CN119587726B - Targeting compound bi-molecular probe and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of nuclear medicine, and discloses a targeting compound double-molecule probe which comprises an A component and a B component marked by radioactive metal nuclides, wherein the A component is a targeting Fibroblast Activation Protein (FAP) molecular probe which comprises a trans-cyclooctene group (TCO), the B component is a targeting integrin alpha_vβ₆ molecular probe which comprises a tetrazine group (Tz), and the radioactivity ratio of the A component to the B component is (1-2) (2-1). The invention has the following technical effects that TCO in the targeting FAP molecular probe and Tz in the targeting integrin alpha_vβ₆ molecular probe can generate biological orthogonal reaction, thereby realizing three-level amplification effect, increasing tumor uptake value, prolonging retention time of the targeting compound double molecular probe, improving target/non-target ratio and enhancing nuclear medicine diagnosis and treatment effect. The invention also discloses a preparation method and application of the targeting compound bimolecular probe.

Description

Targeting compound bi-molecular probe and preparation method and application thereof

Technical Field

The invention belongs to the technical field of nuclear medicine, and particularly relates to a targeting compound bimolecular probe and a preparation method and application thereof.

Background

The occurrence and development of tumors such as tumors of liver and gall system, gastric cancer, lung cancer and the like increasingly threatens the lives of human beings, becomes a serious social problem, the tumors in China are ranked at the second place in various causes of death, and the death rate of the tumors is obviously increased. The key to reducing the death rate of the tumor is to study early diagnosis, specific targeted therapy and an optimal individuation accurate diagnosis and treatment technology for guiding the therapy, the nuclear medicine diagnosis and treatment integration comprises Positron Emission Tomography (PET) imaging, single Photon Emission Computed Tomography (SPECT) imaging and nuclide therapy, and the nuclear medicine diagnosis and treatment integration plays a unique role in tumor diagnosis and treatment, so that the tumor visual diagnosis and treatment problem can be accurately solved. The radioactive targeting molecular probe is used as an important marker in the nuclear medicine diagnosis and treatment integration, and becomes a key for improving the nuclear medicine diagnosis and treatment effect.

The currently adopted radioactive targeting molecular probes are mostly single targets, the diagnostic effect of the single-target radioactive monomer molecular probes on tumors is not ideal, the defects of false negative results and low sensitivity are easy to develop, and the defects of low uptake value of the molecular probes and insufficient retention time of the focus molecular probes still exist in positive focuses.

Bio-orthogonal chemical reactions are widely used for in vivo reactions due to their high specificity, high reaction rate and good biocompatibility. The prior art discloses a method for preparing a TGF beta antibody probe by using a bioorthogonal reaction-click chemistry technology, which comprises the steps of modifying TCO-PEG4-NHS on a TGF beta antibody to obtain the TGF beta antibody-TCO, modifying NOTA on tetrazine small molecule Tz to obtain NOTA-Tz, reacting¹⁸F、Al Cl₃ with the NOTA-Tz to obtain [¹⁸F-AlF₃ ] -NOTA-Tz, incubating the TGF beta antibody-TCO and [¹⁸F-AlF₃ ] -NOTA-Tz ligand together, and generating the bioorthogonal reaction to generate the [¹⁸F-AlF₃ ] -NOTA-Tz-TCO-TG F beta antibody probe. The antibody probe prepared by the method can be used for detecting the expression level of TGF beta in tissues such as tumors, but the antibody probe is still a single-target monomer probe, the imaging of the antibody probe cannot obtain more accurate and comprehensive tumor biological information, and the diagnosis and treatment effects on tumors are still not ideal.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides a targeting compound double-molecule probe, through the combination of a targeting FAP molecular probe and a targeting integrin alpha_vβ₆ molecular probe, the imaging or treatment can be simultaneously carried out on two target targets, the diagnosis and treatment effects of tumors are improved, meanwhile, the TCO in the targeting FAP molecular probe and the Tz in the targeting integrin alpha_vβ₆ molecular probe can generate biological orthogonal reaction, and the three-stage amplification effect is realized. The invention also provides a preparation method and application of the targeting compound bimolecular probe.

The technical effects to be achieved by the invention are realized by the following technical aspects:

in a first aspect, the invention provides a targeting compound bimolecular probe comprising a component a and a component B, respectively labeled with a radiometal nuclide;

the A component 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 targeting integrin alpha_vβ₆ molecular probe, and the targeting integrin alpha_vβ₆ molecular probe comprises a tetrazine group (Tz);

the ratio of the radioactivity of the component A to the radioactivity of the component B are (1-2): (2-1), and preferably 1:1.

It should be noted that the A component is not limited to the targeting FAP molecular probe, but can be other targeting molecular probes containing TCO, and the B component is not limited to the targeting integrin alpha_vβ₆ molecular probe, but can be other targeting molecular probes containing Tz.

As a further description of the technical scheme of the invention, the targeted FAP molecular probe further comprises a FAP inhibitor (FAPI) pharmacophore, a hydrophilic linking group, a metal ion binding chelating group and a radiometal nuclide (^* M), which is abbreviated as^* M-TCO-FAPI;

The targeting integrin alpha_vβ₆ molecular probe also comprises an integrin alpha_vβ₆ receptor ligand (alpha_vβ₆ L) pharmacophore, a hydrophilic linking group, a metal ion binding chelating group and a radioactive metal nuclide (^* M), which is called^*M-Tz-α_vβ₆ L for short.

As a further description of the technical scheme of the present invention, the hydrophilic linking group comprises a di-polyethylene glycol group (PEG 2) and/or a tri-polyethylene glycol group (PEG 3), two aspartic acid groups (Asp 2) and a lysine group (Lys).

As a further description of the technical scheme of the invention, the metal ion-binding chelating group is 1,4, 7-triazacyclononalkyl-N ', N' -diacetyl-N-acetyl (NOTA) or 1,4,7, 10-tetraazacyclododecane-N ', N' -triacetoxy-N-acetyl (DOTA).

As a further description of the technical solution of the present invention, the radionuclide (^* M) is one of¹⁸F、⁶⁸Ga、¹¹¹In、^99mTc、¹⁸⁶Re、¹⁸⁸Re、¹⁷⁵Yb、¹⁵³Sm、¹⁶⁶Ho、⁸⁸Y、⁹⁰Y、¹⁷⁷Lu、⁴⁷Sc、²¹²Bi、²¹³Bi、¹²³I、¹²⁴I、¹³¹I、²¹¹At、¹⁵³Eu、¹⁶⁹Eu、²¹²Pb、⁶⁴Cu、⁶⁷Cu、¹⁸⁸Re、¹⁸⁶Re、¹⁹⁸Au、²²⁵Ac or²²⁷ Th, preferably one of⁶⁸Ga、¹⁸F、⁶⁴ Cu or¹⁷⁷ Lu. More preferably, NOTA binds to [ Al¹⁸F]²⁺、⁶⁸Ga³⁺、⁶⁴Cu²⁺ or¹⁷⁷Lu³⁺ ] and DOTA binds to⁶⁸Ga³⁺、⁶⁴Cu²⁺ or¹⁷⁷Lu³⁺.

As further description of the technical scheme of the invention, the targeting compound double-molecule probe is a mixture of¹⁸ F-marked targeting FAP molecular probe (¹⁸ F-TCO-FAPI) and¹⁸ F-marked targeting integrin alpha_vβ₆ molecular probe (¹⁸F-Tz-α_vβ₆ L), which is called¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L for short (radioactivity ratio is 1:1). The chemical structural formula of¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L is as follows:

It should be noted that, the FAPI, TCO, hydrophilic linking group, metal ion chelating group and radionuclide (^* M) can be freely combined to synthesize different targeted FAP molecular probes according to different types, and the α_vβ₆ L, tz, hydrophilic linking group, metal ion chelating group and radionuclide (^* M) can be freely combined to synthesize different targeted integrin α_vβ₆ molecular probes according to different types.

In a second aspect, the present invention provides a method for preparing a targeting compound bimolecular probe, for preparing the targeting compound bimolecular probe, comprising the steps of:

The preparation of the component A comprises the steps of taking NOTA-TCO-FAPI or DOTA-TCO-FAPI as a precursor raw material, carrying out chelation reaction with radioactive metal nuclide ions, and separating and purifying by a small column to obtain a targeted FAP molecular probe^* M-TCO-FAPI;

The preparation of the component B comprises the steps of taking NOTA-Tz-alpha_vβ₆ L or DOTA-Tz-alpha_vβ₆ L as a precursor raw material, carrying out chelation reaction with radionuclide ions, and separating and purifying by a small column to obtain a targeted integrin alpha_vβ₆ molecular probe^*M-Tz-α_vβ₆ L;

the preparation of the targeting compound bimolecular probe comprises the steps of mixing the component A and the component B according to the radioactivity ratio of (1-2) to (2-1) to obtain the targeting compound bimolecular probe.

It should be noted that, in addition to^* M-TCO-FAPI and^*M-Tz-α_vβ₆ L, the targeting compound bi-molecular probe may further contain two precursor materials NOTA-TCO-FAPI (or DOTA-TCO-FAPI) and NOTA-Tz-alpha_vβ₆ L (or DOTA-Tz-alpha_vβ₆ L), two targeting monomer molecular probes^* M-TCO-FAPI and^*M-Tz-α_vβ₆ L, and molecular probes^*M-α_vβ₆L-Tz-TCO-FAPI、^*M-FAPI-TCO-Tz-α_vβ₆ L and^*M-FAPI-TCO-Tz-α_vβ₆L-^* M where Tz is bound to TCO.

Wherein the precursor raw material is prepared by adopting a solid-phase polypeptide synthesis method. The preparation method comprises the steps of carrying out FAPI pharmacophore modification-PEG 2 to form FAPI pharmacophore-PEG 2, carrying out FAPI pharmacophore-PEG 2 modification-Asp 2 modification, carrying out Lys connection-PEG 3 modification-TCO, carrying out final modification of chelating group-NOTA or-DOTA, carrying out separation and purification by preparative HPLC, and collecting product peaks to obtain purified precursor raw materials NOT A-TCO-FAPI or DOTA-TCO-FAPI, carrying out alpha_vβ₆ L pharmacophore modification-Lys and then connecting with a Tz group, further carrying out-PEG 2 modification to form alpha_vβ₆ L pharmacophore-PEG 2, carrying out alpha_vβ₆ L pharmacophore-PEG 2 modification-Asp 2 and then connecting with chelating group-NOTA or-DOTA, and carrying out separation and purification by preparative HPLC to collect product peaks to obtain purified precursor raw materials NOT A-Tz-alpha_vβ₆ L or DOTA-Tz-alpha_vβ₆ L.

The chemical structural formula of NOTA-TCO-FAPI is as follows:

the chemical structural formula of DOTA-TCO-FAPI is as follows:

The chemical structural formula of NOTA-Tz-alpha_vβ₆ L is as follows:

DOTA-Tz-alpha_vβ₆ L has the chemical structural formula:

As a further description of the technical solution of the present invention, the radionuclide ion is one of⁶⁸Ga³⁺、[Al¹⁸F]²⁺、⁶⁴Cu²⁺ or¹⁷⁷Lu³⁺.

Preferably, the preparation method of the targeting compound bimolecular probe comprises the following steps:

Preparation of (one)^* M-TCO-FAPI

¹⁸ F-TCO-FAPI is prepared by taking NOTA-TCO-FAPI as a precursor raw material, reacting NOTA-TCO-FAPI with¹⁸F^- in an acid solution of AlCl₃ and acetonitrile, heating at 90-100 ℃ for 10-20min, cooling, and separating and purifying by using SEP-PAK C18 small column to obtain¹⁸ F-TCO-FAPI.

⁶⁸ The preparation of Ga-TCO-FAPI comprises the steps of taking NOTA-TCO-FAPI or DOTA-TCO-FAPI as a precursor raw material, reacting the NOTA-TCO-FAPI or DOTA-TCO-FAPI with a weakly acidic⁶⁸Ga³⁺ solution under the heating of 85-110 ℃, and separating and purifying by using an HLB column or an SEP-PAK C18 column to obtain⁶⁸ Ga-TCO-FAPI.

⁶⁴ The preparation of Cu-TCO-FAPI includes taking NOTA-TCO-FAPI or DOTA-TCO-FAPI as precursor raw material, carrying out incubation reaction on the NOTA-TCO-FAPI or DOTA-TCO-FAPI and⁶⁴Cu²⁺ in sodium acetate acid buffer solution for 15min at room temperature, and separating and purifying by HLB column or SEP-PAK C18 column to obtain⁶⁴ Cu-TCO-FAPI.

¹⁷⁷ Preparation of Lu-TCO-FAPI by heating NOTA-TCO-FAPI or DOTA-TCO-FAPI as precursor material in a water bath at 106 deg.C for 15min in a mixed solution of gentisic acid and sodium acetate of NOTA-TCO-FAPI or DOTA-TCO-FAPI and¹⁷⁷ Lu. Separating by HPLC, and purifying by SEP-PAK C18 small column to obtain¹⁷⁷ Lu-TCO-FAPI.

(II) preparation of^*M-Tz-α_vβ₆ L

¹⁸F-Tz-α_vβ₆ Preparation of L¹⁸F-Tz-α_vβ₆ L was obtained by using NOTA-Tz-alpha_vβ₆ L as a precursor material and referring to the preparation method of¹⁸ F-TCO-FAPI.

⁶⁸Ga-Tz-α_vβ₆ Preparation of L by using NOTA-Tz-alpha_vβ₆ L or DOTA-Tz-alpha_vβ₆ L as precursor raw material and referring to a preparation method of⁶⁸ Ga-TCO-FAPI to obtain⁶⁸Ga-Tz-α_vβ₆ L.

⁶⁴Cu-Tz-α_vβ₆ Preparation of L by using NOTA-Tz-alpha_vβ₆ L or DOTA-Tz-alpha_vβ₆ L as precursor raw material and referring to a preparation method of⁶⁴ Cu-TCO-FAPI to obtain⁶⁴Cu-Tz-α_vβ₆ L.

¹⁷⁷Lu-Tz-α_vβ₆ Preparation of L by using NOTA-Tz-alpha_vβ₆ L or DOTA-Tz-alpha_vβ₆ L as precursor raw material, and referring to a preparation method of¹⁷⁷ Lu-TCO-FAPI to obtain¹⁷⁷Lu-Tz-α_vβ₆ L.

And (III) preparing the targeting compound bimolecular probe, namely mixing any^* M-TCO-FAPI with any^*M-Tz-α_vβ₆ L according to a radioactivity ratio of 1:1 to obtain the targeting compound bimolecular probe.

In a third aspect, the invention provides an application of the targeting compound bimolecular probe in preparing tumor diagnosis and treatment agents, including an application in preparing various tumors expressing FAP and/or various tumor diagnosis and treatment agents expressing alpha_vβ₆.

As further description of the technical scheme of the invention, the application of the targeting compound bimolecular probe in preparing a PET imaging agent for tumor, a pre-positioned PET imaging agent and a compound bio-orthogonal PET imaging agent comprises the application of the targeting compound bimolecular probe in preparing various tumors such as lung adenocarcinoma and brain glioma with higher expression of FAP and/or various tumor PET imaging agents such as pancreatic cancer, lung cancer, cholangiocellular carcinoma, gastric cancer, breast cancer or ovarian cancer with higher expression of alpha_vβ₆;

the targeted compound double-molecule probe is applied to the preparation of a SPECT (single photon emission computed tomography) imaging agent of tumors, a pre-positioning SPECT imaging agent and a compound bio-orthogonal SPECT imaging agent;

the targeting compound double-molecule probe is applied to preparation of a targeting radioactive therapeutic agent, a double-targeting radioactive therapeutic agent, a preset targeting radioactive therapeutic agent and a compound bio-orthogonal double-targeting radioactive therapeutic agent.

The application of the targeting compound bimolecular probe in the diagnosis and treatment of tumors can establish a compound biological orthogonal amplification diagnosis and treatment system. The compound biological orthogonal amplification diagnosis and treatment system has the characteristics of compound medicine, multi-targeting property, biological orthogonal reaction and the like, has obvious advantages in the aspects of cost, sensitivity, imaging efficiency and the like, can realize simultaneous imaging of various tumor diseases in one imaging, and has higher clinical value.

In summary, the present invention has at least the following advantages:

1. The targeting compound double-molecule probe provided by the invention can simultaneously carry out imaging or treatment on two target targets by only one administration, increases the total number of binding sites with target molecules, displays more tumor focuses, improves the detection sensitivity and treatment effect of tumors, and simultaneously, the TC O in the targeting FAP molecular probe and the Tz in the targeting integrin alpha_vβ₆ molecular probe can carry out biological orthogonal reaction to realize three-level amplification effect, increase the tumor uptake value, prolong the retention time of the targeting compound double-molecule probe, improve the target/non-target uptake ratio and enhance the diagnosis and treatment effect of nuclear medicine.

2. According to the targeting compound double-molecule probe provided by the invention, the selected targeting FAP molecular probe and targeting integrin alpha_vβ₆ molecular probe are optimized through chemical structures, so that the hydrophilicity is increased, the uptake of a liver and gall system and an intestinal tract system is reduced, the excretion of kidneys is promoted, and the in vivo pharmacokinetics characteristic of the molecular probe is improved. Especially¹⁸ F-TCO-FAPI has high affinity to a tumor FAP target, can be competitively inhibited by an inhibitor, and also has excellent in vivo pharmacokinetics characteristics, wherein¹⁸ F-TCO-FAPI has higher uptake in tumors, long retention time and higher uptake at 6 hours.

3. According to the preparation method of the targeting compound double-molecule probe, the targeting FAP molecular probe and the targeting integrin alpha_vβ₆ molecular probe are respectively prepared by a method of chelating reaction of precursor raw materials and radioactive metal nuclide ions and then separation and purification, and then are mixed according to the optimal radioactivity ratio to prepare the targeting compound double-molecule probe. Can realize the synthesis of the molecular probe with high radiochemical purity and high radiochemical yield, and has simple preparation steps and convenient operation.

4. The application of the targeting compound bimolecular probe provided by the invention applies the targeting compound bimolecular probe to a tumor diagnosis and treatment agent, and provides a novel tumor diagnosis and treatment mode of one-time administration acting on double targets. Is beneficial to promoting the accurate nuclear medicine diagnosis and curative effect evaluation of tumors, solves the difficult problems of accurate visual diagnosis and treatment and evaluation, and has higher clinical value.

5. The invention has the greatest advantages that the invention relates to the three-level amplification effect of a compound biological orthogonal diagnosis and treatment system, and comprises a first-level compound amplification system, a second-level biological orthogonal probe amplification system and a three-level double biological orthogonal reaction amplification system, wherein the diagnosis and treatment amplification system is provided with a predetermined diagnosis and treatment amplification system, and an in-vivo and in-vitro biological activity evaluation verification method is established.

Drawings

FIG. 1 is a schematic diagram of an imaging mode of a targeting compound bilayer probe;

FIG. 2 is a schematic diagram of a three-stage amplification effect mode of a dual-targeting compound bio-orthogonal diagnosis and treatment system;

FIG. 3a is an HPLC analysis chart of precursor raw material NOTA-TCO-FAPI;

FIG. 3b is an HPLC analysis chart of precursor raw material NOTA-Tz-alpha_vβ₆ L;

FIG. 3c is a MS analysis chart of precursor raw material NOTA-TCO-FAPI;

FIG. 3d is a MS analysis profile of precursor raw material NOTA-Tz-alpha_vβ₆ L;

Fig. 4a is a representative radioactive HPLC analysis profile of injection¹⁸ F-TCO-FAPI at a radioactive retention time rt=11.23 min;

Fig. 4b is a representative uv absorption HPLC analysis profile of injection¹⁸ F-TCO-FAPI at uv absorption retention time rt=10.92 min;

fig. 4c is a representative radioactive HPLC analysis profile of injection¹⁸F-Tz-α_vβ₆ L at the radioactivity retention time rt=10.65 min;

fig. 4d is a representative uv absorption HPLC analysis profile of injection¹⁸F-Tz-α_vβ₆ L at uv absorption retention time rt=10.59 min;

FIG. 5a is a 1h representative radioactive HPLC analysis profile of serum of injection¹⁸ F-TCO-FAPI in Kunming mice;

FIG. 5b is a 1h representative radioactive HPLC analysis profile of injection¹⁸ F-TCO-FAPI in urine in Kunming mice;

FIG. 5c is a 2h representative radioactive HPLC analysis profile of injection¹⁸ F-TCO-FAPI in vitro in PBS;

FIG. 5d is a 2h representative radioactive HPLC analysis profile of injection¹⁸ F-TCO-FAPI in vitro fetal bovine serum;

FIG. 6a is a 1h representative radioactive HPLC analysis profile of serum of injection¹⁸F-Tz-α_vβ₆ L in Kunming mice;

FIG. 6b is a 1h representative radioactive HPLC analysis profile of urine from a Kunming mouse in vivo injected¹⁸F-Tz-α_vβ₆ L;

FIG. 6c is a 2h representative radioactive HPLC analysis profile of injection¹⁸F-Tz-α_vβ₆ L in vitro in PBS;

FIG. 6d is a 2h representative radioactive HPLC analysis profile of injection¹⁸F-Tz-α_vβ₆ L in vitro fetal bovine serum;

FIG. 7a is a graph showing the results of cell uptake and inhibition experiments for¹⁸ F-TCO-FAPI;

FIG. 7b is a graph showing the results of a¹⁸ F-TCO-FAPI tumor cell competitive inhibition assay (IC 50 values);

FIG. 8 is a mass spectrometry detection plot of in vitro biorthogonal product FAPI-TCO-Tz-alpha_vβ₆;

FIG. 9a is a diagram showing the biological profile of¹⁸ F-TCO-FAPI in tumor-bearing mice (A549-FAP, A549) for 60min and the biological profile of 60min competitive inhibition in tumor-bearing mice A549-FAP;

FIG. 9b is a diagram showing a biological profile of¹⁸F-Tz-α_vβ₆ L in a tumor-bearing mouse (Capan-2) for 60 min;

FIG. 9c is a graph comparing the in vivo biodistribution of the targeting compound bilayer probe¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L (radioactivity ratio 1:1) with¹⁸ F-TCO-FAPI and¹⁸F-Tz-α_vβ₆ L;

FIG. 10a is a PET/CT image of¹⁸ F-TCO-FAPI at 60min for a549-FAP lung adenocarcinoma tumor-bearing murine model with static uptake (left) and competitive inhibition (right);

FIG. 10b is a PET/CT image of¹⁸ F-TCO-FAPI at 60min of a murine model of U87 glioma tumor-bearing with static uptake (left) and competitive inhibition (right);

FIG. 11a is a bar graph of 18F-Tz- αvβ6L in a Capan-2 tumor-bearing murine model for 2h, 4h, 6 h;

FIG. 11b is a bar graph of 18F-Tz- αvβ6L for 2h, 4h, 6h in BxPC-3 tumor-bearing murine model;

FIG. 11c is a PET/CT image of a targeting compound bimolecular probe¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L (radioactivity ratio 1:1) and¹⁸ F-TCO-FAPI and¹⁸F-Tz-α_vβ₆ L at 30min, 60min, 2h, 4h, 6 h.

Detailed Description

In order that the invention may be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

As a preferred implementation mode, the targeting compound double-molecule probe is a mixture of a targeting FAP molecular probe¹⁸ F-TCO-FAPI and a targeting integrin alpha_vβ₆ molecular probe¹⁸F-Tz-α_vβ₆ L, and the targeting compound double-molecule probe and the targeting integrin alpha_vβ₆ molecular probe are mixed according to a radioactivity ratio of 1:1, and are called¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L for short.

The invention realizes the three-stage amplification effect of the double-targeting compound biological orthogonal diagnosis and treatment system by carrying out biological orthogonal reaction on TCO in the targeting FAP molecular probe and Tz in the targeting integrin alpha_vβ₆ molecular probe in vivo, increases the tumor uptake value and prolongs the retention time of the targeting compound double-molecular probe. The biological orthogonal chemical reaction formula based on IEDDA between the compound biological orthogonal bimolecular probes is as follows:

Wherein R isR' is

The imaging mode of the targeting compound bimolecular probe is shown in figure 1, and the three-level amplification effect mode of the double targeting compound bioorthogonal diagnosis and treatment system is shown in figure 2.

The three-stage amplification effect of the double-targeting compound biological orthogonal diagnosis and treatment system comprises a first-stage compound double-molecular probe amplification system, a second-stage biological orthogonal probe amplification system and a third-stage double-biological orthogonal reaction amplification system, and besides, the double-targeting compound biological orthogonal diagnosis and treatment system is provided with a prepositioning diagnosis and treatment amplification system, and an in-vivo and in-vitro biological activity evaluation verification method can be established.

The primary compound bimolecular probe amplification system is specifically a system for combining target compound bimolecular probe^*M-TCO-FAPI+^*M-Tz-α_vβ₆ L with target FAP and integrin alpha_vβ₆ in tumor respectively. The following experiments prove that^* M-TCO-FAPI specifically targets FAP^* M-TCO-FAPI can be taken up and inhibit PET imaging (the inhibitor is NOTA-FAPI-42) in a FAP positive model (U87 brain glioma, A549-FAP lung adenocarcinoma cells). The following experiments demonstrate that^*M-Tz-α_vβ₆ L of the specific targeting target integrin α_vβ₆：^*M-Tz-α_vβ₆ L uptake and inhibit PET imaging in the α_vβ₆ positive model (cap-2 human pancreatic cancer). Through verification, the targeting compound bi-molecular probe^*M-TCO-FAPI+^*M-Tz-α_vβ₆ L is proved to target two targets respectively.

The secondary bioorthogonal probe amplification system is specifically a system for combining TCO in the targeting compound bimolecular probe^*M-TCO-FAPI+^*M-Tz-α_vβ₆ L with Tz to form^*M-FAPI-TCO-Tz-α_vβ₆L-^*M,^*M-FAPI-TCO-Tz-α_vβ₆L-^*M which is respectively combined with target FAP and integrin alpha_vβ₆ in tumor. The preparation method is verified by mixing a targeting FAP molecular probe¹⁸ F-TCO-FAPI and a targeting integrin alpha_vβ₆ molecular probe¹⁸F-Tz-α_vβ₆ L according to a 1:1 radioactivity ratio to prepare a targeting compound double molecular probe¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L, and verifying that¹⁸F-FAPI-TCO-Tz-α_vβ₆L-¹⁸ F is generated by using an HPLC analysis method. Through molecular docking verification, NOTA-TCO-FAPI, NOTA-Tz-alpha_vβ₆ L and NOTA-FAPI-TCO-Tz-alpha_vβ₆ L-NOTA are combined with FAP and alpha_vβ₆ targets respectively. The tumor-bearing model imaging experiment proves that¹⁸ F-TCO-FAPI is injected into a FAP and alpha_vβ₆ double-target positive model (Capan-2 human pancreatic cancer),¹⁸ F-TCO-FAPI is combined with tumors after a period of time, the radioactive uptake value of the tumors is increased after¹⁸F-Tz-α_vβ₆ L is injected,¹⁸ F-TCO-FAPI is injected firstly after a period of time, NOTA-Asp2-alpha_vβ₆L+¹⁸F-Tz-α_vβ₆ L is injected secondly, tumor uptake is partially inhibited, inhibition of alpha_vβ₆ L in¹⁸F-Tz-α_vβ₆ L and the effect of a target alpha_vβ₆ are indicated,¹⁸ F-TCO-FAPI is injected firstly on the third day, NOTA-Tz+¹⁸F-Tz-α_vβ₆ L is injected after a period of time, and biological orthogonal combination of Tz in¹⁸F-Tz-α_vβ₆ L and TCO in¹⁸ F-TCO-FAPI is indicated.

The three-stage bioorthogonal reaction amplification system specifically comprises a targeting compound bimolecular probe^*M-TCO-FAPI+^*M-Tz-α_vβ₆ L which is combined with a target FAP and integrin alpha_vβ₆ in tumor respectively, and then combined with TCO in^* M-TCO-FAPI and Tz in free^*M-Tz-α_vβ₆ L, And binding of Tz in^*M-Tz-α_vβ₆ L after tumor binding to TCO in free^* M-TCO-FAPI, i.e. TCO-Tz double binding system. The biological orthogonal binding of the tumor-bound targeting single-molecule probe with the free single-molecule probe in vivo can be verified by pre-injecting^* M-TCO-FAP I in a FAP single-cation model (U87 brain glioma), binding the tumor with^* M-TCO-FAPI after a period of time, injecting^*M-Tz-α_vβ₆ L again, increasing the radioactive uptake value of the tumor, pre-injecting^* M-TCO-FAPI the next day, injecting Tz-alpha_vβ₆L+^*M-Tz-α_vβ₆ L again after the same period of time, decreasing the radioactive uptake value of the tumor, demonstrating that TCO in^* M-TCO-FAPI is bound with Tz in the free^*M-Tz-α_vβ₆ L, pre-injecting M-Tz-alpha_vβ₆ L in an alpha_vβ₆ L single-cation model (A549 brain glioma), binding the tumor with M-Tz-alpha_vβ₆ L after a period of time, injecting^* M-FAPI again, pre-injecting^*M-Tz-α_vβ₆ L again after a period of time, and pre-injecting TCO-FAPI +^* M-TCO-3842L again, and demonstrating that binding the tumor with Tz-^*M-Tz-α_vβ₆ L in the free single-cation model (A549 brain glioma).

In addition, the prepositioning diagnosis and treatment amplification system needs to simultaneously give a certain amount of unmarked NOTA-TCO-FAPI +NOTA-Tz-alpha_vβ₆ L and marked^*M-TCO-FAPI+^*M-Tz-α_vβ₆ L, and the action mechanism of the prepositioning diagnosis and treatment amplification system can also relate to the prepositioning diagnosis and treatment amplification effect besides the three-stage diagnosis and treatment amplification effect. The pre-positioning imaging mode can increase tumor uptake and reduce normal tissue uptake through the following experiment, and the pre-positioning diagnosis and treatment system has the amplification potential that^*M-Tz-α_vβ₆ L performs PET imaging in a FAP single-positive model (U87 brain glioma), the tumor does not ingest or ingest is lower, the precursor TCO-FAPI is injected in advance the next day, after a period of time, TCO-FAPI is combined with the tumor, PET imaging is performed after^*M-Tz-α_vβ₆ L of re-injection, the tumor ingest is obviously increased, the precursor TCO-FAPI is injected in advance the next day, and after the same period of time, tz-alpha_vβ₆L+^*M-Tz-α_vβ₆ L is injected, and the radioactive uptake value of the tumor is reduced.^* The M-TCO-FAPI is subjected to PET imaging in an alpha_vβ₆ L single positive model (A549 brain glioma), the tumor does not take or takes less, the precursor Tz-alpha_vβ₆ L is injected in advance the next day, the Tz-alpha_vβ₆ L is combined with the tumor after a period of time, the radioactive uptake value of the tumor is increased by injecting^* M-TCO-FAPI, and the radioactive uptake value of the tumor is reduced by injecting TCO-FAPI +^* M-TCO-FAPI the next day.

The following describes the targeting compound bimolecular probe of the present invention in detail with reference to specific examples, and a preparation method and application thereof.

EXAMPLE 1 preparation of precursor starting materials NOTA-TCO-FAPI and DOTA-TCO-FAPI

After the FAP pharmacophore is modified by-PEG 2 to form the FAP pharmacophore-PEG 2, after the FAP pharmacophore-PEG 2 is modified by Asp2, the FAP pharmacophore-PEG 2 is connected with TCO through Lys and-PEG 3 linker, and finally the chelate group-NOTA is modified, the product peak is collected through separation and purification by preparative HPLC, and then the freeze drying is carried out, so that the purified precursor raw material NOTA-TCO-FAPI is prepared.

The chemical yield of NOTA-TCO-FAPI is high, and the purity is more than 95%. The HPLC and MS measurements of NOTA-TCO-FAPI are shown in FIGS. 3a and 3c, respectively, and the mass spectrum MS (m/z) measured NOTA-TCO-FAPI molecular weight (Mr.) as 1630.76.

The preparation method of DOTA-TCO-FAPI can refer to NOTA-TCO-FAPI, and is not described herein, and the DOTA-TCO-FAPI has higher chemical yield and purity of more than 95% by HPLC and MS measurement.

EXAMPLE 2 preparation of precursor raw materials NOTA-Tz-alpha_vβ₆ L and DOTA-Tz-alpha_vβ₆ L

The targeting alpha_vβ₆ L pharmacophore in the alpha_vβ₆ ligand is modified to be connected with a Tz group, and is further modified by-PEG 2 to form alpha_vβ₆ L pharmacophore-PEG 2, and alpha_vβ₆ L pharmacophore-PEG 2 is modified to be connected with a chelating group-NOTA. The product peak is collected by separation and purification through preparative HPLC, and then the purified precursor raw material NOTA-Tz-alpha_vβ₆ L is prepared through freeze drying.

The chemical yield of NOTA-Tz-alpha_vβ₆ L is high, and the purity is more than 95%. The results of the HPLC and MS measurements of NOTA-Tz-alpha_vβ₆ L are shown in FIG. 3b and FIG. 3d, respectively, and the mass spectrum MS (m/z) measured NOTA-Tz-alpha_vβ₆ L molecular weight (Mr.) as 2150.34.

The preparation method of DOTA-Tz-alpha_vβ₆ L can refer to NOTA-Tz-alpha_vβ₆ L, which is not described herein, and DOTA-Tz-alpha_vβ₆ L has higher chemical yield and purity of more than 95 percent by HPLC and MS measurement.

Example 3 preparation of¹⁸ F-TCO-FAPI and¹⁸F-Tz-α_vβ₆ L

To a reaction flask containing NOTA-TCO-FAPI (or NOTA-Tz-. Alpha._vβ₆ L) (50. Mu.g/. Mu.L, 50. Mu.L) was added 6. Mu.L of 2mM AlCl₃ solution, 5. Mu.L of glacial acetic acid and 300. Mu.L of acetonitrile in this order, followed by mixing.

¹⁸F^-, Produced by a¹⁸O(p,n)¹⁸ F nuclear reaction by a cyclotron, was trapped in a Sep-Pak QMA anion cartridge under an N2 carrier, and¹⁸O^- water was collected in a recovery bottle. Eluting¹⁸F^- of anion (QMA) columns into vials with 0.3-0.4 mL of physiological saline (or sodium acetate buffer), and adding 50 μl of the solution into the reaction vials.

After stirring and mixing evenly, the mixture is heated and reacted for 15min at 100 ℃. Cooling, adding 6-8 mL of water into a reaction bottle, uniformly mixing, and transferring into an HLB column or an SEP-PAK C18 column. After the transfer of the solution in the reaction flask was completed, the column was rinsed with 10ml×3 water for injection and dried.

Finally, 1.5mL of the eluted product was filtered through a sterile filter and collected in a receiving bottle, which was diluted with physiological saline to a 5% ethanol-containing product solution, to give¹⁸ F-TCO-FAPI (or¹⁸F-Tz-α_vβ₆ L) injection.

¹⁸ F-TCO-FAPI and¹⁸F-Tz-α_vβ₆ L have uncorrected radiochemical yields of 20-30% and total radiosynthesis times of 35min.

Example 4 preparation of⁶⁸ Ga-TCO-FAPI and⁶⁸Ga-Tz-α_vβ₆ L

200. Mu.L of 1.25M sodium acetate solution was added sequentially to a reaction flask containing NOTA-TCO-FAPI (or DOTA-TCO-FAPI, or NOTA-Tz-. Alpha._vβ₆ L, or DOTA-Tz-. Alpha._vβ₆ L) (50. Mu.g/. Mu.L, 50. Mu.L).

Eluting⁶⁸GaCl₃ from a⁶⁸Ge/⁶⁸ Ga generator by using 4mL of 0.05M hydrochloric acid into the reaction tube, uniformly mixing, adjusting the pH of the solution to 4.0,100 ℃ and heating for reaction for about 10-15 min. Cooling, adding 4mL of physiological saline into a reaction bottle, uniformly mixing, and transferring into an HLB column or an SEP-PAK C18 column. After the reaction flask had been completely transferred, the column was rinsed with 10ml×2 water for injection and dried.

The product was eluted with 1.5mL of ethanol and collected by sterile filtration into a receiving bottle, which was diluted with physiological saline to a 5% ethanol-containing product solution to give⁶⁸ Ga-TCO-FAPI (or⁶⁸Ga-Tz-α_vβ₆ L) injection.

⁶⁸ The uncorrected radiochemical yields of Ga-TCO-FAPI and⁶⁸Ga-Tz-α_vβ₆ L are 20-50%, and the total radiosynthesis time is 30min.

Example 5 preparation of⁶⁴ Cu-TCO-FAPI and⁶⁴Cu-Tz-α_vβ₆ L

Adding⁶⁴CuCl₂ solution 0.100-1.000mL into a reaction bottle with NOTA-TCO-FAPI (or DOTA-TCO-FAPI, or NOTA-Tz-alpha_vβ₆ L, or DOTA-Tz-alpha_vβ₆ L) (50 mug/mug, 100 mug), adjusting pH to 4.0-5.6 by sodium acetate solution, and reacting for 10-15 min at room temperature. Finally, the mixture is diluted by normal saline and filtered by a sterile filter membrane and then is collected in a receiving bottle, so as to obtain⁶⁴ Cu-TCO-FAPI (or⁶⁴Cu-Tz-α_vβ₆ L) injection.⁶⁴ The uncorrected radiochemical yields of Cu-TCO-FAPI and⁶⁴Cu-Tz-α_vβ₆ L are 50-70%.

Example 6 preparation of¹⁷⁷ Lu-TCO-FAPI and¹⁷⁷Lu-Tz-α_vβ₆ L

Gentisic acid, 160ul sodium acetate and 0.100-1.000mL of free¹⁷⁷ Lu solution were sequentially added to a reaction flask containing NOTA-TCO-FAPI (or DOTA-TCO-FAPI, or NOTA-Tz-. Alpha._vβ₆ L, or DOTA-Tz-. Alpha._vβ₆ L) (50. Mu.g/. Mu.L, 50. Mu.L) and heated in a 106℃water bath for 15min. The reaction effect was monitored by HPLC and isolated by HPLC. The separated product was passed through a C18 column, washed with 30mL of water, and passed through the column with 1mL of 50% ethanol, and the product was collected. Finally, the mixture is diluted by normal saline and filtered by a sterile filter membrane and then is collected in a receiving bottle, so as to obtain¹⁷⁷ Lu-TCO-FAPI (or¹⁷⁷Lu-Tz-α_vβ₆ L) injection.¹⁷⁷ The uncorrected radiochemical yields of Lu-TCO-FAPI and¹⁷⁷Lu-Tz-α_vβ₆ L are 40-60%.

EXAMPLE 7 preparation of Targeted Compound bilayer Probe¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L

¹⁸ F-TCO-FAPI injection and¹⁸F-Tz-α_vβ₆ L injection are mixed according to the radioactivity ratio of 1:1 to prepare the targeting compound bimolecular probe¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L injection.

Example 8 determination of the radiochemical purity and stability of the Targeted Compound bilayer Probe¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L

The radiochemical purity of¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L of the injection was determined by means of High Performance Liquid Chromatography (HPLC).

¹⁸ F-TCO-FAPI and¹⁸F-Tz-α_vβ₆ L HPLC analysis conditions the analytical column was a kromasil 100-5-C18 column. The mobile phase was 0.1% trifluoroacetic acid (TFA) in acetonitrile and 0.1% TFA in water, and the gradient elution was performed for 80/20 of the acetonitrile solution containing 0.1% TFA/0.1% TFA in water at 10/90 and the acetonitrile solution containing 0.1% TFA/0.1% TFA in water at 8 min. The flow rate was 1mL/min, the analysis time was 15min, and the UV detection wavelength was 254nm. The HPLC was co-injected with the corresponding radioactive¹⁸ F-TCO-FAPI injection using a nonradioactive standard¹⁹ F-TCO-FAPI of defined structure to determine if its retention time (Rt) or specific shift value Rf was consistent.

The radiochemical purity of the compounds is more than 95% as measured by an HPLC method. The results of the radioactive HPLC analysis of¹⁸ F-TCO-FAPI injection are shown in FIG. 4a and FIG. 4b (retention times of the radioactive peak and the ultraviolet peak are Rt=11.23 min and Rt=10.92 min, respectively).¹⁸F-Tz-α_vβ₆ As shown in fig. 4c and fig. 4d (radioactive peak and ultraviolet peak retention times rt=10.65 min and rt=10.59 min, respectively), only a single main peak was observed for each of the L injections.

HPLC analysis conditions for targeting compound bilayer probe¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L (1:1) were similar to¹⁸ F-TCO-FAPI and¹⁸F-Tz-α_vβ₆ L, except that the elution gradient was varied from 0min to 35min with 0.1% TFA in acetonitrile/0.1% TFA in water to 55/45 at 20/80. The flow rate was 1mL/min and the analysis time was 40min. Multiple major peaks were observed in the injection by radioactive HPLC. No obvious defluorination or decomposition phenomenon is found in the HPLC method for detecting the stability of¹⁸ F-TCO-FAPI in and out of the body. Wherein¹⁸ F-TCO-FAPI has an amplification purity in serum and urine as shown in FIGS. 5a and 5b, an amplification purity of greater than 90% in PBS buffer and in vitro serum for 2h (see FIGS. 5c and 5 d), an amplification purity of¹⁸F-Tz-α_vβ₆ L in serum and urine as shown in FIGS. 6a and 6b, and an amplification purity of greater than 90% in PBS buffer and in vitro serum for 2h (see FIGS. 6c and 6 d), each only a single major peak was found. The results show that¹⁸ F-TCO-FAPI and¹⁸F-Tz-α_vβ₆ L are stable in vivo and in vitro.

Example 9 Water distribution coefficient determination experiment of ester

The water distribution coefficient of the ester was determined by taking 3ml of n-octanol and water, respectively, and adding¹⁸ F-TCO-FAPI (or¹⁸F-Tz-α_vβ₆ L) drug to the test tube, shaking, centrifuging, taking 100ul of each of the upper and lower layers, and measuring the radioactivity value by using a gamma counter. The measured¹⁸ F-TCO-FAPI lipid water distribution coefficient Log P= -2.961 + -0.203 (n=5),¹⁸F-Tz-α_vβ₆ L lipid water distribution coefficient Log P= -3.36+ -0.20 (n=5) are all of obvious hydrophilicity.

Example 10¹⁸ F-TCO-FAPI in vitro cell experiments

A549-FAP cell uptake and inhibition experiments with FAP high expression:

The tumor cell strain A549 is transfected to obtain the A549-FAP with high FAP expression. Two 24-well plates were spread, randomly divided into five groups of 5min, 15min, 30min, 60min and 120min, the uptake group was added with¹⁸ F-TCO-FAPI and then cultured continuously, the inhibition group was simultaneously added with the NOTA-FAPI-42 inhibitor and¹⁸ F-TCO-FAPI, washed 3 times with PBS respectively, and the cell count was measured with a gamma counter after addition of NaOH-SDS solution.

Cell uptake and inhibition experiments showed that¹⁸ F-TCO-FAPI had relatively high uptake and specificity in A549-FAP cells (as shown in FIG. 7 a).

Affinity assay, tumor cell plating, namely 24-well plate plating, one group of three-well cells and 8 groups of three-well cells. The concentration of the competitive inhibitor (NOTA-FAPI-42) was (0, 10,^-5,10^-6,10^-7,10^-8,10^-9,10-¹⁰,10^-11 M), respectively. Each concentration of inhibitor was dissolved in 1mL of medium, 0.163mL was added to each well, followed by 0.837mL of¹⁸ F-TCO-FAPI (0.5. Mu. Ci/0.5 mL/well) to each well. After incubation for 1h at 37 ℃, wash three times with PBS. Radioactivity was measured by gamma counter after ablating the cells with NaOH-SDS solution.¹⁸ The results of the F-TCO-FAPI competitive binding assay are shown in FIG. 7 b.

Example 11 targeting Compound double molecular Probe¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L in vitro Mass Spectrometry detection test

Precursor materials¹⁸ F-TCO-FAPI and¹⁸F-Tz-α_vβ₆ L of 50ug (1 ml/mg) are mixed and heated for 12 hours, diluted after heating, mass spectrum detection is carried out after dilution to 100ug/ml, and whether the two monomer imaging agents¹⁸ F-TCO-FAPI and¹⁸F-Tz-α_vβ₆ L can generate Diels-Alder (IEDDA) bioorthogonal reaction with inverse electron requirements in vitro is detected, so that a new substance is generated.

The amount of the new substance produced was calculated to be 3748, and compared with the data of mass spectrometry detection as shown in FIG. 8, to confirm that one of the most interesting substances C₁₇₁H₂₅₀N₃₉O₅₄F₂ was produced from outside the body.

Example 12¹⁸ F-TCO-FAPI in vivo biodistribution uptake and inhibition experiments

The establishment method of the subcutaneous tumor model comprises the following steps of culturing A549 and A549-FAP cells by using DMEM culture medium and 10% FBS and 1% double antibody. Nude mice were housed in SPF animal houses, and when nude mice were aged for about 5 weeks, 8 5-week-old nude mice were inoculated 5X 10⁶/A549-FAP cells subcutaneously in the right armpit, and 4 5-week-old nude mice were inoculated 5X 10⁶/A549 cells subcutaneously in the right armpit. About 0.5-1.0cm of tumor grows in the right armpit of the nude mice around 2-3 weeks.

The first group was injected with 0.1-0.2m L of 40-60 μCi¹⁸ F-TCO-FAPI solution by tail vein in 4A 549-FAP tumor-bearing mice, the second group was injected with 0.1-0.2mL of a mixed solution containing 40-60 μCi¹⁸ F-TCO-FAPI and 100ul (1 mg/mL) of inhibitor NOTA-FAPI-42 by tail vein in 4A 549 tumor-bearing mice, and the third group was injected with 0.1-0.2mL of 40-60 μCi¹⁸ F-TCO-FAPI solution by tail vein in 4A 549 tumor-bearing mice. The biodistribution test method is to remove eyeballs from three groups of tumor-bearing mice for 60min after injection, sacrifice the mice from cervical vertebra, dissect and take tissue samples of each organ (blood, brain, heart, lung, liver, gall bladder, kidney, spleen, pancreas, stomach, small intestine, muscle, spine, femur, joint and tumor), weigh, measure the radioactivity count and record the measurement time, and calculate the percentage of radioactive injection dose per gram of tissue for 60min (% ID/g).

The result of the biodistribution experiment shows that¹⁸ F-TCO-FAPI shows excellent pharmacokinetic performance (including targeting, kidney excretion, tumor uptake capacity, intra-tumor residence time and the like) in the preclinical research stage, and the specific expression is that¹⁸ F-TCO-FAPI has specific targeting to FAP targets,¹⁸ F-TCO-FAPI has highest uptake value reaching 17.94+/-8.54% ID/g in a FAP positive A549-FAP tumor-bearing mouse model, and has very low tumor uptake values of 2.23+/-0.68% ID/g and 1.52+/-0.4% ID/g in a FAP positive tumor-bearing mouse inhibition group and a FAP negative A549 tumor-bearing mouse model, and shows high uptake and specific uptake of¹⁸ F-TCO-FAPI by the A549-FAP tumor-bearing mouse model.

The biodistribution results in normal organs and tissues show that¹⁸ F-TCO-FAPI is mainly rapidly excreted through the kidney, and the ingestion is lower at 60min, and only 3.14+/-1.64% ID/g. With the highest radioactive uptake in the joints, the higher radioactive uptake in the spine and bones, the moderately radioactive uptake in the gall bladder, and the uptake in non-target sites such as liver, intestinal tract, lung, pancreas, muscle and blood were at lower levels (results shown in fig. 9 a).

Example 13¹⁸F-Tz-α_vβ₆ L in vivo biodistribution uptake assay

The establishment method of the subcutaneous tumor model comprises taking 95 week old nude mice, inoculating Capan-2 tumor cells under the armpit of the right upper limb according to the method of example 12, and performing in vivo distribution experiment when the tumor grows to about 0.5-1.0 cm.

Tumor-bearing nude mice were randomly divided into 3 groups of 3 mice each, and each Capan-2 tumor-bearing mouse was injected with 0.1-0.2mL containing 40-60 μCi¹⁸F-Tz-α_vβ₆ L via tail vein. The biodistribution test method was the same as in example 12.

The in vivo biodistribution results show that¹⁸F-Tz-α_vβ₆ L has general in vivo activity in the Capan-2 tumor-bearing model, and the in vivo biodistribution of¹⁸F-Tz-α_vβ₆ L is lower in tumor as shown in FIG. 9 b. The kidney has the highest radioactive uptake, the lung and the blood have very high radioactive uptake, and the intestinal tract and the liver have moderate radioactive uptake.

Example 14 in vivo biodistribution comparison experiments targeting Compound bilayer Probe¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L (1:1) with¹⁸ F-TCO-FAPI and¹⁸F-Tz-α_vβ₆ L

A subcutaneous Capan-2 tumor model was established as in example 12, and in vivo distribution experiments were performed when the tumors grew to about 0.5-1.0 cm.

Capan-2 tumor-bearing mice were randomly assigned to groups one, two and three, 6 each. The biological distribution experimental method is the same as that of example 12, wherein the first group is prepared by injecting 0.1-0.2mL of mixed solution containing 40-60 mu Ci of targeting compound bimolecular probe¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L (1:1) into each Capan-2 tumor-bearing mouse through tail vein, the second group is prepared by injecting 0.1-0.2mL of solution containing 40-60 mu Ci¹⁸ F-TCO-FAPI into each Capan-2 tumor-bearing mouse through tail vein, and the third group is prepared by injecting 0.1-0.2mL of solution containing 40-60 mu Ci¹⁸F-Tz-α_vβ₆ L into each Capan-2 tumor-bearing mouse through tail vein.

The in vivo biodistribution results show that the targeting compound double-molecule probe¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L (1:1) combines the in vivo biodistribution of two monomer imaging agents¹⁸ F-TCO-FAPI and¹⁸F-Tz-α_vβ₆ L in the Capan-2 tumor-bearing mice, has very high uptake in tumors, is very close to the uptake value of¹⁸ F-TCO-FAPI tumors, is far higher than the uptake value of¹⁸F-Tz-α_vβ₆ L tumors, is larger than the sum of half uptake values of the two monomer imaging agents (1.43+/-0.07% ID/g, 1.76+/-0.05% ID/g and 0.48+/-0.04% ID/g respectively), and has biological orthogonal effect from the biodistribution level. In normal organs and tissues, the target compound bimolecular probe has the highest uptake value in the kidney, is similar to the biological distribution characteristics of¹⁸F-Tz-α_vβ₆ L in vivo, has high radioactive uptake value in joints, spines and bones, and is similar to the biological distribution characteristics of¹⁸ F-TCO-FAPI in vivo. Notably,¹⁸F-Tz-α_vβ₆ L had very high uptake in lung and blood, but the targeted compound bimolecular probe had very low uptake in lung and blood, which is not completely consistent with the previous conclusion (results shown in fig. 9 c).

Example 15¹⁸ F-TCO-FAPI micro-PET imaging experiment

The establishment method of the subcutaneous tumor model is that 5-week-old nude mice are divided into 3 groups, 9 nude mice in each group are inoculated with A549-FAP lung adenocarcinoma cells, U87 brain glioma cells and A549 lung adenocarcinoma cells respectively according to the method of example 12.

Static uptake and inhibition of PET imaging in tumor-bearing mice, namely, taking 3 mice respectively for weighing in the first group, the second group and the third group, injecting 260 mu Ci imaging agent¹⁸ F-TCO-FAPI into the tail vein, taking 3 mice respectively for weighing in the first group and the second group, injecting a mixture of 260 mu Ci imaging agent¹⁸ F-TCO-FAPI and 100ul (1 mg/ml) NOTA-FAPI-42 inhibitor into the tail vein (diluted by 200ul normal saline), and carrying out PET/CT imaging 60min after injection.

PET imaging results show that¹⁸ F-TCO-FAPI has higher uptake in an A549-FAP lung adenocarcinoma model and a U87 brain glioma model, is stable in 2 hours, is rapidly excreted through kidneys, is not obvious in kidney development after 15 minutes, is not obvious in liver and intestinal tract development, and shows good pharmacokinetic characteristics, and the uptake of tumors is obviously reduced in the A549-FAP model and the U87 model by inhibiting imaging, and is specific uptake (shown in fig. 10a and 10 b), but almost not in the A549 wild lung adenocarcinoma model.

In the contrast study of¹⁸ F-TCO-FAPI and¹⁸ F-FAPI-42PET imaging in the U87 model, 3U 87 tumor-bearing mice are taken, after weighing,¹⁸ F-TCO-FAPI and¹⁸ F-NOTA-FAPI-42 are respectively injected into the same mice, and PET/CT imaging is carried out at 2h, 4h and 6h after injection. The results of contrast imaging of¹⁸ F-TCO-FAPI and¹⁸ F-NOTA-FAPI-42 in the same U87 tumor murine model show that¹⁸ F-TCO-FAPI has high tumor uptake values and long residence time, and that¹⁸ F-TCO-FAPI has high spinal and joint uptake and low intestinal, hepatic and renal uptake in normal organs and tissues, while¹⁸ F-NOTA-FAPI-42 has low tumor uptake values and is seen to be more significantly ingested in joints, spinal and intestinal. As can be seen,¹⁸ F-TCO-FAPI exhibited superior imaging performance to¹⁸ F-NOTA-FAPI-42.

EXAMPLE 16¹⁸F-Tz-α_vβ₆ L micro-PET imaging experiment

The establishment method of the subcutaneous tumor model is that 5-week-old nude mice are divided into 2 groups, 3 nude mice in each group are inoculated with Capan-2 human pancreatic cancer cells and BxPC-3 human in-situ pancreatic cancer cells respectively according to the method of example 12, and when tumors grow to about 0.5-1.0cm, in-vivo micro-PET imaging experiments are carried out.

In vivo static PET imaging of tumor-bearing mice, each mouse is weighed and injected with 260 mu Ci imaging agent¹⁸F-Tz-α_vβ₆ L to the tail vein, and PET/CT imaging is carried out at 30min, 60min and 2h after injection.

The imaging results show that¹⁸F-Tz-α_vβ₆ L is obtained at 30min peak of uptake in tumor, but overall, tumor uptake is low and residence time is not long, mainly by renal excretion, kidney, heart and lung uptake is high, and the bar graph results are shown in FIG. 11a and FIG. 11 b.

Example 17 micro-PET imaging contrast experiments targeting Compound bilayer Probe¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L (1:1) and¹⁸ F-TCO-FAPI and¹⁸F-Tz-α_vβ₆ L

The establishment method of the subcutaneous tumor model comprises taking 3 5-week-old nude mice, inoculating Capan-2 human pancreatic cancer cells to the axilla of the right upper limb according to the method of example 12, and performing in vivo micro-PET imaging experiment when the tumor grows to about 0.5-1.0 cm.

In vivo static PET imaging of tumor-bearing mice in the first experiment, each mouse was weighed and injected into the tail vein with about 240. Mu. Ci imaging agent¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L (1:1) and PET/CT imaging was performed 30min, 60min, 2h, 4h, 6h post injection. In the second experiment, the same mice were weighed and injected into the tail vein with about 240. Mu. Ci of imaging agent¹⁸ F-TCO-FAPI and PET/CT imaging was performed 30min, 60min, 2h, 4h, and 6h after injection. In the third experiment, the same mice were weighed and injected with about 240. Mu. Ci imaging agent into the tail vein, and PET/CT imaging was performed 30min, 60min, 2h, 4h, and 6h after injection.

The PET results are shown in FIG. 11c, and the imaging results show that the tumor uptake in the bio-orthogonal imaging of the targeting compound double molecular probe¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L (1:1) is higher, and the tumor uptake in the imaging of¹⁸ F-TCO-FAPI monomer is not worse in long-time point imaging, and the excretion rate of¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L in the tumor is slower than that of¹⁸ F-TCO-FAPI, probably due to the fact that the binding amount of¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L in the imaging of long-time point is more.¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ The tumor uptake value of L is larger than the sum of half uptake values of¹⁸ F-TCO-FAPI and¹⁸F-Tz-α_vβ₆ L tumors, and the biological orthogonal effect of the experiment is proved from the imaging layer, so that the tumor uptake is increased and the residence time is prolonged.¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L also showed higher uptake in heart, lung, kidney, spine and joints, showing the imaging characteristics of the two monomeric imaging agents¹⁸ F-TCO-FAPI and¹⁸F-Tz-α_vβ₆ L binding. Notably, however,¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L in the imaging showed high uptake in the heart and lungs, not consistent with the performance characteristics of in vivo biodistribution.

The invention establishes a complete and procedural synthesis and quality control method for preparing a targeting FAP molecular probe¹⁸ F-TCO-FAPI, a targeting integrin alpha_vβ₆ molecular probe¹⁸F-Tz-α_vβ₆ L and a targeting compound double molecular probe¹⁸F-TCO-FAPI+¹⁸F-Tz-α_vβ₆ L (1:1), and application thereof in preparing a Positron Emission Tomography (PET) small molecular imaging agent, in constructing a targeting tumor compound medicine bioorthogonal amplification system and in developing a compound medicine bioorthogonal diagnosis and treatment novel technology. The invention can play an important diagnostic role in PET imaging of tumors and other diseases. Modifications and variations of the structure and medicament will occur to those skilled in the art in light of the foregoing description, and all such modifications and variations are intended to be included within the scope of the following claims.

The technical features of the foregoing embodiments may be combined in any manner, and in this specification, for brevity, all of the possible combinations of the technical features of the foregoing embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, it should be considered as the scope described in the present specification. Moreover, the foregoing examples represent only a few of the embodiments of the present invention, which have been described in detail and are not thereby to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.

Claims (9)

1. The targeting compound bimolecular probe is characterized by comprising a component A and a component B which are respectively marked by radioactive metal nuclides;

The component A is a targeted fibroblast activation protein FAP molecular probe, and the targeted fibroblast activation protein FAP molecular probe comprises the following groups:

;

the component B is a targeting integrin alpha_vβ₆ molecular probe, and the targeting integrin alpha_vβ₆ molecular probe comprises 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 of claim 1, wherein the targeted fibroblast activation protein FAP molecular probe further comprises a metal ion-binding chelating group and a radiometal nuclide;

the targeted integrin alpha_vβ₆ molecular probe also comprises a metal ion binding chelating group and a radiometal nuclide.

3. The targeted compound bimolecular probe according to claim 2, wherein the metal ion binding chelating group is 1,4, 7-triazacyclononalkyl-N ', N "-diacetoxy-N-acetyl or 1,4,7, 10-tetraazacyclododecane-N ', N", N ' "-triacetoxy-N-acetyl.

4. The targeted compound bimolecular probe of claim 2, wherein the radionuclide is one of¹⁸F、⁶⁸Ga、¹¹¹In、^99mTc、¹⁸⁶Re、¹⁸⁸Re、¹⁷⁵Yb、¹⁵³Sm、¹⁶⁶Ho、⁸⁸Y、⁹⁰Y、¹⁷⁷Lu、⁴⁷Sc、²¹²Bi、²¹³Bi、¹²³I、¹²⁴I、¹³¹I、²¹¹At、²¹²Pb、⁶⁴Cu、⁶⁷Cu、¹⁹⁸Au、²²⁵Ac or²²⁷ Th.

5. The targeted compound bimolecular probe according to claim 4, wherein the targeted compound bimolecular probe is a mixture of¹⁸ F-labeled targeted fibroblast activation protein FAP molecular probe¹⁸ F-TCO-FAPI and¹⁸ F-labeled targeted integrin α_vβ₆ molecular probe¹⁸F-Tz-α_vβ₆ L,¹⁸F-TCO-FAPI、¹⁸F-Tz-α_vβ₆ L each having the following structural formula:

、。

6. a method for preparing a targeting compound bimolecular probe according to any one of claims 1 to 5, comprising the steps of:

The preparation of the component A comprises the steps of taking NOTA-TCO-FAPI or DOTA-TCO-FAPI as a precursor raw material, carrying out chelation reaction with radioactive metal nuclide ions, and separating and purifying to obtain a targeted fibroblast activation protein FAP molecular probe;

The preparation of the component B comprises the steps of taking NOTA-Tz-alpha_vβ₆ L or DOTA-Tz-alpha_vβ₆ L as a precursor raw material, carrying out chelation reaction with radionuclide ions, and separating and purifying to obtain a targeting integrin alpha_vβ₆ molecular probe;

Preparing a targeting compound bimolecular probe, namely mixing the component A and the component B according to the radioactivity ratio of (1-2) to (2-1) to obtain the targeting compound bimolecular probe;

wherein the precursor raw material is prepared by adopting a solid-phase polypeptide synthesis method;

the NOTA-TCO-FAPI has the following structural formula:

;

the DOTA-TCO-FAPI has the following structural formula:

;

the NOTA-Tz-alpha_vβ₆ L has the following structural formula:

;

The DOTA-Tz-alpha_vβ₆ L has the following structural formula:

。

7. The method of claim 6, wherein the radionuclide ion is one of⁶⁸Ga³⁺、[Al¹⁸F]²⁺、⁶⁴Cu²⁺ or¹⁷⁷Lu³⁺.

8. The use of a targeted compound bimolecular probe according to any one of claims 1 to 5, wherein the targeted compound bimolecular probe is used in the preparation of a tumor diagnosis and treatment agent.

9. The use according to claim 8, wherein the use of the targeted compound bimolecular probe for the preparation of a tumor-targeted PET imaging agent;

The application of the targeting compound double-molecule probe in preparing a tumor-targeted SPECT imaging agent;

the targeting compound double-molecule probe is applied to the preparation of a radioactive therapeutic agent for targeting tumors.