Targeted compound bimolecular probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of nuclear medicine, and discloses a targeted compound bimolecular probe which comprises a component A and a component B which are labeled by radioactive metal nuclides respectively, the component A is a targeted fibroblast activation protein (FAP) molecular probe, and the targeted fibroblast activation protein (FAP) molecular probe comprises a trans-cyclooctene group (TCO); the component B is a targeted integrin alpha v beta 6 molecular probe, and the targeted integrin alpha v beta 6 molecular probe comprises a tetrazine group (Tz); wherein the radioactive activity ratio of the component A to the component B is (1-2): (2-1). The invention has the following technical effects: TCO in the targeted FAP molecular probe and Tz in the targeted integrin alpha v beta 6 molecular probe can be subjected to a biological orthogonal reaction to realize a three-stage amplification effect, increase the tumor uptake value, prolong the residence time of the targeted compound bimolecular probe, increase the target / non-target ratio and enhance the nuclear medicine diagnosis and treatment effects. The invention also discloses a preparation method and application of the targeted compound bimolecular probe.

Description

Technical Field

[0001] The invention belongs to the technical field of nuclear medicine, and specifically relates to a targeted compound bimolecular probe and a preparation method and application thereof. Background Art

[0002] The occurrence and development of tumors such as hepatobiliary system tumors, gastric cancer, and lung cancer increasingly threaten human life and have become a serious social problem. In my country, tumors have ranked second among various causes of death, and the tumor mortality rate has shown a clear upward trend. The key to reducing tumor mortality is to study early diagnosis, specific targeted therapy, and the best individualized precision diagnosis and treatment technology to guide treatment. Integrated nuclear medicine diagnosis and treatment includes positron emission tomography (PET) imaging, single photon emission computed tomography (SPECT), and radionuclide therapy. Integrated nuclear medicine diagnosis and treatment plays a unique role in tumor diagnosis and treatment, and can accurately solve the problem of tumor visualization diagnosis and treatment. Radioactive targeted molecular probes, as important markers in integrated nuclear medicine diagnosis and treatment, have become the key to improving the effectiveness of nuclear medicine diagnosis and treatment.

[0003] The radioactive targeted molecular probes currently used are mostly single-target ones. The diagnostic effect of single-target radioactive monomer molecular probes on tumors is not ideal. Their imaging is prone to false negative results and low sensitivity. In addition, positive lesions still have the disadvantages of low molecular probe uptake values ​​and insufficient retention time of molecular probes in lesions.

[0004] Bioorthogonal chemical reactions are widely used in in vivo reactions due to their high specificity, high reaction rate and good biocompatibility. The prior art discloses a method for preparing a TGFβ antibody probe by bioorthogonal reaction-click chemistry technology, wherein TCO-PEG4-NHS is modified on the TGFβ antibody to obtain TGFβ antibody-TCO; NOTA is modified on the tetrazine small molecule Tz to obtain NOTA-Tz; 18 F、AlCl 3 Reaction with NOTA-Tz to obtain [ 18 F-AlF 3 ]-NOTA-Tz; TGFβ antibody-TCO and [ 18 F-AlF 3 ]-NOTA-Tz ligands were co-incubated to generate [ 18 F-AlF 3 ]-NOTA-Tz-TCO-TG Fβ antibody probe. The antibody probe prepared by this method can be used to detect the expression level of TGFβ in tumors and other tissues, but the antibody probe is still a monomer probe with a single target, and its imaging cannot obtain more accurate and comprehensive tumor biological information, and the diagnosis and treatment effects of tumors are still not ideal. Summary of the invention

[0005] In order to solve the shortcomings of the prior art, the present invention provides a targeted composite bimolecular probe, which targets FAP molecular probes and targets integrin α v β 6 The combination of molecular probes can simultaneously image or treat two target sites, improving the diagnosis and treatment of tumors. At the same time, the TCO in the FAP molecular probe and the integrin α v β 6 The Tz in the molecular probe can undergo a bioorthogonal reaction to achieve a three-stage amplification effect. The present invention also provides a preparation method and application of a targeted composite bimolecular probe.

[0006] The technical effects to be achieved by the present invention are achieved through the following technical aspects:

[0007] In a first aspect, the present invention provides a targeted composite bimolecular probe, comprising a component A and a component B respectively labeled with a radioactive metal nuclide;

[0008] The A component is a molecular probe targeting fibroblast activation protein (FAP), and the molecular probe targeting fibroblast activation protein (FAP) includes a trans-cyclooctene group (TCO);

[0009] The B component targets integrin α v β 6 Molecular probes targeting integrin alpha v β 6 The molecular probe includes a tetrazine group (Tz);

[0010] The radioactivity ratio of the component A to the component B is (1-2):(2-1), preferably 1:1.

[0011] It should be noted that the A component is not limited to targeting FAP molecular probes, but can also be other targeting molecular probes containing TCO; the B component is not limited to targeting integrin α v β 6 The molecular probe may also be other targeted molecular probes containing Tz.

[0012] As a further description of the technical solution of the present invention, the FAP-targeting molecular probe further comprises a FAP inhibitor (FAPI) pharmacophore, a hydrophilic linking group, a metal ion chelating group and a radioactive metal nuclide ( * M), referred to as * M-TCO-FAPI;

[0013] The targeting integrin α v β 6 Molecular probes also include integrin α vβ 6 Receptor ligand (α v β 6 L) pharmacophore, hydrophilic linking group, metal ion chelating group and radioactive metal nuclide ( * M), referred to as * M-Tz-α v β 6 L.

[0014] As a further description of the technical solution of the present invention, the hydrophilic linking group includes a polyethylene glycol group (PEG2) and / or a polyethylene glycol group (PEG3), two aspartic acid groups (Asp2) and a lysine group (Lys).

[0015] As a further description of the technical solution of the present invention, the metal ion chelating group is 1,4,7-triazacyclononacanyl-N',N"-diacetyl-N-acetyl (NOTA) or 1,4,7,10-tetraazacyclododecane-N',N",N"'-triacetyl-N-acetyl (DOTA).

[0016] As a further description of the technical solution of the present invention, the radioactive metal nuclide ( * M) is 18 F. 68 Ga, 111 In, 99m Tc, 186 Re, 188 Re, 175 Yb, 153 Sm, 166 Ho, 88 Y. 90 Y. 177 Lu, 47 Sc, 212 Bi, 213 Bi, 123 I. 124 I. 131 I. 211 At 153 Eu, 169 Eu, 212 Pb, 64 Cu, 67 Cu, 188 Re, 186 Re, 198 Au, 225 Ac or 227 One of Th, preferably 68 Ga, 18 F. 64 Cu or 177More preferably, NOTA and [Al 18 F] 2+ , 68 Ga 3+ , 64 Cu 2+ or 177 Lu 3+ Combining, DOTA and 68 Ga 3+ , 64 Cu 2+ or 177 Lu 3+ Combine.

[0017] As a further description of the technical solution of the present invention, the targeted composite bimolecular probe is 18 F-labeled molecular probe targeting FAP ( 18 F-TCO-FAPI) and 18 F-labeled targeted integrin α v β 6 Molecular probes ( 18 F-Tz-α v β 6 L) mixture, referred to as 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 L (radioactivity ratio is 1:1). 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 The chemical structure of L is:

[0018]

[0019] It should be noted that the above-mentioned FAPI, TCO, hydrophilic linking group, metal ion chelating group and radioactive metal nuclide ( * M) can be freely combined to synthesize different targeted FAP molecular probes according to different types; the above-mentioned α v β 6 L, Tz, hydrophilic linking groups, metal ion chelating groups and radioactive metal nuclides ( * M) can freely combine and synthesize different targeting integrins according to different types v β 6 Molecular probes.

[0020] In a second aspect, the present invention provides a method for preparing a targeted composite bimolecular probe, which is used to prepare the above-mentioned targeted composite bimolecular probe, comprising the following steps:

[0021] Preparation of component A: Using NOTA-TCO-FAPI or DOTA-TCO-FAPI as the precursor material, chelating with radioactive metal nuclide ions, and preparing the targeted FAP molecular probe after separation and purification by a small column * M-TCO-FAPI;

[0022] Preparation of component B: Using NOTA-Tz-α v β 6 L or DOTA-Tz-α v β 6 L is a precursor material, which undergoes a chelation reaction with radioactive metal nuclide ions, and is separated and purified by a small column to prepare the targeted integrin α v β 6 Molecular probes * M-Tz-α v β 6 L;

[0023] Preparation of the targeted compound bimolecular probe: Component A and component B are mixed in a radioactivity ratio of (1-2):(2-1) to obtain the targeted compound bimolecular probe.

[0024] It should be noted that the targeted compound bimolecular probe contains * M-TCO-FAPI and * M-Tz-α v β 6 In addition to L, it may also contain two precursor raw materials NOTA-TCO-FAPI (or DOTA-TCO-FAPI) and NOTA-Tz-α v β 6 L(or DOTA-Tz-α v β 6 L), two targeted monomer molecular probes * M-TCO-FAPI and * M-Tz-α v β 6 L, and molecular probes combining Tz and TCO * M-α v β 6 L-Tz-TCO-FAPI, * M-FAPI-TCO-Tz-α v β 6 L and * M-FAPI-TCO-Tz-α v β 6 L- * M.

[0025] The precursor material is prepared by a solid phase peptide synthesis method. Specifically, the following steps are included: FAPI pharmacophore modification-PEG2 forms FAPI pharmacophore-PEG2, FAPI pharmacophore-PEG2 modifies Asp2, then modifies TCO through Lys-linked-PEG3, and finally modifies the chelating group-NOTA or -DOTA, and separates and purifies the product peaks by preparative HPLC to obtain the purified precursor material NOT A-TCO-FAPI or DOTA-TCO-FAPI; α v β 6 The L pharmacophore is modified with Lys and then connected to the Tz group, which is further modified with -PEG2 to form α v β 6 L-PEG2, α v β 6 After Asp2 is modified with L pharmacophore -PEG2, a chelating group -NOTA or -DOTA is connected. The product peak is collected by preparative HPLC separation and purification to obtain the purified precursor material NOTA-Tz-α v β 6 L or DOTA-Tz-α v β 6 L.

[0026] The chemical structure of NOTA-TCO-FAPI is:

[0027]

[0028] The chemical structure of DOTA-TCO-FAPI is:

[0029]

[0030] NOTA-Tz-α v β 6 The chemical structure of L is:

[0031]

[0032] DOTA-Tz-α v β 6 The chemical structure of L is:

[0033]

[0034] As a further description of the technical solution of the present invention, the radioactive metal nuclide ion is 68 Ga 3+ 、[Al 18 F] 2+ , 64 Cu 2+ or 177 Lu3+ One of them.

[0035] Preferably, the method for preparing the targeted composite bimolecular probe comprises the following steps:

[0036] (one) * Preparation of M-TCO-FAPI

[0037] 18 Preparation of F-TCO-FAPI: Using NOTA-TCO-FAPI as the precursor, NOTA-TCO-FAPI was prepared in the presence of AlCl 3 and acetonitrile in an acidic solution 18 F - The reaction was heated at 90-100°C for 10-20 min and then cooled. The product was separated and purified by SEP-PAK C18 column to obtain 18 F-TCO-FAPI.

[0038] 68 Preparation of Ga-TCO-FAPI: Using NOTA-TCO-FAPI or DOTA-TCO-FAPI as a precursor, NOTA-TCO-FAPI or DOTA-TCO-FAPI and weakly acidic 68 Ga 3+ The solution was heated at 85-110°C for reaction, and separated and purified using an HLB column or a SEP-PAKC18 column to obtain 68 Ga-TCO-FAPI.

[0039] 64 Preparation of Cu-TCO-FAPI: Using NOTA-TCO-FAPI or DOTA-TCO-FAPI as precursor, NOTA-TCO-FAPI or DOTA-TCO-FAPI and 64 Cu 2+ The reaction was incubated in sodium acetate acidic buffer solution at room temperature for 15 min, and separated and purified by HLB column or SEP-PAK C18 column to obtain 64 Cu-TCO-FAPI.

[0040] 177 Preparation of Lu-TCO-FAPI: Using NOTA-TCO-FAPI or DOTA-TCO-FAPI as precursor, NOTA-TCO-FAPI or DOTA-TCO-FAPI and 177 The Lu solution was heated in a mixture of gentisic acid and sodium acetate at 106°C in a water bath for 15 min. After HPLC separation and purification by SEP-PAK C18 column, the 177 Lu-TCO-FAPI.

[0041] (two) * M-Tz-α v β 6 Preparation of L

[0042] 18 F-Tz-α v β 6 Preparation of L: Using NOTA-Tz-α v β 6 L is the precursor material, refer to 18 The preparation method of F-TCO-FAPI is used to prepare the 18 F-Tz-α v β 6 L.

[0043] 68 Ga-Tz-α v β 6 Preparation of L: Using NOTA-Tz-α v β 6 L or DOTA-Tz-α v β 6 L is the precursor material, refer to 68 The preparation method of Ga-TCO-FAPI is used to prepare the 68 Ga-Tz-α v β 6 L.

[0044] 64 Cu-Tz-α v β 6 Preparation of L: Using NOTA-Tz-α v β 6 L or DOTA-Tz-α v β 6 L is the precursor material, refer to 64 The preparation method of Cu-TCO-FAPI is used to prepare the 64 Cu-Tz-α v β 6 L.

[0045] 177 Lu-Tz-α v β 6 Preparation of L: Using NOTA-Tz-α v β 6 L or DOTA-Tz-α v β 6 L is the precursor material, refer to 177 The preparation method of Lu-TCO-FAPI is used to prepare the 177 Lu-Tz-α v β6 L.

[0046] (III) Preparation of targeted compound bimolecular probe: any of the above * M-TCO-FAPI and any of the above * M-Tz-α v β 6 L are mixed at a radioactivity ratio of 1:1 to obtain a targeted composite bimolecular probe.

[0047] In a third aspect, the present invention provides the use of the targeted composite bimolecular probe in the preparation of tumor diagnostic and therapeutic agents, including the use of the targeted composite bimolecular probe in the preparation of multiple tumors with high expression of FAP and (or) expressing α v β 6 Application in various tumor diagnostic and therapeutic agents.

[0048] As a further description of the technical solution of the present invention, the application of the targeted compound bimolecular probe in the preparation of a PET imaging agent for a tumor, a pre-positioned PET imaging agent, and a compound bio-orthogonal PET imaging agent, the application of the targeted compound bimolecular probe in the preparation of a PET imaging agent for a tumor includes the application of the targeted compound bimolecular probe in the preparation of a variety of tumors such as lung adenocarcinoma and brain glioma with high expression of FAP and (or) high expression of α v β 6 Application in PET imaging agents for pancreatic cancer, lung cancer, cholangiocarcinoma, gastric cancer, breast cancer or ovarian cancer;

[0049] Application of the targeted composite bimolecular probe in the preparation of tumor SPECT imaging agents, pre-positioned SPECT imaging agents and composite bio-orthogonal SPECT imaging agents;

[0050] The targeted composite bimolecular probe is used in the preparation of targeted radiotherapeutic agents, dual-targeted radiotherapeutic agents, pre-targeted radiotherapeutic agents, and composite bio-orthogonal dual-targeted radiotherapeutic agents.

[0051] The application of targeted compound bimolecular probes in the diagnosis and treatment of tumors can establish a compound bio-orthogonal amplification diagnosis and treatment system. The compound bio-orthogonal amplification diagnosis and treatment system has the characteristics of compound drugs, multi-targeting and bio-orthogonal reactions, and has obvious advantages in cost, sensitivity, imaging efficiency, etc. It can achieve simultaneous imaging of multiple tumor diseases in one imaging, and has high clinical value.

[0052] In summary, the present invention has at least the following benefits:

[0053] 1. The targeted composite bimolecular probe provided by the present invention can simultaneously image or treat two target points with only one administration, thereby increasing the total number of binding sites with the target molecule, displaying more tumor lesions, and improving the detection sensitivity and treatment effect of tumors; at the same time, the TCO in the FAP molecular probe and the targeting integrin α v β 6 Tz in the molecular probe can undergo bioorthogonal reaction, achieve a tertiary amplification effect, increase tumor uptake value, prolong the retention time of the targeted composite bimolecular probe, improve the target / non-target uptake ratio, and enhance the nuclear medicine diagnosis and treatment effect.

[0054] 2. The targeted composite bimolecular probe provided by the present invention, the selected targeted FAP molecular probe and the targeted integrin α v β 6 Molecular probes are optimized through chemical structure, increased hydrophilicity, reduced uptake by the hepatobiliary system and intestinal system, and promoted renal excretion, thus improving the in vivo pharmacokinetic properties of molecular probes. 18 F-TCO-FAPI has high affinity for tumor FAP targets, can be competitively inhibited by inhibitors, and also exhibits excellent in vivo pharmacokinetic properties. 18 F-TCO-FAPI has a high uptake in tumors and a long retention time, and still has a high uptake at 6 hours.

[0055] 3. The preparation method of the targeted composite bimolecular probe provided by the present invention is to prepare the FAP targeting molecular probe and the integrin α targeting molecular probe by a chelating reaction between the precursor raw material and the radioactive metal nuclide ion and then separating and purifying the precursor raw material and then preparing the FAP targeting molecular probe and the integrin α targeting molecular probe. v β 6 Molecular probes are then mixed according to the optimal radioactivity ratio to form a targeted compound bimolecular probe. The synthesis of molecular probes with high radiochemical yield and high radiochemical purity can be achieved, and the preparation steps are simple and the operation is convenient.

[0056] 4. The application of the targeted compound bimolecular probe provided by the present invention is to apply the targeted compound bimolecular probe to tumor diagnostic and therapeutic agents, providing a new tumor diagnosis and treatment method that acts on "dual targets" with one administration. It is conducive to promoting accurate nuclear medicine diagnosis and efficacy evaluation of tumors, solving the problem of accurate visualization diagnosis and treatment and evaluation, and has high clinical value.

[0057] 5. The greatest advantage of the present invention relates to the three-stage amplification effect of the compound bio-orthogonal diagnostic and therapeutic system, including a first-stage compound amplification system, a second-stage bio-orthogonal probe amplification system and a third-stage dual bio-orthogonal reaction amplification system. There is also a pre-targeted diagnostic and therapeutic amplification system, and an in vivo and in vitro bioactivity evaluation and verification method has been established. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Schematic diagram of the imaging mode of the targeted composite bimolecular probe;

[0059] Figure 2 It is a schematic diagram of the three-stage amplification effect mode of the dual-targeted compound bio-orthogonal diagnostic and therapeutic system;

[0060] Figure 3a This is the HPLC analysis spectrum of the precursor raw material NOTA-TCO-FAPI;

[0061] Figure 3b For the precursor material NOTA-Tz-α v β 6 HPLC analysis spectrum of L;

[0062] Figure 3c This is the MS analysis spectrum of the precursor material NOTA-TCO-FAPI;

[0063] Figure 3d For the precursor material NOTA-Tz-α v β 6 MS analysis spectrum of L;

[0064] Figure 4a For injection 18 Representative radioactive HPLC analysis spectrum of F-TCO-FAPI at radioactive retention time Rt = 11.23min;

[0065] Figure 4b For injection 18 Representative UV absorption HPLC analysis spectrum of F-TCO-FAPI at UV absorption retention time Rt = 10.92min;

[0066] Figure 4c For injection 18 F-Tz-α v β 6 Representative radioactive HPLC analysis spectrum of L at radioactive retention time Rt = 10.65 min;

[0067] Figure 4d For injection 18 F-Tz-α v β 6 Representative UV absorption HPLC analysis spectrum of L at UV absorption retention time Rt = 10.59 min;

[0068] Figure 5a For injection 18 Representative radioactive HPLC analysis of F-TCO-FAPI in serum of Kunming mice for 1 h;

[0069] Figure 5b For injection18 Representative 1-hour radioactive HPLC analysis of F-TCO-FAPI in Kunming rat urine;

[0070] Figure 5c For injection 18 Representative radio-HPLC analysis profile of F-TCO-FAPI in PBS for 2 h in vitro;

[0071] Figure 5d For injection 18 Representative radio-HPLC analysis profile of F-TCO-FAPI in fetal bovine serum for 2 h in vitro;

[0072] Figure 6a For injection 18 F-Tz-α v β 6 Representative radioactive HPLC analysis of L in serum of Kunming mice for 1 h;

[0073] Figure 6b For injection 18 F-Tz-α v β 6 Representative radioactive HPLC analysis of L in urine of Kunming mice for 1 h;

[0074] Figure 6c For injection 18 F-Tz-α v β 6 Representative radio-HPLC analysis profile of L in PBS for 2 h in vitro;

[0075] Figure 6d For injection 18 F-Tz-α v β 6 Representative radioactive HPLC analysis profile of L in fetal bovine serum for 2 h in vitro;

[0076] Figure 7a for 18 Figure 3. Cellular uptake and inhibition experimental results of F-TCO-FAPI;

[0077] Figure 7b for 18 Results of the tumor cell competitive inhibition experiment (IC50 value) of F-TCO-FAPI;

[0078] Figure 8 It is the in vitro bioorthogonal product FAPI-TCO-Tz-α v β 6 Mass spectrometry detection diagram;

[0079] Figure 9a for 18Biodistribution diagram of F-TCO-FAPI in tumor-bearing mice (A549-FAP, A549) for 60 min and biodistribution diagram of competitive inhibition in tumor-bearing mice A549-FAP for 60 min;

[0080] Figure 9b for 18 F-Tz-α v β 6 Biodistribution diagram of L in tumor-bearing mice (Capan-2) for 60 min;

[0081] Fig.9c Targeted compound bimolecular probe 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 L (radioactivity ratio is 1:1) and 18 F-TCO-FAPI and 18 F-Tz-α v β 6 Comparative diagram of in vivo biodistribution of L.

[0082] Fig.10a for 18 PET / CT images of static uptake (left) and competitive inhibition (right) of F-TCO-FAPI in A549-FAP lung adenocarcinoma-bearing mouse model at 60 minutes;

[0083] Fig.10b for 18 PET / CT images of static uptake (left) and competitive inhibition (right) of F-TCO-FAPI in the U87 glioma-bearing mouse model at 60 minutes;

[0084] Fig.11a The bar graphs are for 18F-Tz-αvβ6L at 2h, 4h, and 6h in the Capan-2 tumor-bearing mouse model;

[0085] Fig.11b The bar graphs are for 18F-Tz-αvβ6L at 2h, 4h, and 6h in the BxPC-3 tumor-bearing mouse model;

[0086] Fig.11c Targeted compound bimolecular probe 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 L (radioactivity ratio is 1:1) and 18 F-TCO-FAPI and 18 F-Tz-α v β 6PET / CT images of L at 30min, 60min, 2h, 4h, and 6h. DETAILED DESCRIPTION

[0087] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below in conjunction with specific embodiments and accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0089] As a preferred embodiment, the targeted compound bimolecular probe is a targeted FAP molecular probe. 18 F-TCO-FAPI and targeting integrin α v β 6 Molecular probes 18 F-Tz-α v β 6 L mixture, the two are mixed in a 1:1 radioactivity ratio, referred to as 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 L.

[0090] The present invention targets TCO in FAP molecular probe and targets integrin α v β 6 The Tz in the molecular probe undergoes a bioorthogonal reaction in vivo, achieving the tertiary amplification effect of the dual-targeted compound bioorthogonal diagnostic and therapeutic system, increasing the tumor uptake value and prolonging the retention time of the targeted compound bimolecular probe. The bioorthogonal chemical reaction based on IEDDA between the compound bioorthogonal bimolecular probes is as follows:

[0091]

[0092] Among them, R is R' is

[0093] Schematic diagram of the imaging mode of the targeted composite bimolecular probe Figure 1 As shown, the schematic diagram of the three-stage amplification effect mode of the dual-targeted compound bio-orthogonal diagnostic and therapeutic system is as follows Figure 2 shown.

[0094] The three-stage amplification effect of the dual-targeted compound bio-orthogonal diagnostic and therapeutic system includes a first-stage compound dual-molecular probe amplification system, a second-stage bio-orthogonal probe amplification system and a third-stage dual-bio-orthogonal reaction amplification system. In addition, there is a pre-targeted diagnostic and therapeutic amplification system, and an in vivo and in vitro biological activity evaluation and verification method can be established.

[0095] The first-stage compound bimolecular probe amplification system is specifically a targeted compound bimolecular probe * M-TCO-FAPI+ * M-Tz-α v β 6 L binds to the tumor targets FAP and integrin α v β 6 The combined system was verified by the following experiments. * M-TCO-FAPI specifically targets FAP: * M-TCO-FAPI was taken up and inhibited PET imaging in FAP-positive models (U87 brain glioma, A549-FAP lung adenocarcinoma cells) (the inhibitor was NOTA-FAPI-42). The following experiments were performed to verify * M-Tz-α v β 6 L-specific targeting of integrin α v β 6 : * M-Tz-α v β 6 L in α v β 6 Uptake and inhibition of PET imaging in a positive model (Capan-2 human pancreatic cancer) was performed. * M-TCO-FAPI+ * M-Tz-α v β 6 L targets two targets respectively.

[0096] Secondary bioorthogonal probe amplification system specifically targeted compound bimolecular probe * M-TCO-FAPI+ * M-Tz-α v β 6 After TCO and Tz combine in L, they form * M-FAPI-TCO-Tz-α v β 6 L- * M, * M-FAPI-TCO-Tz-α v β 6 L- * M binds to the tumor targets FAP and integrin α vβ 6 The combined system was verified by the following experiments: the molecular probe targeting FAP 18 F-TCO-FAPI and targeting integrin α v β 6 Molecular probes 18 F-Tz-α v β 6 L was mixed with a 1:1 radioactivity ratio to prepare a targeted composite bimolecular probe 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 L, and the HPLC analysis confirmed the production of 18 F-FAPI-TCO-Tz-α v β 6 L- 18 F. Validated by molecular docking: NOTA-TCO-FAPI, NOTA-Tz-α v β 6 L and NOTA-FAPI-TCO-Tz-α v β 6 L-NOTA and FAP, α v β 6 Target binding. Validated by tumor model imaging experiments: in FAP and α v β 6 In the dual-target positive model (Capan-2 human pancreatic cancer), first inject 18 F-TCO-FAPI, after a period of time 18 F-TCO-FAPI binds to the tumor and is then injected 18 F-Tz-α v β 6 L after, the tumor radioactive uptake value increased; the next day, the first injection 18 F-TCO-FAPI, after a period of time, then inject NOTA-Asp2-α v β 6 L+ 18 F-Tz-α v β 6 L, tumor uptake was partially inhibited, indicating inhibition 18 F-Tz-α v β 6 L in α v β 6 L and target α v β 6 Effect: First injection on the third day 18 F-TCO-FAPI, after a period of time, then inject NOTA-Tz+ 18 F-Tz-αv β 6 L, tumor uptake was partially inhibited, indicating inhibition 18 F-Tz-α v β 6 Tz in L 18 TCO in F-TCO-FAPI undergoes bioorthogonal binding.

[0097] The three-stage dual bioorthogonal reaction amplification system is specifically a targeted compound bimolecular probe * M-TCO-FAPI+ * M-Tz-α v β 6 L binds to the tumor targets FAP and integrin α v β 6 After combination, after combination with tumor * TCO and free * M-Tz-α v β 6 Tz binding in L, and after binding to tumor * M-Tz-α v β 6 Tz and free * The TCO binding in M-TCO-FAPI is a TCO-Tz dual binding system. The following experiments verified that the tumor-binding targeted single-molecule probe can be bio-orthogonally bound to another free single-molecule probe in vivo: pre-injection of FAP single positive model (U87 brain glioma) * M-TCO-FAPI, after a period of time * M-TCO-FAPI binds to the tumor and is then injected * M-Tz-α v β 6 L after, the tumor radioactive uptake value increased, and the next day pre-injection * M-TCO-FAPI, Tz-α injection after the same time v β 6 L+ * M-Tz-α v β 6 L, tumor radioactivity uptake value decreased, proving * TCO and free CO in M-TCO-FAPI * M-Tz-α v β 6 Tz binds in L; in α v β 6 L single positive model (A549 brain glioma) pre-injected with *M-Tz-α v β 6 L, after a period of time *M-Tz-αv β 6 L binds to the tumor and then injects * After M-TCO-FAPI, the tumor radioactivity uptake value increased, and the next day pre-injection * M-Tz-α v β 6 L, after a period of time, inject TCO-FAPI+ * M-TCO-FAPI, tumor radioactivity uptake value decreased, proving * M-Tz-α v β 6 Tz in L and free * TCO combination in M-TCO-FAPI.

[0098] In addition, the pre-positioned diagnosis and treatment amplification system needs to simultaneously administer a certain amount of unlabeled NOTA-TCO-FAPI+NOTA-Tz-α v β 6 L and marked * M-TCO-FAPI+ * M-Tz-α v β 6 L, its mechanism of action involves not only the above-mentioned three-level diagnosis and treatment amplification effect, but also the pre-positioning diagnosis and treatment amplification effect. The following experiments verify that the pre-positioning imaging method can increase tumor uptake and reduce normal tissue uptake, proving that the pre-positioning diagnosis and treatment system has amplification potential: * M-Tz-α v β 6 L performed PET imaging in a FAP single positive model (U87 brain glioma), and the tumor did not take up or took up very little. The next day, the precursor TCO-FAPI was injected in advance. After a period of time, TCO-FAPI combined with the tumor and then injected * M-Tz-α v β 6 PET imaging was performed after L, and tumor uptake increased significantly; the next day, the precursor TCO-FAPI was injected in advance, and Tz-α was injected after the same time. v β 6 L+ * M-Tz-α v β 6 L, decreased tumor radioactivity uptake value. * M-TCO-FAPI in α v β 6 PET imaging was performed in the L single positive model (A549 brain glioma), and the tumor did not take up or took up very little. The next day, the precursor Tz-α was injected in advance. v β 6 L, after a period of time Tz-α v β 6L binds to the tumor and then injects * The radioactive uptake value of the tumor increased with M-TCO-FAPI; the next day, TCO-FAPI+ was injected * M-TCO-FAPI, tumor radioactivity uptake value decreased.

[0099] The targeted composite bimolecular probe of the present invention and its preparation method and application are described in detail below in conjunction with specific embodiments.

[0100] Example 1 Preparation of Precursor Raw Materials NOTA-TCO-FAPI and DOTA-TCO-FAPI

[0101] The FAP pharmacophore-PEG2 is modified with the FAP pharmacophore-PEG2, and the FAP pharmacophore-PEG2 is modified with the Asp2, and then connected with TCO via the Lys and -PEG3 linkers, and finally modified with the chelating group -NOTA. The product peaks are collected and separated and purified by preparative HPLC, and then freeze-dried to obtain the purified precursor raw material NOTA-TCO-FAPI.

[0102] NOTA-TCO-FAPI has a high chemical yield and a purity greater than 95%. The HPLC and MS assay results of NOTA-TCO-FAPI are shown in Figure 2. Figure 3a and Figure 3c As shown, the molecular weight (Mr.) of NOTA-TCO-FAPI determined by mass spectrometry MS (m / z) is 1630.76.

[0103] The preparation method of DOTA-TCO-FAPI can refer to NOTA-TCO-FAPI, which is not described here in detail. DOTA-TCO-FAPI has a high chemical yield and a purity greater than 95% as determined by HPLC and MS.

[0104] Example 2 Precursor material NOTA-Tz-α v β 6 L and DOTA-Tz-α v β 6 Preparation of L

[0105] Targeting α v β 6 α in the ligand v β 6 The L pharmacophore is modified with Lys and then connected to the Tz group, which is further modified with -PEG2 to form α v β 6 L-PEG2, α v β 6The L pharmacophore-PEG2 modifies Asp2 and then connects the chelating group-NOTA. The product peak is separated and purified by preparative HPLC, and then freeze-dried to obtain the purified precursor raw material NOTA-Tz-α v β 6 L.

[0106] NOTA-Tz-α v β 6 L chemical yield is high, purity is greater than 95%. NOTA-Tz-α v β 6 The HPLC and MS results of L were as follows: Figure 3b and Figure 3d As shown, mass spectrometry MS (m / z) was used to determine NOTA-Tz-α v β 6 The molecular weight (Mr.) of L is 2150.34.

[0107] DOTA-Tz-α v β 6 The preparation method of L can refer to NOTA-Tz-α v β 6 L, I will not go into details here, DOTA-Tz-α v β 6 The chemical yield of L was high and the purity was greater than 95% as determined by HPLC and MS.

[0108] Example 3 18 F-TCO-FAPI and 18 F-Tz-α v β 6 Preparation of L

[0109] In the case of NOTA-TCO-FAPI (or NOTA-Tz-α v β 6 L) (50 μg / μL, 50 μL) reaction bottle, and then add 2 mM AlCl 3 6 μL of solution, 5 μL of glacial acetic acid and 300 μL of acetonitrile were mixed.

[0110] Through the cyclotron 18 O(p,n) 18 F produced by nuclear reaction 18 F - , trapped in Sep-PakQMA anion column under N2 carrier, 18 O - The water is collected in the recovery bottle. Use 0.3-0.4 mL of saline (or sodium acetate buffer) to 18 F -Elute into a vial, take 50 μL of it and add it to the above reaction bottle.

[0111] After stirring and mixing, heat the reaction at 100℃ for about 15 minutes. Cool, add 6-8mL of water to the reaction bottle, mix well, and transfer to the HLB column or SEP-PAK C18 column. After all the solutions in the reaction bottle are transferred, rinse the column with 10mL×3 injection water and blow dry the column.

[0112] Finally, the product was eluted with 1.5 mL of ethanol and collected in a receiving bottle after passing through a sterile filter membrane, and diluted with physiological saline to a product solution containing 5% ethanol to obtain 18 F-TCO-FAPI (or 18 F-Tz-α v β 6 L) Injection.

[0113] 18 F-TCO-FAPI and 18 F-Tz-α v β 6 The uncorrected radiochemical yields of L were all 20-30%, and the total radiosynthesis time was all 35 min.

[0114] Example 4 68 Ga-TCO-FAPI and 68 Ga-Tz-α v β 6 Preparation of L

[0115] In the case of NOTA-TCO-FAPI (or DOTA-TCO-FAPI, or NOTA-Tz-α v β 6 L, or DOTA-Tz-α v β 6 L) (50 μg / μL, 50 μL) reaction bottle, and add 200 μL of 1.25M sodium acetate solution in sequence.

[0116] from 68 Ge / 68 Elute with 4 mL of 0.05 M hydrochloric acid in the Ga generator 68 GaCl 3 Add to the above reaction tube, mix well, adjust the solution pH to 4.0, and heat at 100℃ for about 10-15 minutes. Cool, add 4mL of physiological saline to the reaction bottle, mix well, and transfer to the HLB column or SEP-PAK C18 column. After all the solutions in the reaction bottle are transferred, rinse the column with 10mL×2 injection water and blow dry the column.

[0117] The product was eluted with 1.5 mL of ethanol and collected in a receiving bottle after passing through a sterile filter membrane, and diluted with physiological saline to a product solution containing 5% ethanol to obtain 68 Ga-TCO-FAPI (or 68 Ga-Tz-α v β 6 L) Injection.

[0118] 68 Ga-TCO-FAPI and 68 Ga-Tz-α v β 6 The uncorrected radiochemical yields of L were all 20-50%, and the total radiosynthesis time was all 30 min.

[0119] Example 5 64 Cu-TCO-FAPI and 64 Cu-Tz-α v β 6 Preparation of L

[0120] In the case of NOTA-TCO-FAPI (or DOTA-TCO-FAPI, or NOTA-Tz-α v β 6 L, or DOTA-Tz-α v β 6 L) (50 μg / μL, 100 μL) was added to the reaction bottle 64 CuCl 2 0.100-1.000mL of solution was adjusted to pH 4.0-5.6 with sodium acetate solution and reacted at room temperature for 10-15 minutes. Finally, it was diluted with physiological saline and filtered through a sterile filter membrane and collected in a receiving bottle to obtain 64 Cu-TCO-FAPI (or 64 Cu-Tz-α v β 6 L) Injection. 64 Cu-TCO-FAPI and 64 Cu-Tz-α v β 6 The uncorrected radiochemical yields of L were all 50-70%.

[0121] Example 6 177 Lu-TCO-FAPI and 177 Lu-Tz-α v β 6 Preparation of L

[0122] In the case of NOTA-TCO-FAPI (or DOTA-TCO-FAPI, or NOTA-Tz-α v β 6L, or DOTA-Tz-α v β 6 L) (50 μg / μL, 50 μL) was added to the reaction bottle, followed by gentisic acid, 160 μl of sodium acetate and free 177 Lu solution 0.100-1.000mL, heated in a water bath at 106℃ for 15min. Monitor the reaction effect with HPLC and separate by HPLC. After the separated product is passed through a C18 column, rinse with 30ml of water and pass through the column with 1mL of 50% ethanol. The product is collected. Finally, it is diluted with physiological saline and filtered through a sterile filter membrane and collected into a receiving bottle to obtain 177 Lu-TCO-FAPI (or 177 Lu-Tz-α v β 6 L) Injection. 177 Lu-TCO-FAPI and 177 Lu-Tz-α v β 6 The uncorrected radiochemical yields of L were all between 40% and 60%.

[0123] Example 7 Targeted compound bimolecular probe 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 Preparation of L

[0124] Will 18 F-TCO-FAPI injection and 18 F-Tz-α v β 6 L injection was mixed with the radioactivity ratio of 1:1 to prepare the targeted compound bimolecular probe 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 L injection.

[0125] Example 8 Targeted Compound Bimolecular Probe 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 Determination of radiochemical purity and stability

[0126] Determination by radioactive high performance liquid chromatography (HPLC) 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 Radiochemical purity of L injection.

[0127] 18 F-TCO-FAPI and18 F-Tz-α v β 6 L HPLC analysis conditions: The analytical column is a kromasil 100-5-C18 column. The mobile phase is 0.1% trifluoroacetic acid (TFA) in acetonitrile solution: 0.1% TFA in water solution, gradient elution: 0 min, 0.1% TFA in acetonitrile solution / 0.1% TFA in water solution: 10 / 90; 8 min, 0.1% TFA in acetonitrile solution / 0.1% TFA in water solution: 80 / 20. The flow rate is 1 mL / min, the analysis time is 15 min, and the UV detection wavelength is 254 nm. Use a non-radioactive standard substance with a confirmed structure 19 F-TCO-FAPI, and the corresponding radioactive 18 The F-TCO-FAPI injections were co-injected into HPLC to determine whether their retention times (Rt) or ratio shift values ​​(Rf) were consistent.

[0128] The radiochemical purity determined by HPLC was greater than 95%. 18 The radioactive HPLC analysis results of F-TCO-FAPI injection are as follows Figure 4a and Figure 4b (The retention times of the radiation peak and ultraviolet peak are Rt=11.23min and Rt=10.92min respectively). 18 F-Tz-α v β 6 The results of radioactive HPLC analysis of L injection are as follows Figure 4c and Figure 4d As shown in the results (the retention times of the radiation peak and the ultraviolet peak are Rt=10.65min and Rt=10.59min respectively), only a single main peak was observed.

[0129] Targeted compound bimolecular probe 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 The HPLC analysis conditions of L(1:1) and 18 F-TCO-FAPI and 18 F-Tz-α v β 6 L, except that the elution gradient is different: from 0min to 35min, 0.1% TFA acetonitrile solution / 0.1% TFA water solution: 20 / 80 changed to 55 / 45. The flow rate is 1mL / min, and the analysis time is 40min. Multiple main peaks were observed by radioactive HPLC of injection. HPLC method for in vivo and in vitro detection 18 F-TCO-FAPI is stable, and no obvious defluorination and decomposition were found. 18The radiochemical purity of F-TCO-FAPI in serum and urine is Figure 5a and Figure 5b As shown, the radiochemical purity was greater than 90% at 2 h in PBS buffer and in vitro serum (see Figure 5c and Figure 5d ) 18 F-Tz-α v β 6 The radiochemical purity of L in serum and urine is Figure 6a and Figure 6b As shown, the radiochemical purity was greater than 90% at 2 h in PBS buffer and in vitro serum (see Figure 6c and Figure 6d ), only a single main peak was found in the figure. The above results show that 18 F-TCO-FAPI and 18 F-Tz-α v β 6 L is relatively stable both in vivo and in vitro.

[0130] Example 9 Ester-water partition coefficient determination experiment

[0131] The ester-water distribution coefficient is determined as follows: 3 ml of n-octanol and 3 ml of water are added to a test tube, and then 18 F-TCO-FAPI (or 18 F-Tz-α v β 6 L) drug, shake, centrifuge, take 100ul of each of the upper and lower layers, divide into three tubes, and use a gamma counter to measure the radioactivity value. After determination, 18 F-TCO-FAPI lipid-water partition coefficient Log P = -2.961 ± 0.203 (n = 5), 18 F-Tz-α v β 6 The lipid-water partition coefficient of L is Log P = -3.36 ± 0.20 (n = 5), and all of them have obvious hydrophilicity.

[0132] Example 10 18 F-TCO-FAPI in vitro cell experiment

[0133] Uptake and inhibition experiments of A549-FAP cells with high FAP expression:

[0134] Tumor cell line A549 was transfected to obtain A549-FAP with high FAP expression. Two 24-well plates were plated and randomly divided into five groups: 5 min, 15 min, 30 min, 60 min, and 120 min. 18 F-TCO-FAPI was then cultured, and the inhibitor group was simultaneously added with NOTA-FAPI-42 inhibitor and 18F-TCO-FAPI was washed three times with PBS, and the cell count was determined using a γ counter after adding NaOH-SDS solution.

[0135] Cell uptake and inhibition experiment results: 18 F-TCO-FAPI has a relatively high uptake and specificity in A549-FAP cells (e.g. Figure 7a shown).

[0136] Affinity determination: Tumor cell plating: plating a 24-well plate, three wells per group, 8 groups in total. The concentrations of competitive inhibitor (NOTA-FAPI-42) were (0, 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10- 10 , 10 -11 M). Dissolve the inhibitors at each concentration in 1 mL of culture medium and add 0.163 mL to each well. Then add 0.837 mL of 18 F-TCO-FAPI (0.5 μCi / 0.5 mL / well) was incubated at 37°C for 1 hour, and then washed three times with PBS. The cells were ablated with NaOH-SDS solution, and the radioactivity was measured with a γ counter. 18 The results of the F-TCO-FAPI competitive binding experiment are shown in Figure 7b shown.

[0137] Example 11 Targeted compound bimolecular probe 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 L In vitro mass spectrometry test

[0138] The precursor material 18 F-TCO-FAPI and 18 F-Tz-α v β 6 50ug (1ml / mg) of each of L were mixed and heated for 12h, and then diluted to 100ug / ml for mass spectrometry detection to detect the two monomeric imaging agents. 18 F-TCO-FAPI and 18 F-Tz-α v β 6 Whether L can undergo an inverse electron demand Diels-Alder (IEDDA) bioorthogonal reaction in vitro to generate a new substance.

[0139] The amount of the new substance generated was calculated and compared with the data of mass spectrometry detection, confirming that the most concerned substance C was produced in vitro. 171 H 250 N 39 O 54 F 2 The amount of the substance was calculated to be 3748, and the mass spectrum detection diagram was as follows Figure 8 shown.

[0140] Example 12 18 In vivo biodistribution, uptake and inhibition experiments of F-TCO-FAPI

[0141] The method for establishing the subcutaneous tumor model is as follows: A549 and A549-FAP cells were cultured in DMEM medium supplemented with 10% FBS and 1% double antibody. Nude mice were raised in an SPF animal room. When the nude mice were about 5 weeks old, 8 5-week-old nude mice were subcutaneously inoculated with 5×10 6 / A549-FAP cells, 5×10 6 / A549 cells. After 2-3 weeks, a tumor of about 0.5-1.0 cm grew in the right armpit of the nude mouse.

[0142] In the first group, four A549-FAP tumor-bearing mice were injected via the tail vein with 0.1-0.2 mL of 40-60 μCi 18 In the second group, 4 A549-FAP tumor-bearing mice were injected with 0.1-0.2 mL of F-TCO-FAPI solution containing 40-60 μCi 18 A mixed solution of F-TCO-FAPI and 100ul (1mg / ml) inhibitor NOTA-FAPI-42 was injected into the tail vein of four A549 tumor-bearing mice with 0.1-0.2mL containing 40-60μCi 18 The biodistribution test method is to remove the eyeballs of the three groups of tumor-bearing mice and take blood 60 minutes after injection, then kill the mice by cervical vertebrae dissection, dissect and take tissue samples of various organs (blood, brain, heart, lung, liver, gallbladder, kidney, spleen, pancreas, stomach, small intestine, muscle, spine, femur, joints and tumor), weigh them, measure their radioactivity counts and record the measurement time, and calculate the percentage of the radioactivity injected dose per gram of tissue in 60 minutes (%ID / g).

[0143] Biodistribution experiment results: The results showed 18 F-TCO-FAPI showed excellent pharmacokinetic properties (including targeting, renal excretion, tumor uptake ability, tumor retention time, etc.) in preclinical research, as shown below: 18 F-TCO-FAPI has specific targeting to FAP target.18 In the FAP-positive A549-FAP tumor-bearing mouse model, the tumor uptake value of F-TCO-FAPI reached the highest value of 17.94±8.54%ID / g. In the FAP-positive tumor-bearing mouse inhibition group and the FAP-negative A549 tumor-bearing mouse model, the tumor uptake values ​​were very low, 2.23±0.68%ID / g and 1.52±0.4%ID / g, respectively. This shows that the A549-FAP tumor-bearing mouse model is 18 High and specific uptake of F-TCO-FAPI.

[0144] The biodistribution results in normal organs and tissues showed: 18 F-TCO-FAPI is mainly excreted rapidly through the kidneys, with a low uptake of only 3.14±1.64%ID / g at 60 min. The highest radioactive uptake is in the joints, the spine and bones have higher radioactive uptake, the gallbladder has moderate radioactive uptake, and the uptake in non-target sites such as the liver, intestines, lungs, pancreas, muscles and blood is at a lower level (results shown in 9a).

[0145] Example 13 18 F-Tz-α v β 6 In vivo biodistribution uptake experiment

[0146] The method for establishing a subcutaneous tumor model is as follows: 9 5-week-old nude mice were inoculated with Capan-2 tumor cells in the axilla of the right upper limb according to the method of Example 12. When the tumor grew to about 0.5-1.0 cm, an in vivo distribution experiment was performed.

[0147] The tumor-bearing nude mice were randomly divided into 3 groups, with 3 mice in each group. Each Capan-2 tumor-bearing mouse was injected with 0.1-0.2 mL of 40-60 μCi 18 F-Tz-α v β 6 L. The biodistribution experimental method is the same as in Example 12.

[0148] The in vivo biodistribution results showed that 18 F-Tz-α v β 6 The biodistribution of L in the Capan-2 tumor model Figure 9b As shown, 18 F-Tz-α v β 6 L has general biological activity in vivo, and the uptake in tumors is low. The highest radioactive uptake is in the kidney, and the lung and blood also have high radioactive uptake, and the intestine and liver have moderate radioactive uptake.

[0149] Example 14 Targeted compound bimolecular probe 18F-TCO-FAPI+ 18 F-Tz-α v β 6 L(1:1) and 18 F-TCO-FAPI and 18 F-Tz-α v β 6 Comparative experiment on the in vivo biodistribution of L.

[0150] A subcutaneous Capan-2 tumor model was established according to the method of Example 12, and an in vivo distribution experiment was performed when the tumor grew to about 0.5-1.0 cm.

[0151] The Capan-2 tumor-bearing mice were randomly divided into group 1, group 2 and group 3, with 6 mice in each group. In the first group, each Capan-2 tumor-bearing mouse was injected with 0.1-0.2 mL containing 40-60 μCi of the targeted compound bimolecular probe via the tail vein. 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 L (1:1) mixed solution, and in the second group, each Capan-2 tumor-bearing mouse was injected via the tail vein with 0.1-0.2 mL containing 40-60 μCi 18 In the third group, each Capan-2 tumor-bearing mouse was injected with 0.1-0.2 mL of F-TCO-FAPI solution containing 40-60 μCi 18 F-Tz-α v β 6 The biodistribution experimental method of the solution of L is the same as that of Example 12.

[0152] In vivo biodistribution results showed that the targeted compound bimolecular probe 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 Biodistribution of L(1:1) combined with two monomeric imaging agents in Capan-2 tumor-bearing mice 18 F-TCO-FAPI and 18 F-Tz-α v β 6 Advantages and disadvantages of in vivo biodistribution of L: The targeted compound bimolecular probe has a high uptake in tumors and is very close to 18 F-TCO-FAPI tumor uptake value is much higher than 18 F-Tz-α v β 6L tumor uptake value, and greater than the sum of half of the tumor uptake values ​​of the two monomeric imaging agents (1.43±0.07%ID / g, 1.76±0.05%ID / g, 0.48±0.04%ID / g, respectively), confirming the bioorthogonal effect of this experiment from the perspective of biodistribution. In normal organs and tissues, the uptake value of the targeted compound bimolecular probe is highest in the kidney, which is consistent with 18 F-Tz-α v β 6 L has similar biodistribution characteristics in the body, and the joints, spine, and bones also have high radioactivity uptake values. 18 The biodistribution characteristics of F-TCO-FAPI in vivo are similar. It is worth noting that 18 F-Tz-α v β 6 L has a high uptake in the lung and blood, but the uptake values ​​of the targeted composite bimolecular probe in the lung and blood are very low, which is not completely consistent with the previous conclusion (results such as Fig.9c shown).

[0153] Embodiment 15 18 F-TCO-FAPI micro-PET imaging experiment

[0154] The method for establishing a subcutaneous tumor model is as follows: 5-week-old nude mice are divided into 3 groups, each with 9 mice, and each group is inoculated with A549-FAP lung adenocarcinoma cells, U87 brain glioma cells and A549 lung adenocarcinoma cells according to the method of Example 12.

[0155] Static uptake and inhibition of PET imaging in tumor-bearing mice: 3 mice were weighed in the first, second, and third groups, and then 260 μCi of imaging agent was injected into the tail vein. 18 F-TCO-FAPI, and 3 mice in the first and second groups were weighed and injected with 260 μCi of imaging agent into the tail vein 18 A mixture of F-TCO-FAPI and 100ul (1mg / ml) NOTA-FAPI-42 inhibitor (diluted with 200ul saline) was injected and PET / CT imaging was performed 60min after injection.

[0156] PET imaging results showed that: 18 F-TCO-FAPI has a high uptake in A549-FAP lung adenocarcinoma model and U87 brain glioma model and remains stable within 2 hours. 18 F-TCO-FAPI is rapidly excreted through the kidneys. After 15 minutes, the kidneys, liver, and intestines are not clearly visualized, showing good pharmacokinetic properties. In the A549-FAP and U87 models, inhibition imaging showed a significant decrease in tumor uptake, which is specific uptake (such as Fig.10a and Fig.10b ), whereas there was almost no uptake in the A549 wild-type lung adenocarcinoma model.

[0157] In the U87 model 18 F-TCO-FAPI and 18 F-FAPI-42 PET imaging comparison study: Three U87 tumor-bearing mice were weighed and injected into the same mouse 18 F-TCO-FAPI and 18 F-NOTA-FAPI-42, PET / CT imaging was performed at 2h, 4h, and 6h after injection. 18 F-TCO-FAPI and 18 The contrast imaging results of F-NOTA-FAPI-42 show: 18 F-TCO-FAPI has a high tumor uptake value and a long retention time in normal organs and tissues. 18 F-TCO-FAPI had higher uptake in the spine and joints, and lower uptake in the intestine, liver, and kidneys; 18 The tumor uptake value of F-NOTA-FAPI-42 is low, and more obvious uptake can be seen in the joints, spine, and intestines. 18 F-TCO-FAPI performs better than 18 Imaging performance of F-NOTA-FAPI-42.

[0158] Example 16 18 F-Tz-α v β 6 L micro-PET imaging experiment

[0159] The method for establishing a subcutaneous tumor model is as follows: 5-week-old nude mice are divided into 2 groups, each with 3 mice, and each group is inoculated with Capan-2 human pancreatic cancer cells and BxPC-3 human orthotopic pancreatic cancer cells according to the method of Example 12. When the tumor grows to about 0.5-1.0 cm, an in vivo micro-PET imaging experiment is performed.

[0160] Static PET imaging in tumor-bearing mice: Each mouse was weighed and injected with 260 μCi of imaging agent into the tail vein 18 F-Tz-α v β 6 L, PET / CT imaging was performed at 30 min, 60 min, and 2 h after injection.

[0161] The imaging results show that: 18 F-Tz-α v β 6The peak uptake value of L in the tumor was obtained at 30 minutes, but in general, the tumor uptake value was low and the retention time was not long. It was mainly excreted through the kidneys. The uptake values ​​of the kidneys, heart, and lungs were very high. The bar graph results are as follows Fig.11a and Fig.11b shown.

[0162] Example 17 Targeted Compound Bimolecular Probe 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 L(1:1) and 18 F-TCO-FAPI and 18 F-Tz-α v β 6 Comparative experiment of micro-PET imaging of L

[0163] The method for establishing a subcutaneous tumor model is as follows: three 5-week-old nude mice were taken, and Capan-2 human pancreatic cancer cells were inoculated in the right upper armpit of each mouse according to the method of Example 12. When the tumor grew to about 0.5-1.0 cm, an in vivo micro-PET imaging experiment was performed.

[0164] Static PET imaging in tumor-bearing mice: In the first experiment, each mouse was weighed and injected with approximately 240 μCi of imaging agent into the tail vein 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 L (1:1), PET / CT imaging was performed at 30min, 60min, 2h, 4h, and 6h after injection. In the second experiment, the same mouse was weighed and injected with about 240μCi of imaging agent into the tail vein 18 F-TCO-FAPI, PET / CT imaging was performed at 30min, 60min, 2h, 4h, and 6h after injection. In the third experiment, the same mouse was weighed and injected with about 240μCi of imaging agent into the tail vein, and PET / CT imaging was performed at 30min, 60min, 2h, 4h, and 6h after injection.

[0165] PET results are as follows Fig.11c As shown, the imaging results show that: targeted compound bimolecular probe 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 L(1:1) bioorthogonal imaging showed high tumor uptake and was comparable to that of the tumour at long-term imaging. 18 Tumor uptake by F-TCO-FAPI monomer imaging, 18 F-TCO-FAPI+ 18F-Tz-α v β 6 The excretion rate of L in tumors is 18 F-TCO-FAPI is slower, probably due to 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 L was bound more in the long time point imaging. 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 The tumor uptake value of L is greater than 18 F-TCO-FAPI and 18 F-Tz-α v β 6 The sum of the uptake values ​​of half of the L tumor confirmed the bioorthogonal effect of this experiment from the imaging level, increasing tumor uptake and prolonging retention time. 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 L also showed higher uptake in the heart, lungs, kidneys, spine and joints, indicating that the two monomeric imaging agents 18 F-TCO-FAPI and 18 F-Tz-α v β 6 However, it is worth noting that 18 F-TCO-FAPI+ 18 F-Tz-α v β 6 L showed high uptake in the heart and lungs, which was inconsistent with its biodistribution profile in vivo.

[0166] The present invention establishes a method for preparing a molecular probe targeting FAP. 18 F-TCO-FAPI, targeting integrin α v β 6 Molecular probes 18 F-Tz-α v β 6 L and targeted compound bimolecular probe 18 F-TCO-FAPI+ 18 F-Tz-α v β 6A complete and streamlined synthesis and quality control method for L(1:1), and its application in the preparation of positron emission tomography (PET) small molecule imaging agents, in the construction of a bio-orthogonal amplification system for targeted tumor compound drugs, and in the development of new bio-orthogonal diagnosis and treatment technologies for compound drugs. The present invention can play an important diagnostic role in PET imaging of tumors and other diseases. For those skilled in the art, the structure and drugs can be improved or transformed according to the above description, and all these improvements and transformations should fall within the scope of protection of the claims attached to the present invention.

[0167] The technical features of the above embodiments can be combined arbitrarily. In order to make the description concise, this specification does not describe all possible combinations of the technical features in the above embodiments. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. Moreover, the above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as a limitation on the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several modifications and improvements can be made, which all belong to the scope of protection of the present invention.

Claims

1. A targeted compound bimolecular probe, characterized in that: It includes component A and component B respectively labeled with radioactive metal nuclides; The A component is a molecular probe targeting fibroblast activation protein (FAP), and the molecular probe targeting fibroblast activation protein (FAP) includes a trans-cyclooctene group (TCO); The B component targets integrin α v β6 molecular probe, the target integrin α v The β6 molecular probe includes a tetrazine group (Tz); The radioactivity ratio of the component A to the component B is (1-2):(2-1).

2. The targeted composite bimolecular probe according to claim 1, characterized in that: The targeted fibroblast activation protein (FAP) molecular probe further comprises a FAP inhibitor (FAPI) pharmacophore, a hydrophilic linking group, a metal ion chelating group and a radioactive metal nuclide; The targeting integrin α v β6 molecular probes also include integrin α v β6 receptor ligand (α v β6L) pharmacophore, hydrophilic linking group, metal ion chelating group and radioactive metal nuclide.

3. The targeted composite bimolecular probe according to claim 2, characterized in that: The hydrophilic linking groups include a di-polyethylene glycol group (PEG2) and / or a tri-polyethylene glycol group (PEG3), two aspartic acid groups (Asp2) and a lysine group (Lys).

4. The targeted composite bimolecular probe according to claim 2, characterized in that: The metal ion chelating group is 1,4,7-triazacyclononadecyl-N',N"-diacetyl-N-acetyl (NOTA) or 1,4,7,10-tetraazacyclododecane-N',N",N"'-triacetyl-N-acetyl (DOTA).

5. The targeted composite bimolecular probe according to claim 2, characterized in that: The radioactive metal nuclide is 18 F. 68 Ga, 111 In, 99m Tc, 186 Re, 188 Re, 175 Yb, 153 Sm, 166 Ho, 88 Y. 90 Y. 177 Lu, 47 Sc, 212 Bi, 213 Bi, 123 I. 124 I. 131 I. 211 At 153 Eu, 169 Eu, 212 Pb, 64 Cu, 67 Cu, 188 Re, 186 Re, 198 Au, 225 Ac or 227 One of Th.

6. The targeted composite bimolecular probe according to claim 5, characterized in that: The targeted composite bimolecular probe is 18 F-labeled molecular probe targeting fibroblast activation protein (FAP) 18 F-TC O-FAPI) and 18 F-labeled targeted integrin α v β6 molecular probe ( 18 F-Tz-α v β6L) mixture.

7. A method for preparing a targeted composite bimolecular probe, for preparing the targeted composite bimolecular probe according to any one of claims 1 to 6, characterized in that: The following steps are involved: Preparation of component A: Using NOTA-TCO-FAPI or DOTA-TCO-FAPI as a precursor material, a chelation reaction is carried out with radioactive metal nuclide ions, and a molecular probe targeting fibroblast activation protein (FAP) is prepared after separation and purification; Preparation of component B: Using NOTA-Tz-α v β6L or DOTA-Tz-α v β6L is used as a precursor material, which undergoes a chelation reaction with radioactive metal nuclide ions, and the targeted integrin α is prepared after separation and purification. v β6 molecular probe; Preparation of the targeted compound bimolecular probe: Mixing component A and component B according to a radioactivity ratio of (1-2): (2-1) to obtain the targeted compound bimolecular probe; Wherein, the precursor raw material is prepared by a solid phase peptide synthesis method.

8. The preparation method according to claim 7, characterized in that: The radioactive metal nuclide ions are 68 Ga 3+ 、[Al 18 F] 2+ , 64 Cu 2+ or 177 Lu 3+ One of them.

9. The use of the targeted composite bimolecular probe according to any one of claims 1 to 6, characterized in that: The application of the targeted composite bimolecular probe in the preparation of tumor diagnostic and therapeutic agents.

10. The use according to claim 9, characterized in that: Application of the targeted composite bimolecular probe in the preparation of tumor PET imaging agents, pre-positioned PET imaging agents and composite bio-orthogonal PET imaging agents; Application of the targeted composite bimolecular probe in the preparation of tumor SPECT imaging agents, pre-positioned SPECT imaging agents and composite bio-orthogonal SPECT imaging agents; The targeted composite bimolecular probe is used in the preparation of targeted radiotherapeutic agents, dual-targeted radiotherapeutic agents, pre-positioned targeted radiotherapeutic agents, and composite bio-orthogonal dual-targeted radiotherapeutic agents.

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