancer-bearing mice, it is preliminarily judged that the probe has a high uptake at the tumor site, a high signal-to-background ratio, and an enhanced tumor-to-muscle uptake ratio, which is beneficial to the early diagnosis of PSMA-related cancers. Then, through 69 Ga]DOTA-IR-780-C-4 in the fluorescence imaging at the tumor site with high PSMA expression, the uptake at the tumor site increases and the signal-to-background ratio increases, which is beneficial to the fluorescence surgical navigation research of tumor-bearing mice around 24 h to remove prostate cancer lesions. Furthermore, through 177 Lu]DOTA-IR-780-C-4 in the SPECT imaging of tumor-bearing mice with PSMA expression, it is obtained that the probe has a high uptake at the tumor site, a long residence time, and a relatively high signal-to-background ratio, indicating its potential for radiotherapy. Then, 68 Ga]DOTA-IR-780-C-4 is used in prostate cancer patients for clinical PET imaging, and it is concluded that the probe has good specificity and sensitivity. Within two hours, the uptake of both tumors and metastases increases, and the signal-to-background ratio increases, which is beneficial to the diagnosis of prostate cancer. Finally, 69 Ga]DOTA-IR-780-C-4 is used for the rapid detection of prostate cancer tumors and slices in patients to measure the PSMA expression and malignancy degree of prostate cancer tumors, achieving the dual combination of imaging and detection for the purpose of rapid diagnosis of prostate cancer.

[0080] It should be understood that the specific examples shown in the above exemplary embodiments are only for illustrating the principle, but under this principle, a variety of labeling groups, dye skeletons, and ligand groups can be selected, and the probes composed of these different components should all be covered within the protection scope of the present invention.

[0081] For the synthesis of probe molecules by selecting different labeling groups, dye skeletons, and ligand groups, the following general synthesis process can be referred to.

[0082]

[0083] General synthesis process 1

[0084]

[0085] General synthesis process 2

[0086] The above general synthesis process 1 is the synthesis route of the general labeled probe Q1-IR-780-R2-2. Taking the near-infrared fluorescent dye IR-780-Alkyne as an example of the probe skeleton, any of the above alternative binding-type targeting ligands can be connected through the Click reaction (the letter R represents the connected targeting ligand). Then, after connecting N3, the labelable group can be connected again through the Click reaction. For example, after 18 F or 124 / 125 / 131 I-labeled linking group Q1, the final labeled precursor probe Q1-IR-780-R2-2 can be obtained through the above "one-pot three-step" Click reaction.

[0087] The above general synthesis process 2 is the synthesis route of the general labeled probe Q2-IR-780-R2-3. Taking the near-infrared fluorescent dye IR-780-Alkyne as an example of the probe skeleton, any of the above alternative binding-type targeting ligands can be connected through the Click reaction (the letter R represents the connected targeting ligand). Then, after connecting N3, the short linking chain of PG can be connected again through the Click reaction. Finally, the chelating group Q2 can be connected through a condensation reaction. For example, it can chelate 89 Zr, 18 F]AlF3, 68 Ga or 177 chelating groups of metal ions such as Lu to obtain the final labeled precursor probe Q2-IR-780-R2-3.

[0088] In addition to the replaceability of the targeting ligand and the labeling group, those skilled in the art know that there are also various alternatives for the near-infrared fluorescent skeleton in the above synthesis route. For example, in addition to IR-780-Alkyne shown in the above examples, IR780-SO3 can also be selected -, ICG, etc. Those skilled in the art know that cyanine near-infrared fluorescent dyes are double-claw molecules with heterocycles at both ends and a methine conjugated bridge chain in the middle. In the present invention, ligand groups can be connected to the heterocycles at both ends, and labeling groups can be connected to the bridge chain in the middle to form the multimodal probe with a double-ligand structure of the present invention. Therefore, dye molecules with such a double-claw structure can all be used in the present invention. The methine conjugated chain can be a linear carbon chain, such as ICG described above, or a ring structure can be introduced into the carbon chain, such as IR-780 described in the present invention. The substitutions on the methine chain are well-known to those skilled in the art, so they will not be elaborated further below.

[0089] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the specific embodiments and drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0090] In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "front end", "rear end", "both ends", "one end", "the other end", etc. are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be construed as a limitation of the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0091] In the description of the present invention, it should be noted that unless otherwise clearly specified and defined, the terms "installation", "provided with", "connection", etc. should be understood in a broad sense. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be directly connected, or indirectly connected through an intermediate medium, and can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0092] In the following examples, the experimental methods are all conventional methods unless otherwise specified; the reagents and materials can all be obtained from commercial channels unless otherwise specified.

[0093] In the following examples, the "%" is a percentage unless otherwise specified.

[0094] Reagents and Instruments: All chemical reagents and solvents were purchased from Bide, J&K Scientific Ltd. (Shanghai, China), Tokyo Chemical Industry Co., Ltd. (Shanghai, China) and Sigma-Aldrich. Analytical solvents and reagents were of chromatographic purity, and conventional reagents were of analytical purity and were not further purified.

[0095] 1 The 1H-NMR spectra were obtained using a 400 MHz Bruker Avance III 400 nuclear magnetic resonance spectrometer. High performance liquid chromatography (HPLC) was performed using a Thermo Scientific Dionex Ultimate 3000 with eluent of CH3CN / H2O (1‰ CF3COOH). Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis was carried out using an AB SCIEX 4800Plus MALDI TOF / TOF™ mass spectrometer. UV-Vis spectra were measured using an Ocean Optics Maya 2000 Pro spectrometer. Fluorescence spectra were measured using a HORIBA Jobin Yvon Fluoromax-4 fluorometer. Fluorescence images of cells and tissue sections were acquired using an Olympus IX73 inverted fluorescence microscope. In vivo fluorescence images were obtained using an IVIS Lumina XR III system, and the fluorescence intensity was quantified by measuring the signal of the circled area using Living image software (PerkinElmer). Small animal PET imaging was performed using a Siemens PET / CT imager to obtain images, and small animal SPECT imaging was obtained and processed using PMOD software.

[0096] Example 1:

[0097] Design, Synthesis and Characterization of the Probe Labeling Precursor DOTA-IR-780-C-4

[0098] The synthetic route of the intermediate compound cml-2 is as follows:

[0099]

[0100] Reaction conditions: (a) Di-tert-butyl L-glutamate hydrochloride, triethylamine, triphosgene, dichloromethane, -90 °C → 25 °C, 16 h, 70%; (b) 10% palladium on carbon, hydrogen, anhydrous methanol, 25 °C, overnight, 68%;

[0101] Synthesis of Compound cml-1: Dissolve di-tert-butyl L-glutamate hydrochloride (500 mg, 1.6 mmol) and triethylamine (773 μL, 5.5 mmol) in ultradry dichloromethane (30 mL), cool to -90 °C and stir. Then dissolve triphosgene (170.5 mg, 0.58 mmol) in ultradry dichloromethane (20 mL) and add dropwise to the reaction solution at -90 °C. After the addition, continue the reaction at room temperature for 30 min. Then, add tert-butyl (S)-2-amino-6-(((benzyloxy)carbonyl)amino)hexanoate hydrochloride (378 mg, 1 mmol) and triethylamine (141 μL, 1 mmol) dissolved in dichloromethane to the above reaction solution, and continue stirring at room temperature for 16 h. After the reaction, cml-1 (yield 70%) was obtained by column chromatography separation as a transparent colorless oil.

[0102] Synthesis of Compound cml-2: Dissolve compound cml-1 in 10 mL of methanol-free. Then add palladium on carbon and replace with hydrogen overnight. After the reaction, filter through diatomaceous earth to remove palladium on carbon, and obtain colorless oily cml-2 (yield 68%).

[0103]

[0104] Reaction conditions: (a) Fmoc-3-(2-naphthyl)-alanine, DIPEA, dichloromethane, room temperature, 3 h; (b) 20% piperidine; (c) azidoacetic acid, HBTU, DIPEA, DMF, room temperature, 3 h; (d) 1% TFA, 45.7%.

[0105] Synthesis of Compound cml-6: After rinsing the solid-phase peptide synthesis tube twice with 10 mL of dichloromethane, suction filtration was performed to remove the dichloromethane. Weigh 250 mg of 2-chlorotrityl chloride resin (loading rate: 1.1 mmol / g), add it to the solid-phase peptide synthesis tube, add 10 mL of ultra-dry dichloromethane, and shake for 10 min to swell the resin, then perform suction filtration to remove the dichloromethane. (2) Add Fmoc-3-(2-naphthyl)-alanine (131 mg, 0.3 mmol) to the solid-phase peptide synthesis tube, add 10 mL of ultra-dry dichloromethane, and adjust the alkalinity with DIPEA (108 μL, 0.625 mmol) to a pH of 8 - 9, and shake at room temperature for 3 h to allow the amino acid to be loaded onto the resin. (3) After the reaction is completed, perform suction filtration to remove the reaction solution, add 10 mL of dichloromethane, wash the resin, perform suction filtration, repeat 3 times, then add 10 mL of blocking solution (DCM / MeOH / DIPEA = 17 / 2 / 1), and shake for 1 h to block the sites of the resin that have not loaded amino acids. Then take 10 mL of DCM to wash the resin 3 times, perform suction filtration, and then wash the resin 3 times with 10 mL of DMF, and perform suction filtration. (4) Add 10 mL of a 20% piperidine in DMF solution, shake for 10 min, perform suction filtration, repeat 3 times to remove the Fmoc protecting group on the amino acid, then take 10 mL of DMF to wash the resin 3 times to remove piperidine, and perform suction filtration. (5) Weigh azidoacetic acid (22.6 μL, 0.3 mmol) and HBTU (113 mg, 0.3 mmol) and add them to the solid-phase peptide synthesis tube, add 10 mL of ultra-dry DMF, and adjust the alkalinity with DIPEA (130 μL, 0.75 mmol) to a pH of 8 - 9, shake at room temperature for 3 hours, after the reaction is completed, perform suction filtration to remove the reaction solution, add 10 mL of DMF, wash the resin, perform suction filtration, repeat 3 times, and then repeat operation (4) to end the peptide synthesis. Add 10 mL of dichloromethane containing 1% TFA to the solid-phase peptide synthesis tube, shake for 10 min, collect the filtrate, repeat this step until the resin color shows wine red and does not fade. The obtained filtrate was removed of organic solvents under negative pressure by a rotary evaporator, cold ether was added, after a yellow oil was precipitated, it was transferred to a 50 mL centrifuge tube, centrifuged at high speed, the supernatant was removed, and the obtained product was placed in a vacuum drying oven and dried under vacuum for 1 hour to obtain peptide cml-6 (yield 45.7%).

[0106]

[0107] Reaction conditions: (a) cml-2, cml-6, HBTU, DIPEA, THF, r.t., 3 h; (b) TFA:DCM:TiPS = 65:33:2, r.t., 2 h, 74%.

[0108] Synthesis of compound N3-PSMA: Take cml-6 (34 mg, 0.11 mmol), cml-2 (61 mg, 0.13 mmol) and HBTU (50 mg, 0.13 mmol), dissolve them in 5 mL of THF, add DIPEA (60 μL, 0.33 mmol), adjust the pH to 8 - 9, stir the reaction for 3 h under N2 protection and at room temperature, then remove the organic solvent to obtain the crude product cml-7. Next, add TFA:DCM:Tips = 65:33:2 (10 mL), stir the reaction for 2 h at room temperature, and precipitate the white solid N3-PSMA with cold ether.

[0109]

[0110] Synthesis of compound DOTA-IR-780-C-4: First, synthesize the near-infrared fluorescent dye IR-780-Alkyne according to the reported method (J. Am. Chem. Soc. 2020, 142, 2787 - 2794.). (a) Dissolve compound N3-PSMA (308 mg, 0.51 mmol) and IR-780-Alkyne (100 mg, 0.17 mmol) in DMSO. Dissolve CuSO4 (27 mg, 0.17 mmol), sodium ascorbate (67 mg, 0.34 mmol) and THPTA (74 mg, 0.17 mmol) in secondary water, add them to DMSO, and stir at room temperature for 30 minutes to introduce the PSMA group. (b) Subsequently, directly add NaN3 (22.1 mg, 0.34 mmol) dissolved in secondary water to the reaction solution, and stir at room temperature for 12 minutes to introduce the N3 group. (c) Finally, dissolve compound PG (28.6 mg, 0.26 mmol) in DMSO, add it to the reaction solution, and supplement CuSO4 (27 mg, 0.17 mmol), sodium ascorbate (67 mg, 0.34 mmol) and THPTA (74 mg, 0.17 mmol) dissolved in secondary water (200 μL), and continue to stir at room temperature for 30 minutes to introduce the short peptide PG. After the reaction is completed, purify the residue by preparative HPLC and obtain compound IR-780-C-4 as a dark green solid by lyophilization. Yield: 129 mg (40%). MS: calcd. for C 99 H 118 N 21 O 19 + [M + : 1904.8907; found MALDI-MS: m / z 1904.7325.

[0111] Synthesis of Compound DOTA-IR-780-C-4: (d) Dissolve DOTA-NHS (2.6 mg, 0.0057 mmol), Compound IR-780-C-4 (10 mg, 0.0053 mmol) and DIPEA (3 μL, 0.0159 mmol) in DMF (1 mL) solution, stir at room temperature for 2 - 3 hours to introduce the labeling group DOTA. After the reaction, purify the residue by preparative HPLC and obtain Compound DOTA-IR-780-C-4 as a dark green solid by lyophilization. Yield: 10 mg (77%). MS: calcd. for C 116 H 144 N 25 O 28 [M 2 + : 2291.0709; found MALDI-MS: m / z 2291.0310.

[0112] Other synthesis examples:

[0113] In addition to DOTA-IR-780-C-4, probe molecules with different targeting ligands, labeling groups and dye skeletons were also synthesized and are classified and summarized as follows:

[0114] 1) Different ligands can be connected to the skeleton (such as PSMA targeting ligand, FAP targeting ligand, integrin αvβ3 targeting ligand, etc.)

[0115]

[0116] 2) Different 18 F or 124 / 125 / 131 I labeling groups can be connected to the skeleton (such as 18 F-labeled OTS group, 18 F-labeled quaternary ammonium salt group, 124 / 125 / 131 I-labeled phenol group, etc.)

[0117]

[0118] 3) Different 89 Zr, 18 F]AlF3, 68 Ga or 177 chelation groups of metal ions such as Lu (such as DOTA, RESCA-TFP, NOTA, and DFO-NCS, etc.)

[0119]

[0120] 4) Can be connected to different skeletons, such as IR-780-Alkyne and IR-780-SO3 - etc.

[0121]

[0122] Among them, the molar ratio of reactants in the above reaction is as follows:

[0123] Fluorescent dye: Targeting ligand: Sodium azide NaN3: CuSO4•5H2O: Sodium ascorbate: THPTA: 18 F or 124 / 125 / 131 I and other labeling groups = 1: 2.4 - 3: 2 - 2.5: 1: 2 - 2.5: 1: 1.2 - 1.5.

[0124] Fluorescent dye: Targeting ligand: Sodium azide NaN3: CuSO4•5H2O: Sodium ascorbate: THPTA: Propargylamines: Metal chelating group = 1: 2.4 - 3: 2 - 2.5: 1: 2 - 2.5: 1: 1.2 - 1.5: 1.2 - 1.5, and DIPEA is used to adjust the alkaline pH = 8 - 9.

[0125] Probe performance test

[0126] 1. 68 Radiosynthesis and physicochemical property study of [68Ga]DOTA-IR-780-C-4.

[0127] See Figure 2 in a, and use HCl (0.05 M) to elute through 68 Ge / 68 68Ga generator to obtain 68 68Ga, then add sodium acetate buffer solution (0.25 M) to adjust the pH of the reaction solution to 4 - 4.5, and the volume ratio of hydrochloric acid to sodium acetate is 1 mL: 0.285 mL; then add the labeling precursor DOTA-IR-780-C-4 (10 μL, 30 μg) and mix evenly. After reacting at 90 °C for 15 min, perform HPLC analysis and shake once every 5 minutes; finally, after activating the C-18 column (ethanol and water), hang the probe on the C-18 column, wash away the free 68 68Ga ions with water, and then elute the product into a bottle with ethanol to obtain the purified product 68 [68Ga]DOTA-IR-780-C-4. The drug purity was analyzed by radioactive HPLC before and after purification, and it was concluded that the probe had good reactivity, high radiochemical purity and yield ( Figure 2 in b). The purified samples were respectively mixed with PBS ( Figure 2 in c) and serum ( Figure 2After incubation for 1, 2, and 3 h, radio-HPLC was performed to analyze the sample purity to evaluate its stability. The probe showed high stability in both PBS and serum by HPLC, with a radiochemical purity > 97%, indicating good stability. In addition, the probe had good hydrophilicity, which was beneficial for subsequent biological research. Moreover, the cold compound 69 Ga]DOTA-IR-780-C-4 showed good near-infrared optical properties through ultraviolet absorption spectroscopy ( Figure 2 in e) and fluorescence spectroscopy ( Figure 2 in f), which was beneficial for subsequent fluorescence surgical navigation research. The specific result parameters are shown in Table 1 below.

[0128] Table 1 68 Radioactive synthesis and physicochemical property parameters of

[0129]

[0130] 2. Study on the binding affinity of the probe to PSMA

[0131] See Figure 3 in a. Different concentrations of 68 Ga]DOTA-IR-780-C-4 or 68 Ga]PSMA-617 were incubated with adherent 22RV1 cells (250,000 cells / well) at 4 °C for 1 h; and different concentrations of 68 Ga]DOTA-IR-780-C-4 or 68 Ga]PSMA-617 and its 1000-fold concentration inhibitor 2-PMPA were incubated with 22RV1 cells (250,000 cells / well) at 4 °C for 1 h. Both groups of experiments were set up with 3 parallel experiments. After incubation, the supernatant was removed, and the cells were washed twice with PBS and then digested with sodium hydroxide. The radioactive dose was detected by a gamma counter. The measured data were fitted by the software Graphpad to obtain the 68 Ga]DOTA-IR-780-C-4 and 68 Ga]PSMA-617 binding affinities to PSMA on the cell membrane surface of 22RV1. The specific results are shown in Figure 3 in b, showing that the introduction of dual PSMA targeting significantly enhanced the binding affinity of the probe to the target, increasing by about 2-fold, as shown in Figure 3 in c.

[0132] 3. Specificity study of the probe 68 / 69 Ga]DOTA-IR-780-C-4 in prostate cancer cells

[0133] (A) 68Ga]DOTA-IR-780-C-4 or 68 Ga]PSMA-617 (5 μCi) was incubated with 22RV1, 22RV1 + 2-PMPA (2-PMPA was incubated with 22RV1 cells 0.5 h in advance), and PC-3 cells (250,000 cells / well) at 37 °C for 0.5 h, 1 h, and 2 h respectively. Each group had 3 parallel groups. After incubation, the supernatant was removed, and the cells were washed twice with PBS and then digested with trypsin. The radioactive dose was detected by a gamma counter. Finally, the relative uptake of the probe by the cells was calculated (Uptake % = Radioactivity count value of cells / Radioactivity count value of added drug * 100%). The results are shown as Figure 4 shown in a of 68 Ga]DOTA-IR-780-C-4 had good targeting specificity and could significantly distinguish prostate cancer cells with different PSMA expressions. In addition, at 2 h, the uptake of 68 Ga]DOTA-IR-780-C-4 by 22RV1 cells was 68 2 times higher than that of

[0134] (B) 69 After incubating Ga]DOTA-IR-780-C-4 (5 μM) with 22RV1, 22RV1 + 2-PMPA (2-PMPA was incubated with 22RV1 cells 0.5 h in advance), and PC-3 cells at 37 °C for 2 h, the supernatant was removed, Hoechst was added and incubation continued for 15 min. Then the supernatant was removed, the cells were washed twice with PBS, and serum-free medium was added for fluorescence imaging of the cells. The results are shown as Figure 4 shown in b of 69 Ga]DOTA-IR-780-C-4 also had good targeting specificity and could significantly distinguish prostate cancer cells with different PSMA expressions, and had good near-infrared optical properties, which was beneficial for subsequent in vivo fluorescence imaging.

[0135] 4. PET imaging of the probe on tumor-bearing mice with different PSMA expressions at the in vivo level

[0136] See Figure 5 shown in a of. Fifteen 5 - 6-week-old male NCG mice were taken and a tumor-bearing mouse model with different PSMA expressions was constructed by injecting prostate cancer tumor cells into the right axilla. When the tumor diameter was about 0.6 - 0.8 cm, it could be used for imaging studies. The one containing 68 Ga]DOTA-IR-780-C-4 or 68Normal saline containing Ga]PSMA-617 (100 μCi) was injected into tumor-bearing mice via the tail vein. Whole-body images were acquired using a PET imager at the time points of 1 h, 2 h, and 3 h after injection.

[0137] After intravenous injection of 68 Ga]DOTA-IR-780-C-4, obvious radioactive signals could be rapidly detected at the site of PSMA-overexpressing Lncap tumors, and over time, the tumor uptake ( Figure 5 b in Figure 5 and the signal-to-background ratio ( 68 c in 68 Ga]PSMA-617 at the 3-h time point, the tumor uptake was 2.6 times higher (14.27 ± 1.10 % ID / g vs 5.40 ± 1.64 % ID / g), and the signal-to-background ratio was 1.8 times higher (22.80 ± 1.23 vs 12.41 ± 1.52).

[0138] After the 3-h PET imaging was completed, the tumors and major tissues and organs of the tumor-bearing mice were removed and detected for their radioactive content using a gamma counter. As can be seen from Figure 6 , compared with other groups, the tumor sites of Lncap tumor-bearing mice injected with 68 Ga]DOTA-IR-780-C-4 showed higher radioactive uptake, which was significantly higher than that of 22RV1 tumor-bearing mice with medium expression and PC-3 tumor-bearing mice with low expression. In addition, 68 the uptake of Ga]DOTA-IR-780-C-4 at the tumor site of Lncap tumor-bearing mice was 2.8 times higher than that of 68 Ga]PSMA-617 ( Figure 6 a in Figure 6 ), and the signal-to-background ratio was 1.5 times higher (

[0139] 5. Near-infrared fluorescence imaging and fluorescence surgical navigation applications in tumor-bearing mice with different PSMA expressions at the in vivo level

[0140] See Figure 7 a in 69Ga]DOTA-IR-780-C-4 (50 μM, 200 μL) was subjected to near-infrared fluorescence imaging before injection and at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after injection. Figure 7 As can be observed in Figure 7 , this fluorescent probe also has good targeting specificity and can distinguish tumor-bearing mice with different PSMA expressions. At the same time, in the tumors of positive Lncap tumor-bearing mice, in addition to monitoring a high fluorescence intensity at the tumor site, this probe has a long retention effect at the tumor site, and still has a high fluorescence intensity at 24 h ( Figure 7 b), and the signal-to-background ratio continuously increases, reaching the highest at 24 h ( Figure 8 c), which is beneficial for subsequent research on fluorescence surgical navigation applications. After completing the near-infrared fluorescence imaging, the tumors of the mice were dissected and taken out for near-infrared fluorescence imaging, HE staining, and IHC immunohistochemistry studies of tissue sections. According to

[0141] After completing the above fluorescence imaging, next, the near-infrared fluorescent probe 69 Ga]DOTA-IR-780-C-4 was applied to surgical navigation applications. 24 h after tail vein injection of 69 Ga]DOTA-IR-780-C-4 (50 μM, 200 μL), fluorescence surgical navigation was performed. From Figure 9 , we can observe that obvious near-infrared fluorescence signals can only be monitored at the tumor site, and the tumor margin is clearly outlined, assisting in quickly cutting the tumor to reduce damage to normal tissues. After completing the surgical navigation experiment, the mice were dissected to detect the fluorescence distribution in the tumors and normal tissues. As Figure 10 shown, this probe is mainly distributed in the tumors and kidneys, and almost no obvious near-infrared fluorescence can be detected in other normal tissues ( Figure 10 a and b). Since the probe has a long circulation time in the blood and there is a certain expression of PSMA in the renal tubules, there is a phenomenon of renal tubular reabsorption. Therefore, its metabolism in the kidneys is slightly slower, but it does not affect the application performance of the probe (as Figure 11 shown).

[0142] 6. 177 Radiosynthesis and in vitro stability study of

[0143] Due to 68 / 69Ga]DOTA-IR-780-C-4 has high uptake and long retention at positive Lncap tumors, which is beneficial for the translational research of radiotherapy probes to study their radiotherapy potential. Therefore, the diagnostic probe 68 Ga]DOTA-IR-780-C-4 was transformed into a radiotherapy probe 177 Lu]DOTA-IR-780-C-4 to study its potential for radiotherapy applications. See Figure 12 a. First, 177 Lu was added to HCl (0.05 M), and sodium acetate buffer (0.25 M) was added to adjust the pH of the reaction solution to 4 - 4.5. The volume ratio of hydrochloric acid to sodium acetate was 1 mL:0.285 mL. Then, the labeling precursor DOTA-IR-780-C-4 (10 μL, 30 μg) was added and mixed evenly. After reacting at 90 °C for 15 min, HPLC analysis was performed, and the mixture was shaken every 5 minutes. Finally, after activating the C-18 column (with ethanol and water), the probe was loaded onto the C-18 column, and the free 177 Lu ions were washed away with water, and then the product was eluted into a bottle with ethanol to obtain the purified product 177 Lu]DOTA-IR-780-C-4. The purity of the drug was analyzed by radioactive HPLC before and after purification, and it was found that the probe had good reactivity, high radiochemical purity and yield ( Figure 12 b). The purified samples were incubated with PBS ( Figure 12 c) and serum ( Figure 12 d) for 1, 3, and 7 days, and then radioactive HPLC was used to detect and analyze the sample purity to evaluate its stability. HPLC showed that the probe had high stability in both PBS and serum, with radiochemical purity > 98%, indicating good stability, which was beneficial for subsequent biological research. The specific result parameters are shown in Table 2 below.

[0144] Table 2 177 Radioactive synthesis and in vitro stability parameters of

[0145]

[0146] 7. SPECT imaging of Lncap tumor-bearing mice at the in vivo level

[0147] See Figure 13 a. Six 5 - 6-week-old male NCG mice were selected and prostate cancer tumor cells were injected subaxillarily on the right side to establish a Lncap tumor-bearing mouse model. SPECT imaging studies could be performed when the tumor diameter was about 0.6 - 0.8 cm. The solution containing 177 Lu]DOTA-IR-780-C-4 or 177Saline solution containing [[Lu]]PSMA-617 (300 μCi) was injected into tumor-bearing mice via the tail vein. Whole-body images were acquired using a SPECT imager at 24 h, 48 h, and 72 h after injection.

[0148] As Figure 13 shown in b and c, after intravenous injection of 177 Lu]DOTA-IR-780-C-4, obvious radioactive signals could be monitored at the site of Lncap tumors with overexpressed PSMA. 177 For [[Lu]]DOTA-IR-780-C-4 at the positive Lncap tumors, the tumor uptake was significantly higher than that of 177 Lu]PSMA-617, and it had long retention at the tumor site and a high signal-to-background ratio.

[0149] After SPECT imaging, the tumors and major tissues and organs of Lncap tumor-bearing mice at 84 h after injection of the probe were removed and detected for their radioactive content using a gamma counter. The uptake of this probe at the tumor site was as high as 93.7% ID / g at 84 h. In addition, as Figure 14 we could monitor from 177 Lu]DOTA-IR-780-C-4 had a higher uptake at the tumor site of Lncap tumor-bearing mice than 68 Ga]PSMA-617 by 21.8 times ( Figure 14 as shown in a), and the signal-to-background ratio was 2.8 times higher ( Figure 14 as shown in b). At the same time, except for the kidneys, the uptake ratios of the probe in the tumors compared with the heart, liver, and muscle were all relatively high, indicating that 177 Lu]DOTA-IR-780-C-4 had the potential for radiotherapy applications.

[0150] Details not described in this invention are all well-known techniques to those skilled in the art.

[0151] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified and equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A dual-targeted multimodal diagnosis and treatment integrated probe, characterized in that, The probe includes the following parts: A labeled tracer part, a target-binding ligand, and a cyanine fluorescent dye part, wherein the labeled tracer part is a group labeled with a radioisotope or a group capable of chelating a radioisotope, the cyanine fluorescent dye is a double-claw structure group having heterocycles at both ends and a bridging chain in the middle, the labeled tracer part is connected to the bridging chain of the cyanine fluorescent dye molecular backbone, and a target-binding ligand is connected to each of the heterocycles at both ends of the cyanine fluorescent dye molecule to form a double-ligand structure.

2. The dual-targeted multimodal diagnosis and treatment integrated probe according to claim 1, wherein The radiolabeled group is selected from 18 F]AlF-labeled RSECA-TFP, 18 F-labeled N-propynyl-N,N-dimethylammonium methyl boron trifluoride, 18 F-labeled OTS group, 18 F-labeled quaternary ammonium salt group, 124 / 125 / 131 I-labeled phenol group, 89 Zr-labeled DFO group, or any one or more thereof.

3. The dual-targeted multi-modal diagnosis and treatment integrated probe according to claim 1, characterized in that, The group capable of chelating a radioisotope is selected from those capable of undergoing 68 / 69 Ga or 177 any one or more of the DOTA, NOTA, NODA, NODAGA, DOTP, TETA, NOTAM, DO3AP chelating groups labeled with Lu.

4. The dual-targeted multimodal diagnosis and treatment integrated probe according to claim 1, wherein The cyanine fluorescent dye is a cyanine fluorescent dye having near-infrared fluorescence.

5. The dual-targeted multimodal diagnosis and treatment integrated probe according to claim 1, wherein The target-binding ligand is any one or more bindable ligands selected from ligands targeting PSMA for prostate cancer, FAPI ligands targeting tumor-targeting fibroblasts FAP, ligands targeting somatostatin receptor SSTR for neuroendocrine tumors, RGD ligands targeting tumor-targeting integrin αvβ3, and ligands targeting GPC-3 for liver cancer or hepatocellular carcinoma.

6. The dual-targeted multimodal diagnosis and treatment integrated probe according to any one of claims 1 to 5, characterized in that, The labeled tracer part is DOTA, the target-binding ligand is a PSMA ligand, and the cyanine fluorescent dye is IR-780.

7. The dual-targeted multi-modal diagnosis and treatment integrated probe according to claim 6, wherein, It has the following molecular structure: 。 8. The preparation method of the dual-targeted multimodal diagnosis and treatment integrated probe according to any one of claims 1 to 7, characterized in that, The method includes: First, reacting the target-binding ligand with the cyanine fluorescent dye and connecting it to the heterocycles at both ends of the dye molecule, and then reacting the labeled tracer part with the cyanine fluorescent dye and connecting it to the bridging chain of the dye molecule, or first reacting the labeled tracer part with the cyanine fluorescent dye and connecting it to the bridging chain of the dye molecule, and then reacting the target-binding ligand with the cyanine fluorescent dye and connecting it to the heterocycles at both ends of the dye molecule, to form a dual-targeted multimodal diagnosis and treatment integrated probe having dual target-binding ligands and simultaneously having radioactive tracer and fluorescence tracer capabilities.

9. The method according to claim 8, characterized in that, The reaction is a click chemical reaction, wherein an alkyne group and an azide group are respectively introduced at the reaction sites of the target-binding ligand and the cyanine fluorescent dye, and after the reaction, an alkyne group and an azide group are respectively introduced at the reaction sites of the labeled tracer part and the cyanine fluorescent dye and then continue to react, or an alkyne group and an azide group are respectively introduced at the reaction sites of the labeled tracer part and the cyanine fluorescent dye, and after the reaction, an alkyne group and an azide group are respectively introduced at the reaction sites of the target-binding ligand and the cyanine fluorescent dye and then continue to react.

10. The method according to claim 9, characterized in that, The cyanine fluorescent dye used is IR-780, the labeled tracer part is DOTA, and the target-binding ligand is PSMA. The specific reaction is as follows: (a) Performing a click chemical reaction on the compound IR-780-Alkyne and the compound N3-PSMA to introduce dual PSMA targeting groups; (b) After step (a) is completed, the product is directly subjected to a substitution reaction without purification to introduce an azide group; (c) The reaction product of step (b) is directly subjected to a click chemical reaction with the compound PG without purification to introduce an alkynylamine short chain to obtain the compound IR-780-C-4; (d) Condensing the compound IR-780-C-4 with DOTA-NHS to introduce a labeling group to obtain the labeled precursor DOTA-IR-780-C-4.

11. The method according to claim 10, wherein The method further includes the step of radioisotope labeling the obtained labeled precursor.

12. Use of the dual-targeted multimodal diagnosis and treatment integrated probe according to any one of claims 1 to 7 or the dual-targeted multimodal diagnosis and treatment integrated probe prepared by the method according to any one of claims 8 to 11 in imaging of target tissues.

13. The use according to claim 12, wherein the tissue imaging method is PET or SPECT.

14. The use according to claim 12 or 13, wherein the target tissue is tumor tissue.

15. Use of the dual-targeted multimodal diagnosis and treatment integrated probe according to any one of claims 1 to 7 or the dual-targeted multimodal diagnosis and treatment integrated probe prepared by the method according to any one of claims 8 to 11 in guiding fluorescence surgical navigation.

Images