Targeting complex double-molecule probe and preparation method and application thereof

Abstract

The present invention relates to the field of nuclear medicine technology, and discloses a targeted compound bimolecular probe, comprising a component A and a component B respectively labeled with radioactive metal nuclides; component A is a molecular probe targeting fibroblast activation protein (FAP), and the molecular probe targeting fibroblast activation protein (FAP) includes a trans-cyclooctene group (TCO); component B is a molecular probe targeting integrin α v β6 molecular probe, targeting integrin α v The β6 molecular probe includes a tetrazine group (Tz); wherein the radioactivity ratio of component A to component B is (1-2): (2-1). The present invention has the following technical effects: the TCO in the FAP-targeting molecular probe and the integrin α v The Tz in the β6 molecular probe can undergo a bioorthogonal reaction, achieving a tertiary amplification effect, increasing tumor uptake, prolonging the retention time of the targeted compound bimolecular probe, improving the target / non-target ratio, and enhancing nuclear medicine diagnostic and therapeutic effects. The present invention also discloses a method for preparing and using the targeted compound bimolecular probe.

Description

Technical Field

[0001] The present invention belongs to the technical field of nuclear medicine, and in particular 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 are increasingly threatening 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 is showing 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 technologies 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 using 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, AlCl3 react with NOTA-Tz to obtain [ 18 F-AlF3]-NOTA-Tz; TGFβ antibody-TCO and [ 18 F-AlF3]-NOTA-Tz ligands were co-incubated to produce a bioorthogonal reaction. 18 The antibody probe prepared by this method can be used to detect the expression level of TGFβ in tumors and other tissues. However, this antibody probe is still a monomeric probe with a single target, and its imaging cannot obtain more accurate and comprehensive tumor biological information, and its effect on tumor diagnosis and treatment is still not ideal. Summary of the Invention

[0005] In order to solve the deficiencies of the prior art, the present invention provides a targeted compound bimolecular probe, which targets FAP molecular probes and targets integrin α v The combination of the β6 molecular probe 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 The Tz in the β6 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 compound 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 component A 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 β6 molecular probe targeting integrin α v The β6 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 The β6 molecular probe can 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 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 ( * M), referred to as *M-Tz-a v β6L.

[0014] As a further description of the technical solution of the present application, the hydrophilic linking group comprises a dimeric ethylene glycol group (PEG2) and / or a trimeric ethylene 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 application, the metal ion binding chelating group is 1,4,7-triazacyclononane-N',N"-diacetic acid-N-acetyl (NOTA) or 1,4,7,10-tetraazacyclododecane-N',N",N"' -triacetic acid-N-acetyl (DOTA).

[0016] As a further description of the technical solution of the present application, the radioactive metal isotope (M) is one of * M) is one of 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 Th, preferably one of 68 Ga, 18 F, 64 Cu or 177 Lu. More preferably, NOTA is combined with [Al 18 F] 2+ , 68 Ga 3+ , 64 Cu 2+ or 177 Lu 3+Combined with DOTA 68 Ga 3+ 、 64 Cu 2+ or 177 Lu 3+ combination.

[0017] As a further description of the technical solution of the present invention, the targeted compound bimolecular probe is 18 F-labeled molecular probe targeting FAP ( 18 F-TCO-FAPI) and 18 F-labeled targeted integrin α v β6 molecular probe ( 18 F-Tz-α v β6L) mixture, referred to as 18 F-TCO-FAPI+ 18 F-Tz-α v β6L (radioactivity ratio is 1:1). 18 F-TCO-FAPI+ 18 F-Tz-α v The chemical structure of β6L 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 targeting FAP molecular probes according to different types; the above-mentioned α v β6L, Tz, hydrophilic linking group, metal ion chelating group and radioactive metal nuclide ( * M) can be freely combined to synthesize different targeting integrins according to different types v β6 molecular probe.

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

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

[0022] Preparation of component B: Using NOTA-Tz-α v β6L or DOTA-Tz-α vβ6L is a precursor raw material, which is chelated with radioactive metal ion, and the target integrin α v β6 molecular probe * M-Tz-α v β6L;

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

[0024] It should be noted that, in addition to containing * M-TCO-FAPI and * M-Tz-α v β6L, the targeting compound double molecular probe can also contain two precursor raw materials NOTA-TCO-FAPI (or DOTA-TCO-FAPI) and NOTA-Tz-α v β6L (or DOTA-Tz-α v β6L, two targeting monomer molecular probes * M-TCO-FAPI and * M-Tz-α v β6L, and molecular probes combined with Tz and TCO * M-α v β6L-Tz-TCO-FAPI, * M-FAPI-TCO-Tz-α v β6L and * M-FAPI-TCO-Tz-α v β6L- * M.

[0025] The precursor raw material is prepared by a solid-phase polypeptide synthesis method. Specifically, the following steps are included: FAPI pharmacophore group modification-PEG2 to form FAPI pharmacophore group-PEG2, modification of Asp2 by FAPI pharmacophore group-PEG2, modification of TCO by Lys connection-PEG3, and finally modification of chelating group-NOTA or-DOTA. The product peak is collected after separation and purification by preparative HPLC, and the purified precursor raw material NOTA-TCO-FAPI or DOTA-TCO-FAPI is obtained. v β6L pharmacophore group modification Lys, and further connection of Tz group, and further modification of α v β6L pharmacophore group-PEG2, α v β6L pharmacophore group-PEG2 modification of Asp2, and connection of chelating group-NOTA or-DOTA. The product peak is collected after separation and purification by preparative HPLC, and the purified precursor raw material NOTA-Tz-α is obtained.v β6L or DOTA-Tz-alpha v β6L.

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

[0027]

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

[0029]

[0030] NOTA-Tz-alpha v The chemical structural formula of β6L is:

[0031]

[0032] DOTA-Tz-alpha v The chemical structural formula of β6L is:

[0033]

[0034] As a further description of the technical scheme of the present application, the radioactive metal ion is one of 68 Ga 3+ , [Al 18 F] 2+ , 64 Cu 2+ or 177 Lu 3+ .

[0035] Preferably, the preparation method of the targeting complex double-molecule probe comprises the following steps:

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

[0037] 18 Preparation of F-TCO-FAPI: using NOTA-TCO-FAPI as a precursor raw material, NOTA-TCO-FAPI reacts with 18 F - in an acidic solution of AlCl3 and acetonitrile, heated at 90-100℃ for 10-20min, then cooled, separated and purified by a SEP-PAK C18 column to obtain 18 F-TCO-FAPI.

[0038] 68Preparation of Ga-TCO-FAPI: using NOTA-TCO-FAPI or DOTA-TCO-FAPI as precursor raw material, NOTA-TCO-FAPI or DOTA-TCO-FAPI is reacted with weak acid 68 Ga 3+ solution is heated at 85-110℃, and purified by HLB column or SEP-PAK C18 column to obtain 68 Ga-TCO-FAPI.

[0039] 64 Preparation of Cu-TCO-FAPI: using NOTA-TCO-FAPI or DOTA-TCO-FAPI as precursor raw material, NOTA-TCO-FAPI or DOTA-TCO-FAPI is reacted with 64 Cu 2+ incubated at room temperature in sodium acetate acid buffer solution for 15min, 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 raw material, NOTA-TCO-FAPI or DOTA-TCO-FAPI is reacted with 177 Lu solution is heated in a mixture of gentisic acid and sodium acetate at 106℃ for 15min. Purified by HPLC and SEP-PAK C18 column to obtain 177 Lu-TCO-FAPI.

[0041] (II) * M-Tz-α v Preparation of β6L

[0042] 18 F-Tz-α v Preparation of β6L: using NOTA-Tz-α v β6L as precursor raw material, prepared according to the preparation method of 18 F-TCO-FAPI. 18 F-Tz-α v β6L.

[0043] 68 Ga-Tz-α v Preparation of β6L: using NOTA-Tz-α v β6L or DOTA-Tz-α v β6L as precursor raw material, prepared according to the preparation method of 68Ga-TCO-FAPI is prepared by the preparation method of Ga-TCO-FAPI, and Ga-TCO-FAPI is obtained. 68 Ga-Tz-alpha v β6L.

[0044] 64 Cu-Tz-alpha v The preparation of β6L is as follows: taking NOTA-Tz-alpha v β6L or DOTA-Tz-alpha v β6L as the precursor raw material, referring to 64 Cu-TCO-FAPI is prepared by the preparation method of Cu-TCO-FAPI, and Cu-TCO-FAPI is obtained. 64 Cu-Tz-alpha v β6L.

[0045] 177 Lu-Tz-alpha v The preparation of β6L is as follows: taking NOTA-Tz-alpha v β6L or DOTA-Tz-alpha v β6L as the precursor raw material, referring to 177 Lu-TCO-FAPI is prepared by the preparation method of Lu-TCO-FAPI, and Lu-TCO-FAPI is obtained. 177 Lu-Tz-alpha v β6L.

[0046] (III) Preparation of the targeted compound double-molecule probe: any one of the above * M-TCO-FAPI and any one of the above * M-Tz-alpha v β6L is mixed in a radioactivity ratio of 1:1 to obtain the targeted compound double-molecule probe.

[0047] In a third aspect, the application provides the use of the targeted compound double-molecule probe in the preparation of a tumor diagnosis and treatment agent, including the use in the preparation of a tumor diagnosis and treatment agent for a plurality of tumors with higher expression of FAP and / or a plurality of tumors expressing alpha v β6.

[0048] As a further description of the technical scheme of the application, the use of the targeted compound double-molecule probe in the preparation of a PET imaging agent for a tumor, a pre-positioning PET imaging agent, and a compound biological orthogonal PET imaging agent, the use of the targeted compound double-molecule probe in the preparation of a PET imaging agent for a tumor includes the use of the targeted compound double-molecule probe in the preparation of a PET imaging agent for a plurality of tumors with higher expression of FAP and / or a plurality of tumors with higher expression of alpha v β6.

[0049] The application of the targeted compound bimolecular probe in preparing a SPECT imaging agent for tumors, a pre-positioning SPECT imaging agent, and a compound bio-orthogonal SPECT imaging agent.

[0050] The application of the targeted compound bimolecular probe in preparing a targeted radiotherapy agent, a double-targeted radiotherapy agent, a pre-positioning targeted radiotherapy agent, and a compound bio-orthogonal double-targeted radiotherapy agent.

[0051] The application of the targeted compound bimolecular probe 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 and imaging efficiency.

[0052] In summary, the present application has at least the following advantages:

[0053] 1. The targeted compound bimolecular probe provided by the present application can simultaneously image or treat two target points through one administration, increases the total number of binding sites with target molecules, displays more tumor lesions, and improves the detection sensitivity and treatment effect of tumors. v The TCO in the targeted FAP molecular probe and the Tz in the targeted integrin alpha v beta6 molecular probe can undergo bio-orthogonal reactions, realize a three-level amplification effect, increase the tumor uptake value, prolong the retention time of the targeted compound bimolecular probe, improve the target / non-target uptake ratio, and enhance the effect of nuclear medicine diagnosis and treatment.

[0054] 2. The targeted compound bimolecular probe provided by the present application is selected from the targeted FAP molecular probe and the targeted integrin alpha v beta6 molecular probe, both of which are optimized in chemical structure to increase hydrophilicity, reduce uptake by the liver and gallbladder systems and the intestinal system, promote kidney excretion, and improve the in vivo pharmacokinetic properties of the molecular probe. 18 In particular, F-TCO-FAPI has a high affinity for the tumor FAP target point, can be competitively inhibited by an inhibitor, and also has excellent in vivo pharmacokinetic properties, 18 F-TCO-FAPI has high uptake in tumors and a long retention time, and still has a high uptake at 6h.

[0055] 3. The preparation method of the targeted compound bimolecular probe provided by the present application, which is prepared by chelating reaction of precursor raw materials with radioactive metal ion and then separation and purification, respectively prepares the targeted FAP molecular probe and the targeted integrin alpha vThe β6 molecular probe is mixed again according to the optimal radioactivity ratio to prepare the targeted compound double molecular probe. The synthesis of the molecular probe can realize high radiochemical yield and high radiochemical purity, and the preparation steps are simple and convenient to operate.

[0056] 4. The application of the targeted compound double molecular probe provided by the application is applied to a tumor diagnosis and treatment agent, a novel tumor diagnosis and treatment mode of acting on 'double targets' by one-time administration is provided, and the application is beneficial to promoting precise nuclear medical diagnosis and efficacy evaluation of tumors, solving the problems of precise visual diagnosis and evaluation, and has high clinical value.

[0057] 5. The biggest advantage of the application relates to the three-stage amplification effect of the compound biological orthogonal diagnosis and treatment system, including a first-stage compound amplification system, a second-stage biological orthogonal probe amplification system and a third-stage double biological orthogonal reaction amplification system, there is also a predetermined positioning diagnosis and treatment amplification system, and an in-vivo and in-vitro biological activity evaluation verification method is established. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 It is an imaging mode schematic diagram of the targeted compound double molecular probe;

[0059] Figure 2 It is a three-stage amplification effect mode schematic diagram of the double-targeted compound biological orthogonal diagnosis and treatment system;

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

[0061] Figure 3b It is an HPLC analysis spectrum of the precursor raw material NOTA-Tz-α v β6L;

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

[0063] Figure 3d It is an MS analysis spectrum of the precursor raw material NOTA-Tz-α v β6L;

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

[0065] Figure 4b It is an injection 18 It is a representative ultraviolet absorption HPLC analysis spectrum of F-TCO-FAPI at an ultraviolet absorption retention time Rt=10.92 min;

[0066] Figure 4c for injection 18 F-Tz-α v Representative radio-HPLC analysis profile of β6L at a radio-retention time Rt = 10.65 min;

[0067] Figure 4d for injection 18 F-Tz-α v Representative UV-absorption HPLC analysis profile of β6L at a UV-absorption retention time Rt = 10.59 min;

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

[0069] Figure 5b for injection 18 Representative radio-HPLC analysis profile of F-TCO-FAPI in urine of Kunming mice at 1 h;

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

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

[0072] Figure 6a for injection 18 F-Tz-α v Representative radio-HPLC analysis profile of β6L in serum of Kunming mice at 1 h;

[0073] Figure 6b for injection 18 F-Tz-α v Representative radio-HPLC analysis profile of β6L in urine of Kunming mice at 1 h;

[0074] Figure 6c for injection 18 F-Tz-α v Representative radio-HPLC analysis profile of β6L in PBS at 2 h in vitro;

[0075] Figure 6d for injection 18 F-Tz-α v Representative radio-HPLC analysis profile of β6L in fetal bovine serum at 2 h in vitro;

[0076] Figure 7a To 18 Figure of cell uptake and inhibition experiment results of F-TCO-FAPI;

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

[0078] Figure 8 FAPI-TCO-Tz-α v Figure of mass spectrum detection of β6;

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

[0080] Figure 9b To 18 F-Tz-α v Figure of biodistribution of β6L in tumor-bearing mice (Capan-2) at 60 min;

[0081] Figure 9c Targeting compound double-molecule probe 18 F-TCO-FAPI+ 18 F-Tz-α v β6L (radioactive activity ratio of 1:1) and 18 F-TCO-FAPI and 18 F-Tz-α v Figure of comparison of in vivo biodistribution of β6L;

[0082] Figure 10a To 18 Figure of PET / CT imaging of F-TCO-FAPI in A549-FAP lung adenocarcinoma tumor-bearing mouse model at 60 min of static uptake (left) and competitive inhibition (right);

[0083] Figure 10b To 18 Figure of PET / CT imaging of F-TCO-FAPI in U87 brain glioma tumor-bearing mouse model at 60 min of static uptake (left) and competitive inhibition (right);

[0084] Figure 11a Figure of 18F-Tz-αvβ6L in Capan-2 tumor-bearing mouse model at 2h, 4h, 6h;

[0085] Figure 11bColumn chart of 18F-Tz-αvβ6L in BxPC-3 tumor-bearing mouse model at 2h, 4h, 6h;

[0086] Figure 11c Targeting compound bimolecular probe 18 F-TCO-FAPI+ 18 F-Tz-α v β6L (radioactivity ratio 1:1) and 18 F-TCO-FAPI and 18 F-Tz-α v β6L PET / CT imaging images at 30min, 60min, 2h, 4h, 6h. DETAILED DESCRIPTION

[0087] In order to facilitate the understanding of the present application, the present application will be described more fully below in conjunction with specific embodiments and drawings. However, the present application can be realized 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 application more thorough and comprehensive.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the specification of the present application herein is only for the purpose of describing specific embodiments and is not intended to limit the present application.

[0089] As a preferred embodiment, the targeting compound bimolecular probe is a targeting FAP molecular probe 18 F-TCO-FAPI and targeting integrin α v β6 molecular probe 18 F-Tz-α v β6L, both mixed in a radioactivity ratio of 1:1, referred to as 18 F-TCO-FAPI+ 18 F-Tz-α v β6L.

[0090] The present application realizes the three-stage amplification effect of the double-targeting compound bio-orthogonal diagnosis and treatment system by the bio-orthogonal reaction of TCO in the targeting FAP molecular probe and Tz in the targeting integrin α v β6 molecular probe in vivo, increases the tumor uptake value, and prolongs the retention time of the targeting compound bimolecular probe. The bio-orthogonal chemical reaction formula based on IEDDA between the compound bio-orthogonal bimolecular probe is as follows:

[0091]

[0092] wherein R is R' is

[0093] The imaging mode schematic diagram of the targeted compound bimolecular probe is shown in Figure 1 The three-stage amplification effect mode schematic diagram of the double-targeted compound bio-orthogonal diagnosis and treatment system is shown in Figure 2

[0094] The three-stage amplification effect of the double-targeted compound bio-orthogonal diagnosis and treatment system includes the first-stage compound bimolecular probe amplification system, the second-stage bio-orthogonal probe amplification system, and the third-stage double bio-orthogonal reaction amplification system. In addition, there is a pre-positioning diagnosis and treatment amplification system, and an in vivo and in vitro bioactivity evaluation verification method can be established.

[0095] The first-stage compound bimolecular probe amplification system is the targeted compound bimolecular probe * M-TCO-FAPI+ * M-Tz-α v β6L respectively targets FAP and integrin α v β6 in the tumor. The following experiments verify that * M-TCO-FAPI specifically targets target FAP: * M-TCO-FAPI is taken up in FAP-positive models (U87 brain glioma, A549-FAP lung adenocarcinoma cells) and inhibits PET imaging (the inhibitor is NOTA-FAPI-42). The following experiments verify that * M-Tz-α v β6L specifically targets target integrin α v β6: * M-Tz-α v β6L in α v β6 positive model (Capan-2 human pancreatic cancer) is taken up and inhibits PET imaging. It is verified that the targeted compound bimolecular probe * M-TCO-FAPI+ * M-Tz-α v β6L respectively targets two targets.

[0096] The second-stage bio-orthogonal probe amplification system is the targeted compound bimolecular probe * M-TCO-FAPI+ * M-Tz-α v β6L in which TCO and Tz are combined to form * M-FAPI-TCO-Tz-α v β6L- * M, * M-FAPI-TCO-Tz-α v β6L- * ​M binds to the target FAP and integrin alpha v β6 in the tumor. This is verified by the following experiment: the FAP-targeting molecular probe 18 F-TCO-FAPI and the integrin alpha v β6 molecular probe 18 F-Tz-alpha v β6L is mixed in a 1:1 radioactivity ratio to prepare a targeting compound bimolecular probe 18 F-TCO-FAPI+ 18 F-Tz-alpha v β6L, which is verified by HPLC analysis to produce 18 F-FAPI-TCO-Tz-alpha v β6L- 18 F. It is verified by molecular docking: NOTA-TCO-FAPI, NOTA-Tz-alpha v β6L and NOTA-FAPI-TCO-Tz-alpha v β6L-NOTA binds to FAP, alpha v β6 target. It is verified by tumor-bearing model imaging experiments: in the FAP and alpha v β6 dual-targeting 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 then inject 18 F-Tz-alpha v β6L, the tumor radioactivity uptake value increases; the next day, first inject 18 F-TCO-FAPI, after a period of time, inject NOTA-Asp2-alpha v β6L+ 18 F-Tz-alpha v β6L, the tumor uptake is partially inhibited, indicating that the inhibition 18 F-Tz-alpha v β6L in alpha v β6L and the target alpha v β6 effect; the third day, first inject 18 F-TCO-FAPI, after a period of time, inject NOTA-Tz+ 18 F-Tz-alpha v β6L, the tumor uptake is partially inhibited, indicating that the inhibition 18 F-Tz-alpha v β6L in Tz and 18 TCO in F-TCO-FAPI occurs bio-orthogonal binding.

[0097] The three-stage double-bioorthogonal reaction amplification system is specifically a targeted compound double-molecule probe * M-TCO-FAPI * M-Tz-α v β6L respectively binds to the target FAP and integrin α v β6 in the tumor after binding, and * TCO in M-TCO-FAPI binds to free * M-Tz-α v Tz in β6L binds to, and * M-Tz-α v Tz in β6L binds to free * TCO in M-TCO-FAPI binds, that is, the TCO-Tz double-binding system. The following experiment verifies that the targeted single-molecule probe that binds to the tumor can bioorthogonally combine with another single-molecule probe in free state in vivo: in a FAP single-positive model (U87 brain glioma), *M-TCO-FAPI is pre-injected * M-TCO-FAPI, after a period of time * M-TCO-FAPI binds to the tumor, and then *M-Tz-α is injected * M-Tz-α v β6L, the next day, *M-TCO-FAPI is pre-injected * M-TCO-FAPI, after the same period of time, *Tz-α is injected v β6L * M-Tz-α v β6L, the tumor radioactivity uptake value decreases, proving that * TCO in M-TCO-FAPI binds to free * M-Tz-α v Tz in β6L binds; in α v β6L single-positive model (A549 brain glioma), *M-Tz-α is pre-injected v M-Tz-α v β6L binds to the tumor, and then *M-TCO-FAPI is injected * M-TCO-FAPI, the next day, *M-Tz-α is pre-injected * M-Tz-α v β6L, after a period of time, *TCO-FAPI is injected * M-TCO-FAPI, the tumor radioactivity uptake value decreases, proving that * M-Tz-α v Tz in β6L binds to free * TCO in M-TCO-FAPI binds.

[0098] In addition, the pre-positioning diagnosis and treatment amplification system needs to give a certain amount of unmarked NOTA-TCO-FAPI+NOTA-Tz-alpha v β6L and labeled * M-TCO-FAPI+ * M-Tz-alpha v β6L, the mechanism of action involves not only the above-mentioned three-stage 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-alpha v β6L in FAP single-positive model (U87 brain glioma) for PET imaging, the tumor does not uptake or uptake is low, the next day, the precursor TCO-FAPI is injected in advance, after a period of time, TCO-FAPI binds to the tumor, and then * M-Tz-alpha v β6L is injected, and PET imaging is performed, and the tumor uptake is significantly increased; the next day, the precursor TCO-FAPI is injected in advance, and after the same period of time, Tz-alpha v β6L+ * M-Tz-alpha v β6L, the tumor radioactivity uptake value is reduced. * M-TCO-FAPI in alpha v β6L single-positive model (A549 brain glioma) for PET imaging, the tumor does not uptake or uptake is low, the next day, the precursor Tz-alpha is injected in advance v β6L, after a period of time, Tz-alpha v β6L binds to the tumor, and then * M-TCO-FAPI tumor radioactivity uptake value is increased; the next day, TCO-FAPI+ * M-TCO-FAPI, the tumor radioactivity uptake value is reduced.

[0099] The targeted compound double-molecule probe, its preparation method and application are described in detail in the following specific examples.

[0100] Example 1 Preparation of precursor raw material NOTA-TCO-FAPI and DOTA-TCO-FAPI

[0101] After modification of the FAP pharmacophore group-PEG2, the FAP pharmacophore group-PEG2 is modified Asp2, and then connected to TCO through Lys and a-PEG3 linker. Finally, the chelating group-NOTA is modified, the product peak is collected after separation and purification by preparative HPLC, and then freeze-dried to obtain the purified precursor raw material NOTA-TCO-FAPI.

[0102] The chemical yield of NOTA-TCO-FAPI is high, and the purity is greater than 95%. The HPLC and MS determination results of NOTA-TCO-FAPI are shown in Figure 3a and Figure 3c The mass spectrum MS (m / z) determines the molecular weight (Mr.) of NOTA-TCO-FAPI as 1630.76.

[0103] The preparation method of DOTA-TCO-FAPI can refer to NOTA-TCO-FAPI, which is not described here. The chemical yield of DOTA-TCO-FAPI is high, and the purity is greater than 95% after HPLC and MS determination.

[0104] Example 2 precursor raw material NOTA-Tz-α v β6L and DOTA-Tz-α v Preparation of β6L

[0105] Targeting α v α in β6 ligand v After modifying Lys in the β6L pharmacophore group, the Tz group is connected, and further modified by-PEG2 to form α v β6L pharmacophore group-PEG2, α v After modifying Asp2 in the β6L pharmacophore group-PEG2, the chelating group-NOTA is connected. The product peak is collected after separation and purification by preparative HPLC, and then freeze-dried to obtain the purified precursor raw material NOTA-Tz-α v β6L.

[0106] NOTA-Tz-α v The chemical yield of β6L is high, and the purity is greater than 95%. NOTA-Tz-α v The HPLC and MS determination results of β6L are shown in Figure 3b and Figure 3d The mass spectrum MS (m / z) determines the molecular weight (Mr.) of NOTA-Tz-α v β6L as 2150.34.

[0107] DOTA-Tz-α v The preparation method of DOTA-Tz-α v β6L can refer to NOTA-Tz-α v β6L, which is not described here. The chemical yield of DOTA-Tz-α 18 is high, and the purity is greater than 95% after HPLC and MS determination.

[0108] Example 3 18 F-Tz-α v Preparation of β6L

[0109] In the reaction bottle with NOTA-TCO-FAPI (or NOTA-Tz-α v β6L) (50 μg / μL, 50 μL), 6 μL of 2 mM AlCl3 solution, 5 μL of glacial acetic acid and 300 μL of acetonitrile were sequentially added, and mixed.

[0110] produced by cyclotron via 18 O (p, n) 18 F nuclear reaction 18 F - Under N2 carrier, it was captured in Sep-Pak QMA anion column, 18 O - Water was collected in a recovery bottle. 0.3-0.4 mL of normal saline (or sodium acetate buffer) was used to elute the anion (QMA) column into a vial, and 50 μL of which was added to the above reaction bottle. 18 F - Eluted into a vial, and 50 μL of which was added to the above reaction bottle.

[0111] After stirring and mixing, the reaction was heated at 100°C for about 15 min. After cooling, 6-8 mL of water was added to the reaction bottle, mixed, and transferred to the HLB column or SEP-PAK C18 column. After the solution in the reaction bottle was completely transferred, the column was washed with 10 mL of water for injection 3 times, and the column was blown dry.

[0112] Finally, the product was eluted with 1.5 mL of ethanol, collected in a receiving bottle after passing through a sterile filter membrane, and diluted with normal saline to obtain a product solution containing 5% ethanol. 18 F-TCO-FAPI (or 18 F-Tz-α v β6L) injection.

[0113] 18 F-TCO-FAPI and 18 F-Tz-α v β6L The uncorrected radiochemical yield was 20-30%, and the total radiosynthesis time was 35 min.

[0114] Example 4 68 Ga-TCO-FAPI and 68 Ga-Tz-α v β6L

[0115] In the reaction bottle with NOTA-TCO-FAPI (or DOTA-TCO-FAPI, or NOTA-Tz-α v β6L, or DOTA-Tz-α v β6L) (50 μg / μL, 50 μL), 200 μL of 1.25 M sodium acetate solution was sequentially added.

[0116] From 68 Ge / 68 Ga generator eluted with 0.05M hydrochloric acid 4mL 68 GaCl3 into the above reaction vial, mixed well, adjusted the pH of the solution to 4.0, heated the reaction at 100℃ for 10-15min. Cooled, added normal saline 4mL into the reaction vial, mixed well, transferred to HLB cartridge or SEP-PAK C18 cartridge. After the solution in the reaction vial was completely transferred, the cartridge was washed with 10mL x 2 water for injection, and the cartridge was blown dry.

[0117] The product was eluted with ethanol 1.5mL and collected in a receiving vial after sterile filter membrane, and diluted with normal saline to obtain a product solution containing 5% ethanol, to obtain 68 Ga-TCO-FAPI (or 68 Ga-Tz-α v β6L) injection.

[0118] 68 Ga-TCO-FAPI and 68 Ga-Tz-α v β6L uncorrected radiochemical yield was 20-50%, and the total radiosynthesis time was 30min.

[0119] Example 5 64 Cu-TCO-FAPI and 64 Cu-Tz-α v β6L preparation

[0120] In the reaction vial containing NOTA-TCO-FAPI (or DOTA-TCO-FAPI, or NOTA-Tz-α v β6L, or DOTA-Tz-α v β6L) (50μg / μL, 100μL), added 64 CuCl2 solution 0.100-1.000mL, adjusted to pH 4.0-5.6 with sodium acetate solution, reacted at room temperature for 10-15min. Finally diluted with normal saline and collected in a receiving vial after sterile filter membrane filtration, to obtain 64 Cu-TCO-FAPI (or 64 Cu-Tz-α v β6L) injection. 64 Cu-TCO-FAPI and 64 Cu-Tz-α v β6L uncorrected radiochemical yield was 50-70%.

[0121] Example 6 177 Lu-TCO-FAPI and177 Lu-Tz-α v Preparation of β6L

[0122] In the reaction bottle of β6L (50 μg / μL, 50 μL) added gentisic acid, 160 ul sodium acetate and free v β6L, or DOTA-Tz-α v β6L) (50 μg / μL, 50 μL) were added gentisic acid, 160 ul sodium acetate and free 177 Lu solution 0.100-1.000 mL, 106 ℃ water bath heating for 15 min. The reaction effect was monitored by HPLC, and separated by HPLC. The separation product was passed through C18 column, then washed with 30 ml water, passed through the column with 1 mL 50% ethanol, and the product was collected. Finally, it was diluted with physiological saline and filtered through a sterile filter membrane into a receiving bottle to obtain 177 Lu-TCO-FAPI (or 177 Lu-Tz-α v β6L) injection. 177 Lu-TCO-FAPI and 177 Lu-Tz-α v β6L uncorrected radiochemical yield was 40-60%.

[0123] Example 7 Targeting compound double-molecular probe 18 F-TCO-FAPI+ 18 F-Tz-α v Preparation of β6L

[0124] The 18 F-TCO-FAPI injection was mixed with 18 F-Tz-α v β6L injection at a radioactivity ratio of 1:1 to prepare a targeting compound double-molecular probe 18 F-TCO-FAPI+ 18 F-Tz-α v β6L injection.

[0125] Example 8 Targeting compound double-molecular probe 18 F-TCO-FAPI+ 18 F-Tz-α v Determination of radiochemical purity and stability of β6L

[0126] The radiochemical purity of 18 F-TCO-FAPI+ 18 F-Tz-α v β6L injection was determined by radio high performance liquid chromatography (HPLC).

[0127] 18 F-TCO-FAPI and 18 F-Tz-α v β6L HPLC analysis conditions: analytical column kromasil 100-5-C18 column. Mobile phase 0.1% trifluoroacetic acid (TFA) in acetonitrile: 0.1% TFA in water, gradient elution: 0 min, 0.1% TFA in acetonitrile / 0.1% TFA in water: 10 / 90; 8 min, 0.1% TFA in acetonitrile / 0.1% TFA in water: 80 / 20. Flow rate 1 mL / min, analysis time 15 min, UV detection wavelength 254 nm. Non-radioactive standard of defined structure 19 F-TCO-FAPI, with the corresponding radioactive 18 F-TCO-FAPI injection was co-injected into HPLC to determine whether its retention time (Rt) or the ratio of migration Rf was consistent.

[0128] The radiochemical purity was determined by HPLC method and was greater than 95%. Among them, 18 The results of radio-HPLC analysis of F-TCO-FAPI injection are shown in Figure 4a and Figure 4b (Rt of radioactive peak and UV peak were Rt=11.23 min and Rt=10.92 min, respectively). 18 F-Tz-α v The results of radio-HPLC analysis of β6L injection are shown in Figure 4c and Figure 4d (Rt of radioactive peak and UV peak were Rt=10.65 min and Rt=10.59 min, respectively), only a single main peak was observed.

[0129] Targeting complex double-molecule probe 18 F-TCO-FAPI+ 18 F-Tz-α v β6L (1:1) HPLC analysis conditions were similar to 18 F-TCO-FAPI and 18 F-Tz-α v β6L, except that the elution gradient was different: 0 min to 35 min, 0.1% TFA in acetonitrile / 0.1% TFA in water: 20 / 80 changed to 55 / 45. Flow rate 1 mL / min, analysis time 40 min. Injection radio-HPLC observed multiple main peaks. In vitro and in vivo 18 F-TCO-FAPI stability, no obvious defluorination and decomposition were found. Among them, 18 The radiochemical purity of F-TCO-FAPI in serum and urine is as followsFigure 5a and Figure 5b The radiochemical purity of F-TCO-FAPI and F-Tz-α at 2h in PBS buffer and in vitro serum was greater than 90% (see Figure 1 and Figure 2) and only one main peak was found in the figure. Figure 5c and Figure 5d The radiochemical purity of F-TCO-FAPI and F-Tz-α at 2h in PBS buffer and in vitro serum was greater than 90% (see Figure 1 and Figure 2) and only one main peak was found in the figure. 18 F-Tz-α v The radiochemical purity of F-TCO-FAPI and F-Tz-α at 2h in PBS buffer and in vitro serum was greater than 90% (see Figure 1 and Figure 2) and only one main peak was found in the figure. Figure 6a and Figure 6b The radiochemical purity of F-TCO-FAPI and F-Tz-α at 2h in PBS buffer and in vitro serum was greater than 90% (see Figure 1 and Figure 2) and only one main peak was found in the figure. Figure 6c and Figure 6d The radiochemical purity of F-TCO-FAPI and F-Tz-α at 2h in PBS buffer and in vitro serum was greater than 90% (see Figure 1 and Figure 2) and only one main peak was found in the figure. 18 F-TCO-FAPI and 18 F-Tz-α v F-TCO-FAPI and F-Tz-α were stable in vitro and in vivo.

[0130] Example 9 Determination of the octanol-water partition coefficient of the ester

[0131] The octanol-water partition coefficient of the ester was determined as follows: 3ml of n-octanol and water were added to a test tube, then the drug F-TCO-FAPI (or F-Tz-α, F-TCO-FAPI) was added, shaken, centrifuged, and 100ul of the upper and lower layers were taken and divided into three tubes. The radioactivity value was measured using a gamma counter. It was determined that the octanol-water partition coefficient of F-TCO-FAPI was -2.961 ± 0.203 (n = 5), and the octanol-water partition coefficient of F-Tz-α was -3.36 ± 0.20 (n = 5), both of which were significantly hydrophilic. 18 F-TCO-FAPI (or 18 F-Tz-α v F-TCO-FAPI) was added, shaken, centrifuged, and 100ul of the upper and lower layers were taken and divided into three tubes. The radioactivity value was measured using a gamma counter. It was determined that the octanol-water partition coefficient of F-TCO-FAPI was -2.961 ± 0.203 (n = 5), and the octanol-water partition coefficient of F-Tz-α was -3.36 ± 0.20 (n = 5), both of which were significantly hydrophilic. 18 F-TCO-FAPI was -2.961 ± 0.203 (n = 5), and the octanol-water partition coefficient of F-Tz-α was -3.36 ± 0.20 (n = 5), both of which were significantly hydrophilic. 18 F-Tz-α v F-TCO-FAPI was -2.961 ± 0.203 (n = 5), and the octanol-water partition coefficient of F-Tz-α was -3.36 ± 0.20 (n = 5), both of which were significantly hydrophilic.

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

[0133] FAP high expression A549-FAP cell uptake and inhibition experiment:

[0134] The tumor cell line A549 was treated by transfection to obtain FAP high expression A549-FAP. Two 24-well plates were prepared and randomly divided into five groups: 5min, 15min, 30min, 60min, and 120min. The uptake group was added with F-TCO-FAPI and then cultured, and the inhibition group was added with NOTA-FAPI-42 inhibitor and F-TCO-FAPI at the same time, and then washed with PBS for three times. After adding NaOH-SDS solution, the cell count was measured using a gamma counter. 18 F-TCO-FAPI was -2.961 ± 0.203 (n = 5), and the octanol-water partition coefficient of F-Tz-α was -3.36 ± 0.20 (n = 5), both of which were significantly hydrophilic. 18 F-TCO-FAPI was -2.961 ± 0.203 (n = 5), and the octanol-water partition coefficient of F-Tz-α was -3.36 ± 0.20 (n = 5), both of which were significantly hydrophilic.

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

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

[0137] Example 11 Targeting complex double-molecular probe 18 F-TCO-FAPI+ 18 F-Tz-α v β6L in vitro mass spectrometry test

[0138] The precursor raw materials 18 F-TCO-FAPI and 18 F-Tz-α v β6L were mixed at 50 ug each (1 ml / mg) and heated for 12 h. After heating, dilution was performed, and after dilution to 100 ug / mL, mass spectrometry was performed to detect whether the two monomer imaging agents 18 F-TCO-FAPI and 18 F-Tz-α v β6L could undergo an inverse electron demand Diels-Alder (IEDDA) bio-orthogonal reaction in vitro to generate a new substance.

[0139] The amount of substance of the generated new substance was calculated and compared with the mass spectrometry data to confirm that the most concerned substance C 171 H 250 N 39 O 54 F2 was generated in vitro. The amount of substance of the substance was calculated to be 3748, and the mass spectrometry detection chart is shown in Figure 8 .

[0140] Example 12 18 F-TCO-FAPI in vivo biodistribution uptake and inhibition experiment

[0141] The method for establishing a subcutaneous tumor model is as follows: A549, A549-FAP cells are cultured with DMEM medium plus 10% FBS and 1% double-antibiotic. The nude mice are raised in the SPF animal room, and when the nude mice are about 5 weeks old, 8 nude mice of 5 weeks old are subcutaneously inoculated with 5x10 6 A549-FAP cells in the right axillary fossa, and 4 nude mice of 5 weeks old are subcutaneously inoculated with 5x10 6 A549 cells in the right axillary fossa. About 2-3 weeks later, the nude mice grow tumors of about 0.5-1.0 cm in the right axillary fossa.

[0142] The first group is injected with 0.1-0.2 mL of a solution containing 40-60 μCi of F-TCO-FAPI via the tail vein of 4 A549-FAP tumor-bearing mice, the second group is injected with 0.1-0.2 mL of a mixed solution containing 40-60 μCi of F-TCO-FAPI and 100 ul (1 mg / ml) of the inhibitor NOTA-FAPI-42 via the tail vein of 4 A549-FAP tumor-bearing mice, and the third group is injected with 0.1-0.2 mL of a solution containing 40-60 μCi of F-TCO-FAPI via the tail vein of 4 A549 tumor-bearing mice. 18 18 18 The biodistribution experiment method is as follows: 60 min after injection, the eyeballs of the three groups of tumor-bearing mice are removed for blood removal, and the mice are sacrificed by cervical dislocation, the tissue samples of the organs (blood, brain, heart, lung, liver, gallbladder, kidney, spleen, pancreas, stomach, small intestine, muscle, spine, femur, joint, and tumor) are dissected, weighed, measured for radioactivity count, and recorded for the measurement time, and the percentage of radioactivity injection dose per gram of tissue (%ID / g) at 60 min is calculated.

[0143] The biodistribution experiment results show that 18 F-TCO-FAPI exhibits excellent pharmacokinetic performance (including targeting, kidney excretion, tumor uptake capacity, intratumoral retention time, etc.) in the preclinical research stage, which is specifically manifested as follows: 18 F-TCO-FAPI has specific targeting to the FAP target, 18 F-TCO-FAPI has the highest tumor uptake value of 17.94±8.54 %ID / g in the FAP-positive A549-FAP tumor-bearing mouse model, and the tumor uptake values in the FAP-positive tumor-bearing mouse inhibition group and the FAP-negative A549 tumor-bearing mouse model are very low, being 2.23±0.68 %ID / g and 1.52±0.4 %ID / g, respectively, which shows that the A549-FAP tumor-bearing mouse model has high specificity for​​18 High uptake and specific uptake of F-TCO-FAPI.

[0144] The biodistribution results in normal organs, tissues showed that: 18 F-TCO-FAPI was mainly excreted through the kidney, and had low uptake at 60 min, only 3.14±1.64%ID / g. It had the highest radioactive uptake in joints, higher radioactive uptake in spine and bone, moderate radioactive uptake in gallbladder, and low uptake in non-target sites such as liver, intestine, lung, pancreas, muscle and blood (results shown in 9a).

[0145] Example 13 18 F-Tz-α v Biodistribution uptake experiment of β6L in vivo

[0146] The method for establishing a subcutaneous tumor model is as follows: 9 five-week-old nude mice are inoculated with Capan-2 tumor cells in the right upper limb armpit according to the method of Example 12, and when the tumor grows to about 0.5-1.0 cm, the in vivo distribution experiment is performed.

[0147] The tumor-bearing nude mice are randomly divided into 3 groups, 3 in each group, and 0.1-0.2 mL of 40-60 μCi of F-TCO-FAPI, F-Tz-α or β6L is injected into the tail vein of each Capan-2 tumor-bearing mouse. 18 F-Tz-α v Biodistribution experiment method of β6L is the same as Example 12.

[0148] The results of in vivo biodistribution show that, 18 F-Tz-α v The in vivo biodistribution of β6L in Capan-2 tumor-bearing model is as shown in Figure 9b , 18 F-Tz-α v β6L has general in vivo activity, and has low uptake in tumors. It has the highest radioactive uptake in the kidney, and also has very high radioactive uptake in the lung and blood, and moderate radioactive uptake in the intestine and liver.

[0149] Example 14 Targeting complex double-molecule probe 18 F-TCO-FAPI+ 18 F-Tz-α v β6L (1:1) and 18 F-TCO-FAPI and 18 F-Tz-α v β6L in vivo biodistribution comparison experiment

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

[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 β6L (1:1) mixed solution, 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 via the tail vein. 18 F-Tz-α v The biodistribution experimental method of the β6L solution was 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 Biodistribution of β6L (1:1) combined with two monomeric imaging agents in Capan-2 tumor-bearing mice 18 F-TCO-FAPI and 18 F-Tz-α v Advantages and disadvantages of in vivo biodistribution of β6L: 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 The tumor uptake value of β6L was higher than the sum of half the tumor uptake values ​​of the two monomeric imaging agents (1.43±0.07%ID / g, 1.76±0.05%ID / g, and 0.48±0.04%ID / g, respectively), which confirmed the bioorthogonal effect of this experiment from the perspective of biodistribution. Among normal organs and tissues, the uptake value of the targeted compound bimolecular probe was the highest in the kidney, which was consistent with the results of the previous study. 18 F-Tz-α v The biodistribution characteristics of β6L in the body are similar, and the uptake values ​​of joints, spine, and bones also have high radioactive uptake, which is similar to 18 The biodistribution characteristics of F-TCO-FAPI in vivo are similar. 18 F-Tz-α v β6L has a high uptake in the lung and blood, but the uptake of the targeted compound bimolecular probe in the lung and blood is very low, which is not completely consistent with the previous conclusion (results such as Figure 9c shown).

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

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

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

[0156] The PET imaging results showed that: 18 F-TCO-FAPI had high uptake in A549-FAP lung adenocarcinoma model and U87 brain glioma model, and remained stable within 2h, 18 F-TCO-FAPI was rapidly excreted through the kidneys, and there was no obvious kidney imaging at 15 min, and there was also no obvious liver and intestinal imaging, showing good pharmacokinetic properties; inhibition imaging of A549-FAP and U87 model showed that the tumor uptake decreased significantly, which was specific uptake (as shown in Figure 10a and Figure 10b ), while there was almost no uptake in A549 wild-type lung adenocarcinoma model.

[0157] In the U87 model 18 F-TCO-FAPI and 18 F-FAPI-42 PET imaging comparative study: 3 U87 tumor-bearing mice were taken, weighed, and then 18 F-TCO-FAPI and 18 F-NOTA-FAPI-42 were injected into the same mouse respectively, and PET / CT imaging was performed at 2h, 4h and 6h after injection. In the same U87 tumor mouse model 18 F-TCO-FAPI and 18 F-NOTA-FAPI-42 comparative imaging results showed that: 18 F-TCO-FAPI had high tumor uptake value and long retention time, and in normal organs and tissues, 18F-TCO-FAPI showed higher uptake in spine and joints, and lower uptake in intestine, liver and kidney. 18 F-NOTA-FAPI-42 showed lower uptake in tumor, and obvious uptake in joints, spine and intestine. 18 F-TCO-FAPI showed better imaging performance than 18 F-NOTA-FAPI-42.

[0158] Example 16 18 F-Tz-α v β6L micro-PET imaging experiment

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

[0160] Tumor-bearing mouse in vivo static PET imaging: each mouse is weighed and injected with 260 μCi of imaging agent into the tail vein 18 F-Tz-α v β6L, PET / CT imaging is performed at 30 min, 60 min, and 2 h after injection.

[0161] The imaging results show that: 18 F-Tz-α v β6L The peak uptake in tumor is obtained at 30 min, but overall, the tumor uptake is low and the retention time is not long, mainly excreted through the kidneys, with high uptake in the kidneys, heart and lungs, and the column chart results are shown in Figure 11a and Figure 11b .

[0162] Example 17 Targeting Compound Bimolecular Probe 18 F-TCO-FAPI+ 18 F-Tz-α v β6L (1:1) and 18 F-TCO-FAPI and 18 F-Tz-α v β6L micro-PET imaging comparison experiment

[0163] The method for establishing subcutaneous tumor model is as follows: 3 five-week-old nude mice are taken, and each mouse is inoculated with Capan-2 human pancreatic cancer cells in the right upper limb axilla according to the method of Example 12. When the tumor grows to about 0.5-1.0 cm, in vivo micro-PET imaging experiment is performed.

[0164] In vivo static PET imaging of tumor-bearing mice: In the first experiment, each mouse was weighed and injected with about 240 μCi of imaging agent via the tail vein 18 F-TCO-FAPI 18 F-Tz-α v β6L (1:1) showed higher tumor uptake and showed no difference in long time point imaging than 18 F-TCO-FAPI, PET / CT imaging was performed at 30 min, 60 min, 2 h, 4 h, 6 h after injection. In the third experiment, the same mouse was weighed and injected with about 240 μCi of imaging agent via the tail vein, and PET / CT imaging was performed at 30 min, 60 min, 2 h, 4 h, 6 h after injection.

[0165] The PET results are shown in Figure 11c The imaging results show that the targeting compound 18 F-TCO-FAPI 18 F-Tz-α v β6L (1:1) showed higher tumor uptake and showed no difference in long time point imaging than 18 F-TCO-FAPI monomer imaging, 18 F-TCO-FAPI 18 F-Tz-α v β6L excreted more slowly in the tumor than 18 F-TCO-FAPI, which may be due to 18 F-TCO-FAPI 18 F-Tz-α v β6L bound more in long time point imaging. 18 F-TCO-FAPI 18 F-Tz-α v The tumor uptake of β6L was greater than 18 F-TCO-FAPI and 18 F-Tz-α v β6L tumor half-uptake value, which confirmed from the imaging level that this experiment had a biological orthogonal effect, increased tumor uptake, and prolonged retention time. However 18 F-TCO-FAPI 18 F-Tz-α v β6L also showed higher uptake in the heart, lungs, kidneys, spine, and joints, showing the imaging characteristics of the binding of the two monomer imaging agents 18 F-TCO-FAPI and 18 F-Tz-α v β6L. However, it is worth noting that in the imaging18 F-TCO-FAPI 18 F-Tz-alpha v Beta6L shows high uptake in heart and lung, which is inconsistent with the performance characteristics of in vivo biodistribution.

[0166] The present application establishes a set of complete and streamlined synthesis and quality control methods for preparing a FAP-targeted molecular probe 18 F-TCO-FAPI, targeting integrin alpha v Beta6 molecular probe 18 F-Tz-alpha v Beta6L and a compound-targeted dual-molecular probe 18 F-TCO-FAPI 18 F-Tz-alpha v A set of complete and streamlined synthesis and quality control methods for Beta6L (1:1), and its application in the preparation of positron emission tomography (PET) small molecule imaging agents, in the construction of tumor-targeted compound drug biological orthogonal amplification systems, and in the development of compound drug biological orthogonal diagnosis and treatment new technologies. The present application can play an important diagnostic role in tumor and other disease PET imaging. For those skilled in the art, the structure and drugs can be improved or changed according to the above description, and all these improvements and changes should belong to the protection scope of the appended claims of the present application.

[0167] The technical features of the above embodiments can be combined in any way. The present description is brief and does not describe all possible combinations of the technical features in the above embodiments. However, as long as the combinations of the technical features do not contradict, they should be considered within the scope of the present description. Moreover, the above-described embodiments only express some of the embodiments of the present application, which are described in more detail and in detail, but should not be construed as limiting the scope of the patent. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application.

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 the following groups: ； The B component targets integrin α v β6 molecular probe targeting integrin α v β6 molecular probes include the following groups: ； The radioactivity ratio of the component A to the component B is (1-2): (2-1).

2. The targeted compound bimolecular probe according to claim 1, characterized in that: The molecular probe targeting fibroblast activation protein FAP further comprises a metal ion chelating group and a radioactive metal nuclide; The targeting integrin α v The β6 molecular probe also includes a metal ion chelating group and a radioactive metal nuclide.

3. The targeted compound bimolecular probe according to claim 2, characterized in that: The metal ion binding chelating group is 1,4,7-triazacyclononaalkyl-N',N"-diacetyl-N-acetyl or 1,4,7,10-tetraazacyclododecane-N',N'',N'''-triacetyl-N-acetyl.

4. The targeted compound 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 212 Pb, 64 Cu, 67 Cu, 198 Au, 225 Ac or 227 One of Th.

5. The targeted compound bimolecular probe according to claim 4, characterized in that: The targeted compound bimolecular probe is 18 F-labeled molecular probe targeting fibroblast activation protein FAP 18 F-TCO-FAPI and 18 F-labeled targeted integrin α v β6 molecular probe 18 F-Tz-α v A mixture of β6L, 18 F-TCO-FAPI, 18 F-Tz-α v β6L has the following structural formula: 、 。 6. A method for preparing a targeted compound bimolecular probe, for preparing the targeted compound bimolecular probe according to any one of claims 1 to 5, characterized in that: The following steps are involved: Preparation of component A: Using NOTA-TCO-FAPI or DOTA-TCO-FAPI as a precursor, a chelation reaction occurs with radioactive metal nuclide ions, and after separation and purification, a molecular probe targeting fibroblast activation protein FAP is prepared; 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 is separated and purified to prepare the targeted integrin α v β6 molecular probe; Preparation of the targeted compound bimolecular probe: Component A and component B are mixed 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 solid phase peptide synthesis method; The NOTA-TCO-FAPI has the following structural formula: ； The DOTA-TCO-FAPI has the following structural formula: ； The NOTA-Tz-α v β6L has the following structural formula: ； The DOTA-Tz-α v β6L has the following structural formula: 。 7. The preparation method according to claim 6, 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.

8. The use of the targeted composite bimolecular probe according to any one of claims 1 to 5, characterized in that: Application of the targeted compound bimolecular probe in the preparation of tumor diagnostic and therapeutic agents.

9. The use according to claim 8, characterized in that Application of the targeted compound bimolecular probe in the preparation of a tumor-targeting PET imaging agent; Application of the targeted composite bimolecular probe in the preparation of a tumor-targeting SPECT imaging agent; The targeted compound bimolecular probe is used in the preparation of a radioactive therapeutic agent targeting tumors.

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