阅读说明：本技术 靶向成纤维细胞活化蛋白探针、制备方法及其在制备pet显像剂中的应用 (Targeted fibroblast activation protein probe, preparation method and application thereof in preparation of PET (polyethylene terephthalate) imaging agent ) 是由 唐刚华 黄佳文 胡孔珍 韩彦江 傅丽兰 于 2021-06-01 设计创作，主要内容包括：本发明公开了一种靶向成纤维细胞活化蛋白(FAP)探针、制备方法及其在制备正电子发射断层(PET)显像剂中的应用。本发明的FAP探针为具有优良药代动力学特性的含双配体新型FAP探针[X～(n+)]NOTA-FAPT,其中,FAPT由FAP药效基团配体、吡喃葡萄糖胺基配体以及联结基团构成,NOTA为1,4,7-三氮杂环九烷基-N’,N”-二乙酸基-N-乙酰基类似物,X～(n+)为～(68)Ga～(3+)、[Al～(18)F]～(2+)、～(64)Cu～(2+)或其它放射性金属离子。本发明的FAP探针通过优化化学结构,改善了探针的体内药代动力学特性和靶向性,增加亲水性并减少了肝脏系统、胆道系统以及肠道系统的摄取,可实现自动化高产率放射合成,可应用于制备肿瘤和多种疾病的生物学功能显像剂。(The invention discloses a target Fibroblast Activation Protein (FAP) probe, a preparation method and application thereof in preparation of a Positron Emission Tomography (PET) developer. The FAP probe of the invention is a novel FAP probe [ X ] containing double ligands with excellent pharmacokinetic characteristics n+ ]NOTA-FAPT, wherein FAPT is composed of FAP pharmacophore ligand, glucopyranose amido ligand and linking group, NOTA is 1,4, 7-triazacyclononaalkyl-N ', N' -diacetoxy-N-acetyl analogue, X n+ Is composed of 68 Ga 3+ 、[Al 18 F] 2+ 、 64 Cu 2+ Or other radioactive metal ions. The FAP probe improves the in vivo pharmacokinetic characteristic and targeting property of the probe and increases the hydrophilicity by optimizing the chemical structureAnd the uptake of a liver system, a biliary tract system and an intestinal tract system is reduced, the automatic high-yield radiosynthesis can be realized, and the method can be applied to the preparation of biological function imaging agents for tumors and various diseases.)

1. A target Fibroblast Activation Protein (FAP) probe is characterized by comprising an FAP pharmacophore, a glucopyranose amido, a linking group, a chelating group for bonding metal ions and a radioactive metal nuclide Xⁿ⁺And (4) forming.

2. The targeted Fibroblast Activation Protein (FAP) probe of claim 1, wherein the glucopyranosylamine group comprises a 2-acetamido-2-deoxy- β -D-glucopyranosylamine group or a glucosaminyl group;

and/or, the linking group comprises a polyethylene glycol group (PEG2), an aspartic acid group, and a glutamic acid group;

and/or, the metal ion-binding chelating group is 1,4, 7-triazacyclononalkyl-N ', N "-diacetoxy-N-acetyl (-NOTA) or an analog thereof, and the analog of 1,4, 7-triazacyclononalkyl-N', N" -diacetoxy-N-acetyl (-NOTA) includes 2-S- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (p-SCN-Bn-NOTA) or 1,4,7, 10-tetraazacyclododecane-N ', N "N'" -triacetoxy-N-acetyl (-DOTA);

and/or the radioactive metal species Xⁿ⁺Is composed of⁶⁸Ga³⁺、[Al¹⁸F]²⁺、⁶⁴Cu²⁺Or other radioactive metal ions.

3. The Fibroblast Activation Protein (FAP) -targeting probe according to claim 1 or 2, characterized in that it is represented by [ X ]ⁿ⁺]NOTA-FAPT, having a chemical formula as shown in formula I:

wherein R isXⁿ⁺Is composed of⁶⁸Ga³⁺、[Al¹⁸F]²⁺、⁶⁴Cu²⁺Or other radioactive metal ions.

4. A preparation method of a target Fibroblast Activation Protein (FAP) probe is characterized in that NOTA-FAPT or an analogue DOTA-FAPT thereof is taken as a precursor raw material and is respectively mixed with a radioactive metal nuclide Xⁿ⁺Chelating reaction and separating and purifying with small column to obtain FAP probe [ X ]ⁿ⁺]NOTA-FAPT or analogues thereof [ Xⁿ⁺]DOTA-FAPT。

5. The process according to claim 4, wherein said precursor starting material, NOTA-FAPT or its analog DOTA-FAPT, is prepared by the following method:

FAP pharmacophore ligand-PEG 2 is formed after FAP pharmacophore ligand modification-PEG 2, Asp2, Glu and glucopyranose amido are modified by FAP pharmacophore ligand-PEG 2, chelating group-NOTA or-DOTA is finally modified, and product peaks are collected by preparative HPLC separation and purification, thus obtaining the purified precursor raw material NOTA-FAPT or DOTA-FAPT.

6. The method of claim 4 or 5, wherein the fibroblast activation protein-targeting probe is [ 2 ]⁶⁸Ga]NOTA-FAPT or⁶⁸Ga]DOTA-FAPT, said⁶⁸Ga]NOTA-FAPT or⁶⁸Ga]DOTA-FAPT was prepared as follows:

NOTA-FAPT or an analogue thereof DOTA-FAPT is taken as a precursor raw material, and the precursor raw material NOTA-FAPT or DOTA-FAPT is weakly acidic⁶⁸GaCl₃The solution is heated at 85-110 deg.C for reaction, and separated and purified by HLB column or SEP-PAK C18 column to obtain the product⁶⁸Ga]NOTA-FAPT or⁶⁸Ga]DOTA-FAPT injection.

7. The method of claim 4 or 5, wherein the fibroblast activation protein-targeting probe is [ 2 ]¹⁸F]AlF-NOTA-FAPT, the [ alpha ], [ alpha ] and [ alpha ] and¹⁸F]the AlF-NOTA-FAPT is prepared by the following method:

NOTA-FAPT is taken as a precursor raw material, and the precursor raw material NOTA-FAPT is added in AlCl₃And acetonitrile in an acidic solution¹⁸F^-Reacting, separating and purifying by using an HLB column or an SEP-PAK C18 column to obtain the product¹⁸F]AlF-NOTA-FAPT injection.

8. The method of claim 4 or 5, wherein the fibroblast activation protein-targeting probe is [ 2 ]⁶⁴Cu]NOTA-FAPT or⁶⁴Cu]DOTA-FAPT, said⁶⁴Cu]NOTA-FAPT or⁶⁴Cu]DOTA-FAPT was prepared as follows:

NOTA-FAPT or an analogue thereof DOTA-FAPT is taken as a precursor raw material, and the precursor raw material NOTA-FAPT or DOTA-FAPT and⁶⁴CuCl₂in acetic acidIncubating in sodium acid buffer solution at room temperature for 15min, separating and purifying with HLB column or SEP-PAK C18 column to obtain the product⁶⁴Cu]NOTA-FAPT or⁶⁴Cu]DOTA-FAPT injection.

9. Use of a targeted Fibroblast Activation Protein (FAP) probe in the preparation of a PET imaging agent, wherein the targeted fibroblast activation protein probe is the targeted fibroblast activation protein probe according to any one of claims 1 to 3.

10. The use of claim 9, in the preparation of a PET imaging agent targeting high expression of tumor FAP, comprising: the application in preparing PET imaging agent for lung adenocarcinoma and brain glioma, or in preparing PET imaging agent for other tumors and diseases.

Technical Field

The invention relates to the technical field of target cell probes, in particular to a target fibroblast activation protein probe, a preparation method and application thereof in preparation of a PET (positron emission tomography) developer.

Background

In the traditional diagnostic and therapeutic methods, the target of tumor parenchymal cells is mainly aimed. Recently, the tumor microenvironment and tumor stroma have become the focus of increasing attention, which has made possible new diagnostic and therapeutic modalities. Cancer-associated fibroblasts (CAF) are the major component of tumors, accounting for 90% of the total tumor mass that can be achieved. CAF differs from normal fibroblasts in the relative specific expression of Fibroblast Activation Protein (FAP), often associated with poor prognosis and outcome of the respective cancer. FAP has dipeptidyl peptidase and collagenase activities, can cleave substrates of various dipeptidyl peptidase activities including gelatin and denatured type I collagen in a matrix, participate in the degradation of extracellular matrix (ECM), and promote the detachment, invasion and metastasis of tumor cells from a primary site. FAP is rarely expressed in normal tissues, but is highly expressed in cancer-associated fibroblasts (CAFs) of various tumors, such as ovarian, pancreatic and hepatocellular carcinomas. Therefore, FAP is expected to become a new target for tumor imaging.

The results of the preliminary clinical study showed that the probe was compatible with the most commonly used Positron Emission Tomography (PET) imaging agent (probe)¹⁸F]Fluorodeoxyglucose ([ alpha ], [ beta ] and [ beta ], [ beta ] and [ beta ] a¹⁸F]FDG) of the samples was compared with each other,⁶⁸ga or¹⁸The F-labeled FAP inhibitor (FAPI) developer (also called probe) has obvious advantages in the imaging effect of gastric cancer and malignant tumor of biliary system, has certain advantages in the boundary tumor of most tumor primary focuses, malignant tumor liver metastasis and bone metastasis, and has general detection value in common solid malignant tumor applicationValue and¹⁸F]FDG is similar. Is more commonly used⁶⁸Ga-labeled FAPI imaging agent [ alpha ], [ alpha ]⁶⁸Ga]FAPI-04, shows high tumor/target organ radioactive uptake ratio and rapid clearance characteristics in preclinical and preliminary clinical experiments. However, the term⁶⁸Ga]FAPI-04 can be ingested in a diversity way in the pancreas, the ingestion is relatively unstable, and certain difficulty is brought to the interpretation of good and malignant diagnosis of the pancreas unless the clear pathology of the pancreas is confirmed; the biliary system of a small part of patients⁶⁸Ga]FAPI-04 physiological uptake; in addition to this, the present invention is,⁶⁸ga has a short half-life (67.71min) and is not suitable for long-distance transportation. Due to the fact that¹⁸The F half-life (109.8min) is longer, and is suitable for long-distance transportation and delayed imaging. In recent times, it has been desired to develop,¹⁸f-labeled FAPI imaging agents develop faster and are rapidly converting clinically. Based on preclinical and clinical PET imaging studies findings: [¹⁸F]FAPI-42 has physiological uptake in the biliary, pancreatic and gall bladder systems, with minor intestinal uptake occurring in a small percentage of patients. The recent foreign scholars successfully develop the novel model based on Click chemical reaction¹⁸F-labeled FAPI imaging agent¹⁸F]FGlc-FAPI, preliminary biological evaluation indicates that, is¹⁸F]FGlc-FAPI exhibits the same meaning as⁶⁸Ga]FAPI-04 has the same or even higher tumor uptake value. However, the term¹⁸F]FGlc-FAPI is excreted through the metabolic pathways of liver, gallbladder and intestinal tract, which causes the detection rate of abdominal tumor detection to be reduced. Furthermore, in terms of the synthetic method, the existing FAPI imaging agent, in particular, [ 2 ]¹⁸F]FGlc-FAPI, the labeling process is complicated, the time consumption is long, and the radiochemical yield is low.

Disclosure of Invention

In order to solve the defects of poor targeting and pharmacokinetic properties of the existing FAPI imaging agent and overcome the defect of high physiological uptake of the existing FAPI imaging agent in a liver and gall system, a biliary tract system, a pancreas and an intestinal tract system, the invention provides a targeted Fibroblast Activation Protein (FAP) probe, in particular to a novel FAPI probe (imaging agent) containing double ligands and having excellent pharmacokinetic properties.

In addition, in order to solve the problems of complicated synthetic labeling process, long time consumption and low radiochemical yield of the conventional FAPI imaging agent, the invention also provides a method for preparing the target fibroblast activation protein probe.

The invention also aims to provide application of the targeted Fibroblast Activation Protein (FAP) probe, in particular application in preparing biological functional imaging agents for tumors and various diseases.

The purpose of the invention is realized by the following technical scheme.

A target fibroblast activation protein probe, in particular to a positron nuclide labeled target fibroblast activation protein small molecule inhibitor (FAPI) probe, which is composed of FAP pharmacophore ligand, glucopyranose amido ligand (R), a linking group, a chelating group combined with metal ions and radioactive metal nuclide (X)ⁿ⁺) And (4) forming.

In the targeting FAP probe, the double ligands are FAP pharmacophore and glucopyranose amido. Wherein, FAP pharmacophore is a targeting FAP ligand; glucopyranose amino group of [ 2 ]¹⁸F]FDG analog groups, including 2-acetamido-2-deoxy- β -D-glucopyranosylamino or glucosaminyl groups, may have mechanisms of action involving glucose transporters and certain enzymes, and may also improve the pharmacokinetic properties of the targeted FAP ligands.

In a preferred embodiment, the glucopyranosamino group comprises a 2-acetamido-2-deoxy- β -D-glucopyranosamino group or a glucosaminyl group;

and/or, the linking group comprises a polyethylene glycol group (PEG2), an aspartic acid group (Asp2), and a glutamic acid group (Glu), the linking group can improve and enhance the pharmacokinetic properties of the targeting FAP ligand;

and/or the chelating group for binding metal ions is 1,4, 7-triazacyclononaalkyl-N ', N' -diacetoxy-N-acetyl (-NOTA) or an analogue thereof (-DOTA) which can specifically bind radioactive metal nuclides;

and/or the radioactive metal species Xⁿ⁺Is composed of⁶⁸Ga³⁺、[Al¹⁸F]²⁺、⁶⁴Cu²⁺Or other radioactive metal ions.

In more preferred embodiments, the 1,4, 7-triazacyclononanyl-N ', N "-diacetoxy-N-acetyl analog comprises 2-S- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (p-SCN-Bn-NOTA) or 1,4,7, 10-tetraazacyclododecane-N ', N" -N ' "-triacetic acid-N-acetyl (-DOTA).

In a preferred embodiment, the Fibroblast Activation Protein (FAP) -targeting probe of any one of the above is represented by [ X [ ]ⁿ⁺]NOTA-FAPT, wherein the linking group comprises two polyethylene glycol groups, two aspartic acid groups and a glutamic acid group, and the chemical structural formula is shown as the formula I:

wherein R isXⁿ⁺Is composed of⁶⁸Ga³⁺、[Al¹⁸F]²⁺、⁶⁴Cu²⁺Or other radioactive metal ions.

The invention provides the target Fibroblast Activation Protein (FAP) probe ([ X)ⁿ⁺]NOTA-FAPT and analogs thereof [ Xⁿ⁺]DOTA-FAPT) preparation method, NOTA-FAPT or DOTA-FAPT is taken as precursor raw material, and the NOTA-FAPT or DOTA-FAPT is respectively mixed with the precursor raw material⁶⁸Ga³⁺、[Al¹⁸F]²⁺、⁶⁴Cu²⁺Chelating reaction, separating and purifying with column to obtain target Fibroblast Activation Protein (FAP) probe [ X ]ⁿ⁺]NOTA-FAPT; DOTA-FAPT as precursor material is reacted with⁶⁸Ga³⁺、⁶⁴Cu²⁺、¹⁷⁷Lu, etc. are chelated and separated and purified by a small column to prepare a target Fibroblast Activation Protein (FAP) probe [ Xⁿ⁺]DOTA-FAPT。

A method for preparing a target Fibroblast Activation Protein (FAP) probe comprises using NOTA-FAP T or its analog DOTA-FAP T as precursor raw material, and mixing with radioactive metal nuclide Xⁿ⁺Chelating reaction and separating and purifying with small column to obtain FAP probe [ X ]ⁿ⁺]NOTA-FAPT or analogues thereof [ Xⁿ⁺]DOTA-FAPT。

Radiosynthesis targeting FAP probes [ Xⁿ⁺]The key to NOTA-FAPT is the preparation of NOTA-FAPT or-DOTA as a precursor material. In a preferred embodiment, the precursor material NOTA-FAPT or its analog DOTA-FAPT is prepared by the following method:

FAP pharmacophore ligand modification-PEG 2 forms FAP pharmacophore ligand-PEG 2, FAP pharmacophore ligand-PEG 2 modifies Asp2, Glu and glucopyranose amino ligand, and finally modifies chelating group-NOTA or-DOTA, and product peaks are collected by preparative HPLC separation and purification, thus obtaining purified precursor raw material NOTA-FAPT or DOTA-FAPT.

In a preferred embodiment, the fibroblast activation protein-targeting probe is [ alpha ], [ beta ] -an⁶⁸Ga]NOTA-FAPT or⁶⁸Ga]DOTA-FAPT, said⁶⁸Ga]NOTA-FAPT or⁶⁸Ga]DOTA-FAPT was prepared as follows:

NOTA-FAPT or an analogue thereof DOTA-FAPT is taken as a precursor raw material, and the precursor raw material NOTA-FAPT or DOTA-FAPT is weakly acidic⁶⁸GaCl₃The solution is heated at 85-110 deg.C for reaction, and separated and purified by HLB column or SEP-PAK C18 column to obtain the product⁶⁸Ga]NOTA-FAPT or⁶⁸Ga]DOTA-FAPT injection.

In a preferred embodiment, the fibroblast activation protein-targeting probe is [ alpha ], [ beta ] -an¹⁸F]AlF-NOTA-FAPT, the [ alpha ], [ alpha ] and [ alpha ] and¹⁸F]the AlF-NOTA-FAPT is prepared by the following method:

NOTA-FAPT is taken as a precursor raw material, and the precursor raw material NOTA-FAPT is added in AlCl₃And acetonitrile in an acidic solution¹⁸F^-Reacting, separating and purifying by using an HLB column or an SEP-PAK C18 column to obtain the product¹⁸F]AlF-NOTA-FAPT injection.

In a preferred embodiment, the fibroblast activation protein-targeting probe is [ alpha ], [ beta ] -an⁶⁴Cu]NOTA-FAPT or⁶⁴Cu]DOTA-FAPT, said⁶⁴Cu]NOTA-FAPT or⁶⁴Cu]DOTA-FAPT was prepared as follows:

NOTA-FAPT or an analogue thereof DOTA-FAPT is taken as a precursor raw material, and the precursor raw material NOTA-FAPT or DOTA-FAPT and⁶⁴CuCl₂incubating in sodium acetate acidic buffer solution at room temperature for 15min, separating and purifying with HLB column or SEP-PAK C18 column to obtain the product⁶⁴Cu]NOTA-FAPT or⁶⁴Cu]DOTA-FAPT injection.

The invention provides the target Fibroblast Activation Protein (FAP) probe ([ X)ⁿ⁺]NOTA-FAPT and analogs thereof [ Xⁿ⁺]DOTA-FAPT) in Positron Emission Tomography (PET) imaging of tumors, comprising [ X [ ]ⁿ⁺]NOTA-FAPT or [ X^{n +}]The DOTA-FAPT is applied to PET imaging of various solid tumors such as lung adenocarcinoma, brain glioma and the like, can be further applied to the curative effect evaluation of various solid tumors such as lung adenocarcinoma, brain glioma and the like, and can also be applied to PET imaging of other tumors and diseases.

Use of a targeted Fibroblast Activation Protein (FAP) probe in the preparation of a PET imaging agent, wherein the targeted FAP probe is the targeted FAP probe described in any one of the above.

In a preferred embodiment, the application of the targeted Fibroblast Activation Protein (FAP) in preparing a PET imaging agent with high expression of the targeted tumor FAP is provided.

In a more preferred embodiment, the targeting FAP probe is used for preparing a PET imaging agent for lung adenocarcinoma and brain glioma.

In a more preferred embodiment, the targeting FAP probe is used for preparing PET imaging agents of other tumors and diseases.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) according to the FAP probe, FAP pharmacophore ligand-PEG 2 is formed after FAP pharmacophore ligand modification-PEG 2, after Asp2, Glu and glucopyranose amido are modified by the FAP pharmacophore ligand-PEG 2, chelating group-NOTA or-DOTA is finally modified, the in vivo pharmacokinetic characteristic and targeting property of the probe are improved by optimizing a chemical structure, the hydrophilicity is increased, and the uptake of a liver and gall system, a biliary tract system, a pancreatic system and an intestinal tract system is reduced.

(2) The FAP probe comprises [ X ]ⁿ⁺]NOTA-FAPT and analogs thereof [ Xⁿ⁺]DOTA-FAPT, showing excellent in vivo drugThe pharmacokinetics characteristic is that the composition can be taken up in the tumor at 15min after administration, and is convenient for the early development of the tumor.

(3) In the preparation method of the FAP probe, NOTA-FAPT precursor raw material can be used as nuclide⁶⁸Ga-labelled and available with nuclides having a longer half-life¹⁸F-labelled and may further be labelled with longer half-life nuclides such as⁶⁴Cu²⁺Isotags, are highly advantageous for commercial transport and delayed imaging, resulting in the formation of probes [ X ]ⁿ⁺]NOTA-FAPT; the precursor raw material DOTA-FAPT can be formed by positron nuclide⁶⁸Ga³⁺、⁶⁴Cu²⁺The same mark can also be made of diagnostic nuclides¹⁷⁷Lu labeling to form a probe [ X ]ⁿ⁺]DOTA-FAPT. In addition, the invention solves the preparation of a precursor raw material NOTA-FAPT or DOTA-FAPT, can obtain a product after one-step reaction with positron nuclide and separation and purification by a small column, can realize high-yield and high-purity automatic synthesis, and meets the requirement of clinical PET imaging.

(4) The FAP probe can be applied to the preparation of PET imaging agents, including the application in the preparation of PET imaging agents with high expression of targeting tumor FAP, the application in the preparation of PET imaging agents for lung adenocarcinoma and brain glioma, or the application in the preparation of PET imaging agents for other tumors and diseases, and the detection efficiency of the tumors and the diseases is improved.

Drawings

Fig. 1a is an HPLC analysis profile of NOTA-FAPT (R ═ 2-acetylamino-2-deoxy- β -D-glucopyranosylamino) as the precursor starting material;

fig. 1b is a MS analysis of the precursor starting material NOTA-FAPT (R ═ 2-acetylamino-2-deoxy- β -D-glucopyranosylamino);

FIG. 2a is [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ] is¹⁸F]A representative radioactive HPLC analysis pattern of the AlF-NOTA-FAPT injection at the radioactive retention time Rt of 8.38 min;

FIG. 2b is a representative UV absorption HPLC analysis profile of precursor NOTA-FAPT at a UV absorption retention time Rt of 8.17 min;

FIG. 3a is [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ] is¹⁸F]Performing radioactive HPLC analysis chromatogram of AlF-NOTA-FAPT injection in serum for 1h in vivo;

FIG. 3b is [ 2 ]¹⁸F]Performing radioactive HPLC analysis chromatogram of AlF-NOTA-FAPT injection in 1h urine in vivo;

FIG. 4a is [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ] is¹⁸F]Performing radioactive HPLC analysis pattern of AlF-NOTA-FAPT injection in vitro serum for 2 h;

FIG. 4b is [ 2 ]¹⁸F]Performing radioactive HPLC analysis pattern of AlF-NOTA-FAPT injection in PBS buffer solution for 2 h;

FIG. 5 is a schematic view of a term¹⁸F]Uptake and inhibition patterns of the AlF-NOTA-FAPT injection at different time points in A549-FAP lung adenocarcinoma cells with high FAP expression and uptake patterns at different time points in wild-type A549 lung adenocarcinoma cells;

FIG. 6 is [ 2 ]¹⁸F]Uptake and inhibition profiles of AlF-NOTA-FAPT injection at 60min in 293T-FAP cells highly expressing FAP, and at 60min in wild-type 293T cells;

FIG. 7 is [ 2 ]¹⁸F]Schematic tumor cell plating for AlF-NOTA-FAPT affinity assay;

FIG. 8 is a cross section of a word "2¹⁸F]A graph of AlF-NOTA-FAPT affinity assay results;

FIG. 9a is [ alpha ], [ beta ]¹⁸F]Biodistribution of AlF-NOTA-FAPT at 3 time points 30min, 60min, 90min in Kunming mice;

FIG. 9b is a value of 60min¹⁸F]P-FAPI(¹⁸F-P-FAPI)、[¹⁸F]P-FAPI(¹⁸F-P-FAPI competitive inhibition and [ 2 ]¹⁸F]FAPI-42(¹⁸F-FAPI-42));

FIG. 10 is a cross section of [ 2 ]¹⁸F]AlF-NOTA-FAPT in FAP high expression U87 glioma model at 60min in small animal PET/CT image (left), competition inhibition visualization image (right);

FIG. 11 is [ 2 ]¹⁸F]AlF-NOTA-FAPT in FAP high expression A549-FAP lung adenocarcinoma model at 60min in small animal PET/CT picture (left), competition inhibition visualization picture (right);

FIG. 12 is a cross section of [ 2 ]¹⁸F]AlF-NOTA-FAPT A549-FAP high expression lung adenocarcinoma model at 60min small animal PET/CT visualization (left), wild type A549 lung adenocarcinoma model at 60min small animal PET/CT visualization (right);

FIG. 13a shows the sameModel [ 2 ]¹⁸F]AlF-NOTA-FAPT (FAPT) and [, [ 2 ]¹⁸F]PET imaging quantitative comparison graph of FAPI-42(FAPI 42);

FIG. 13b is the same model [ 2 ]¹⁸F]AlF-NOTA-FAPT (NOTA-FAPT) and¹⁸F]PET visualization of FAPI-42 (NOTA-FAPI-42);

FIG. 14a is [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ], [¹⁸F]PET/CT profiles of AlF-NOTA-FAPT at various times in a549-FAP lung adenocarcinoma model in small animals;

FIG. 14b is [ 2 ]¹⁸F]Graphs of% ID changes over time for different organs of small animals at different times in the A549-FAP lung adenocarcinoma model for AlF-NOTA-FAPT;

FIG. 14c is [ 2 ]¹⁸F]A plot of the ratio of the% ID value of tumor to muscle in small animals at different times over time in the A549-FAP lung adenocarcinoma model for AlF-NOTA-FAPT;

FIG. 15a is [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ], [¹⁸F]PET/CT visualizations of AlF-NOTA-FAPT in the U87 glioma model at different times in the small animals;

FIG. 15b is [ 2 ]¹⁸F]Time-dependent plots of% ID values for different organs in AlF-NOTA-FAPT in the U87 glioma model for different time-dependent animals;

FIG. 16a is a graph of a healthy volunteer at 30 minutes¹⁸F]AlF-NOTA-FAPT(¹⁸F-AlF-FAPT and¹⁸F]FAPI-42(¹⁸F-AlF-API-42) PET imaging result comparison graph;

FIG. 16b is a set of data sets of 60 minutes in a healthy volunteer¹⁸F]AlF-NOTA-FAPT(¹⁸F-AlF-FAPT and¹⁸F]FAPI-42(¹⁸F-AlF-API-42) PET imaging result comparison graph;

FIG. 16c is a graph of time 60 minutes for a healthy volunteer¹⁸F]AlF-NOTA-FAPT(¹⁸F-AlF-FAPT and¹⁸F]FAPI-42(¹⁸F-AlF-API-42) PET imaging result comparison graph;

FIG. 17a is a photograph of a patient suffering from lung cancer¹⁸F]An AlF-NOTA-FAPT PET/CT visualization map;

FIG. 17b shows a patient with gastric cancer¹⁸F]An AlF-NOTA-FAPT PET/CT visualization map;

FIG. 17c is the product of treatment of the patient with signet ring of appendix cell carcinoma¹⁸F]AlF-NOTA-FAPT PET/CT imaging map。

Detailed Description

The technical solution of the present invention is further described in detail with reference to the following specific examples, but the scope and implementation of the present invention are not limited thereto. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Also, 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 in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The invention relates to a targeted Fibroblast Activation Protein (FAP) probe, in particular to a positron nuclide labeling targeted FAP small molecule inhibitor (FAPI) probe, which is composed of FAP pharmacophore ligand, glucopyranose amido ligand (R), a linking group, a metal ion-binding chelating group and radioactive metal nuclide Xⁿ⁺And (4) forming.

In a preferred embodiment, the glucopyranosamino group comprises a 2-acetamido-2-deoxy- β -D-glucopyranosamino group or a glucosaminyl group;

and/or, the linking group comprises a polyethylene glycol group (PEG2), an aspartic acid group (Asp2), and a glutamic acid group (Glu);

and/or the metal ion-binding chelating group is 1,4, 7-triazacyclononaalkyl-N ', N' -diacetoxy-N-acetyl (-NOTA) or an analogue thereof; in more preferred embodiments, the 1,4, 7-triazacyclononanyl-N ', N "-diacetoxy-N-acetyl analog comprises 2-S- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (p-SCN-Bn-NOTA) or 1,4,7, 10-tetraazacyclododecane-N ', N" -N ' "-triacetic acid-N-acetyl (-DOTA).

And/or the radioactive metal species Xⁿ⁺Is composed of⁶⁸Ga³⁺、[Al¹⁸F]²⁺、⁶⁴Cu²⁺Or other radioactive metal ions.

In a particularly preferred embodiment, the targeted FAP probe is represented by [ Xⁿ⁺]NOTA-FAPT, wherein the linking group comprises two polyethylene glycol groups, two aspartic acid groups and a glutamic acid group, and the chemical structural formula is shown as the formula I:

wherein R isXⁿ⁺Is composed of⁶⁸Ga³⁺、[Al¹⁸F]²⁺、⁶⁴Cu²⁺Or other radioactive metal ions.

The preparation of the targeting FAP probe of the invention.

First, the precursor raw material is prepared. FAP pharmacophore modifies ligand-PEG 2 to form FAP pharmacophore ligand-PEG 2, FAP pharmacophore-PEG 2 modifies Asp2, Glu and glucopyranose amino ligand, finally modifies chelating group-NOTA or-DOTA, product peaks are collected by preparative HPLC separation and purification, and the purified precursor raw material NOTA-FAPT or DOTA-FAPT can be obtained by freeze drying.

The preparation of NOTA-FAPT or DOTA-FAPT serving as a precursor raw material aims at further solving the problem of one-step automated synthesis of [ Xⁿ⁺]NOTA-FAPT and analogues thereof probes [ Xⁿ⁺]The DOTA-FAPT lays a foundation.

Then, a radiosynthesis of a probe is carried out, wherein the radioactive metal nuclide Xⁿ⁺＝⁶⁸Ga³⁺,[Al¹⁸F]²⁺,64Cu²⁺Or other radioactive metal ions. NOTA-FAPT as precursor material is respectively reacted with⁶⁸Ga³⁺、[Al¹⁸F]²⁺、⁶⁴Cu²⁺Or other radioactive metal ions, adjusting the reaction solution to a suitable pH, and completing the reaction⁶⁸Ga³⁺、[Al¹⁸F]²⁺、⁶⁴Cu²⁺Or chelating other metal ions, separating and purifying with small column to obtain [ X ]ⁿ⁺]A NOTA-FAPT probe; DOTA-FAPT as precursor material is reacted with⁶⁸Ga³⁺、⁶⁴Cu²⁺、¹⁷⁷Lu and the like are subjected to chelation reaction and separated and purified by a small column to prepare the targeting FAP probe [ X ]ⁿ⁺]DOTA-FAPT。

In a preferred embodiment, NOTA-FAPT (R ═ 2-acetamido-2-deoxy- β -D-glucopyranosylamino) as the precursor is reacted with a starting material under weakly acidic conditions (pH 3.5-4.5) and at a temperature of 85-110 deg.C, preferably at a pH of 4.0 and 100 deg.C⁶⁸GaCl₃After chelation reaction, the product is separated and purified by Sep-Pak plus C18 column or HLB column to prepare the product⁶⁸Ga]The synthesis reaction of the NOTA-FAPT injection is shown as a synthetic scheme 1.

In a preferred embodiment, NOTA-FAPT as a precursor material is reacted with Al under weakly acidic conditions (pH 3.6-4.4) and at 85-100 deg.C, preferably at pH4.0 and 95 deg.C¹⁸After the chelation reaction of F, the product is separated and purified by a Sep-Pak plus C18 column or an HLB column to obtain the product of [ 2 ], [ 2 ]¹⁸F]The synthesis reaction of the AlF-NOTA-FAPT injection is shown as a synthetic route 2.

Further, in another preferred embodiment, NOTA-FAPT as a precursor material is reacted with a NOTA-FAPT precursor at a mildly acidic pH of 5.0-6.5 and at a temperature of 25-90 deg.C, preferably at a pH of 5.6 and 80 deg.C⁶⁴CuCl₂After the chelation reaction, the pure substances are separated by a Sep-Pak plus C18 small column or an HLB small columnCan be converted into the term⁶⁴Cu]The synthesis reaction of the AlF-NOTA-FAPT injection is shown as a synthetic route 3.

The FAP probe of the present invention, its preparation method and its application are described in detail below with reference to specific embodiments.

EXAMPLE 1 preparation of precursor NOTA-FAPT

The precursor starting material NOTA-FAPT (R ═ 2-acetylamino-2-deoxy- β -D-glucopyranosylamino) was prepared by the following method:

FAP pharmacophore ligand modification-PEG 2 forms FAP pharmacophore ligand-PEG 2, FAP pharmacophore ligand-PEG 2 modifies ASP2, Glu and 2-acetamido-2-deoxy-beta-D-glucopyranosyl amine ligand, and finally the NOTA is combined with NOTA to generate NOTA-FAPT, and the product peak is separated, purified and collected by preparative HPLC, thus obtaining the purified precursor product NOTA-FAPT.

The NOTA-FAPT has high chemical yield and purity of more than 95 percent. The results of HPLC and MS measurements of NOTA-FAPT are shown in FIG. 1a and FIG. 1b, respectively, and the molecular weight (Mr.) of NOTA-FAPT as determined by mass spectrometry MS (m/z) is 1478.48.

The precursor raw materials DOTA-FAPT (R ═ 2-acetylamino-2-deoxy- β -D-glucopyranosylamino, or glucosylamino) and NOTA-FAPT (R ═ glucosylamino) were prepared by reactions in a similar manner as described above, and the products were determined by HPLC and MS.

Example 2[ alpha ], [ alpha ] an¹⁸F]Radiosynthesis of AlF-NOTA-FAPT

To a reaction flask containing NOTA-FAPT (R ═ 2-acetylamino-2-deoxy- β -D-glucopyranosylamino) (50 μ g/. mu.l, 50 μ L) was added 2mM AlCl in sequence₃mu.L of the solution, 5. mu.L of glacial acetic acid and 300. mu.L of acetonitrile are mixed uniformly. By a cyclotron¹⁸O(p,n)¹⁸Produced by reaction of F nuclei¹⁸F^-In N at₂The carrier band is collected in a Sep-Pak QMA anion column,¹⁸the O-water was collected in a recovery bottle. Adding 0.3-0.4 mL of physiological saline (or sodium acetate buffer) into the anion (QMA) column¹⁸F^-The mixture was eluted into a vial, and 50. mu.L of the eluate was taken and added to the above reaction flask. Stirring and mixing uniformly, and heating and reacting at 100 ℃ for about 10-15 min. And cooling, adding 6-8 mL of water into a reaction bottle, uniformly mixing, and transferring to an HLB column or an SEP-PAK C18 column. After the solution in the reaction flask was completely transferred, the column was rinsed with 10mL of X3 water for injection and then blown dry. Finally, the product eluted by 1.5mL of ethanol is collected in a receiving bottle after passing through a sterile filter membrane, and is diluted into a product solution containing 5 percent of ethanol by using normal saline to obtain the product solution meeting the requirement¹⁸F]An AlF-NOTA-FAPT (R ═ 2-acetylamino-2-deoxy- β -D-glucopyranosylamino) injection. [¹⁸F]The uncorrected radiochemical yield of AlF-NOTA-FAPT is 10-30%, and the total radiosynthesis time is 35 min.

[¹⁸F]AlF-labeled NOTA-FAPT (R ═ glucosaminyl) can be prepared by analogy with the methods described above.

Example 3[ alpha ], [ alpha ] an⁶⁸Ga]NOTA-FAPT and [ [ alpha ] ]⁶⁴Cu]Radiosynthesis of NOTA-FAPT

To a 50. mu.L reaction tube containing the precursor NOTA-FAPT (R ═ 2-acetylamino-2-deoxy- β -D-glucopyranosylamino) (50. mu.g/. mu.L) was added 200. mu.L of a 1.25M sodium acetate solution. From⁶⁸Ge/⁶⁸Elution in Ga Generator with 4mL of 0.05M hydrochloric acid⁶⁸GaCl₃And (3) uniformly mixing the mixture into the reaction tube, adjusting the pH value of the solution to 4.0, and heating the solution at 100 ℃ for reaction for about 10-15 min. Cooling, adding 4mL of physiological saline into a reaction bottle, mixing uniformly, and transferring to an HLB column or an SEP-PAK C18 column. After the solution in the reaction flask is completely transferred, the column is washed with 10mL of X2 water for injection, and then dried. Then, the product is eluted with 1.5mL of ethanol and collected into a receiving bottle after passing through a sterile filter membrane, and the product solution containing 5% ethanol is diluted with physiological saline to obtain the product solution meeting the requirements⁶⁸Ga]NOTA-FAPT (R ═ 2-acetamido-2-deoxy-beta-D-glucopyranosylamino) injection. [⁶⁸Ga]The uncorrected radiochemical yield of NOTA-FAPT is 20-50%, and the total radiosynthesis time is 30 min.

To a reaction tube were added 100. mu.L of NOTA-FAPT (R ═ 2-acetylamino-2-deoxy-beta-D-glucopyranosylamino) (50. mu.g/. mu.L) and⁶⁴CuCl₂0.100-1.000mL of the solution is dissolved in sodium acetateAdjusting the pH value to 4.0-5.6, and reacting at room temperature or for 10-15 min. Finally, the solution is diluted by normal saline and collected into a receiving bottle after being filtered by a sterile filter membrane to obtain the product meeting the requirement⁶⁴Cu]NOTA-FAPT (R ═ 2-acetamido-2-deoxy-beta-D-glucopyranosylamino) injection. [⁶⁴Cu]The NOTA-FAPT uncorrected radiochemical yield is 50-70%.

⁶⁸Ga and⁶⁴the Cu-labeled NOTA-FAPT (R ═ glucosaminyl) can be prepared by the similar method as above.

Example 4[ alpha ], [ alpha ] an⁶⁸Ga]DOTA-FAPT and [ [ alpha ] ]⁶⁴Cu]Radiosynthesis of DOTA-FAPT

To a reaction tube were added 50 μ L DOTA-FAPT (R ═ 2-acetylamino-2-deoxy- β -D-glucopyranosylamino) (50 μ g/. mu.l) and 200 μ L1.25M sodium acetate solution in this order. From⁶⁸Ge/⁶⁸Elution in Ga Generator with 4mL of 0.05M hydrochloric acid⁶⁸GaCl₃Mixing the solution with the reaction tube, adjusting the pH value of the solution to 4.0, and heating the solution at 100 ℃ for reaction for 10 min. After cooling, 4mL of physiological saline was added to the reaction flask, mixed well and transferred to an HLB column. After the solution in the reaction flask is completely transferred, the column is washed with 10mL of X2 water for injection, and then dried. Then eluting the product with ethanol 1.5, collecting the product in a receiving bottle after passing through a sterile filter membrane, and diluting the product with normal saline to obtain a product solution containing 5% ethanol, thereby obtaining the product meeting the requirements⁶⁸Ga]DOTA-FAPT (R ═ 2-acetylamino-2-deoxy- β -D-glucopyranosylamino) injection. [⁶⁸Ga]The uncorrected radiochemical yield of DOTA-FAPT is 30-60%, and the total radiosynthesis time is about 30 min.

To a reaction tube were added DOTA-FAPT (R ═ 2-acetylamino-2-deoxy- β -D-glucopyranosylamino) (50 μ g/μ L)100 μ L and⁶⁴CuCl₂0.100-1.0mL of solution, adjusting the pH value to 4.0-5.6 by using sodium acetate solution, and reacting for 10-15 min at room temperature-90 ℃. Finally, the solution is diluted by normal saline and collected into a receiving bottle after being filtered by a sterile filter membrane to obtain the product meeting the requirement⁶⁴Cu]DOTA-FAPT (R ═ 2-acetylamino-2-deoxy- β -D-glucopyranosylamino) injection. [⁶⁴Cu]The uncorrected radiochemical yield of DOTA-FAPT is 30-70%.

⁶⁸Ga and⁶⁴the Cu-labeled DOTA-FAPT (R ═ glucosaminyl) can be prepared by an analogous method as above. In addition, DOTA-FAPT (R ═ 2-acetylamino-2-deoxy- β -D-glucopyranosylamino, glucosaminyl) is used as a precursor, and a conventional method is used¹⁷⁷LuCl₃The labeling method can prepare the antibody in a suitable yield¹⁷⁷Lu]DOTA-FAPT。

EXAMPLE 5 determination of the radiochemical purity and stability of the product

Radiochemical purity of the drug injection was determined by means of radioactive High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC). Conditions for HPLC analysis: the analytical column is a Zorbax eclipse xdb-c18 column. Mobile phase 0.1% trifluoroacetic acid (TFA) in acetonitrile: aqueous 0.1% TFA, gradient elution: 0min, acetonitrile/0.1% TFA in water: 10/90, respectively; gradually increasing to 10min, 0.1% TFA in acetonitrile/0.1% TFA in water: 80/20. The flow rate was 1mL/min, and the UV detection wavelengths were 210nm and 254 nm. By using a non-radioactive standard of definite structure¹⁹F]AlF-NOTA-FAPT、[Ga³⁺]NOTA-FAPT、[Cu²⁺]NOTA-FAPT、[Ga³⁺]DOTA-FAPT、[Cu²⁺]DOTA-FAPT and [ Lu³⁺]DOTA-FAPT, each with a corresponding radioactive probe¹⁸F]AlF-NOTA-FAPT、[⁶⁸Ga]NOTA-FAPT、[⁶⁴Cu]NOTA-FAPT、[⁶⁸Ga]DOTA-FAPT、[⁶⁴Cu]DOTA-FAPT and [ [ alpha ] ]¹⁷⁷Lu]DOTA-FAPT injections were co-injected into HPLC, or co-spotted to perform TLC to determine if their retention times (Rt) or specific shift values Rf were consistent and to confirm the authenticity of the prepared probes. The radiochemical purity of the product is more than 95 percent by HPLC method. Wherein, the representative example [ 2 ]¹⁸F]The results of the radioactive HPLC analysis of AlF-NOTA-FAPT injection are shown in fig. 2a and fig. 2b (emission peak and uv peak retention time Rt 8.38mi and Rt 8.17, respectively) with a radiochemical purity of greater than 95%.

TLC method for detecting target Fibroblast Activation Protein (FAP) [ X ]]Radiochemical purity of NOTA-FAPT injection. Taking a silica gel plate, placing behind the shielding lead glass, sucking a small amount of radioactive sample and a standard substance (with the concentration of 0.5mg/mL) thereof by using a capillary tube, lightly dropping the radioactive sample and the standard substance on the silica gel plate at a position 1.5cm away from one end, and drying by using an electric blower. In thatCarrying out chromatography in a chromatography cylinder, wherein a developing solvent is methanol: 1.0M ammonium acetate 50:50(V/V), dried with hot air after chromatography and scanned in thin layers using a radioactive TLC scanner. After scanning, TLC plates were stained with iodine and the shift values (Rf) of radioactive samples and standards were determined. Representative [ 2 ]¹⁸F]Rf of AlF-NOTA-FAPT injection was 0.45.

HPLC method for detecting in vivo and in vitro [ X ]]NOTA-FAPT stability. A representative example is the detection of neutralization of urine in serum of an animal by HPLC¹⁸F]AlF-NOTA-FAPT in vivo stability. [¹⁸F]The radiochemical purity of AlF-NOTA-FAPT in serum and urine is shown in FIGS. 3a and 3 b. As can be seen from FIGS. 3a and 3b, at 60min, only a single major peak was observed in urine, and no other minor peaks were detected, indicating that¹⁸F]AlF-NOTA-FAPT did not defluorinate and decompose within 1h in urine (FIG. 3 b); no significant defluorination was observed in the serum (FIG. 3 a). [¹⁸F]AlF-NOTA-FAPT has an in vitro stability as shown by the fact that the in vitro stability is good when the in vitro purity of the AlF-NOTA-FAPT is more than 90% in PBS buffer and in vitro serum for 2h (shown in figure 4a and figure 4 b).

Detection of other [ X ] s by the same methodⁿ⁺]NOTA-FAPT is stable in vitro, and only a single main peak is found. These results indicate that the other [ X ]ⁿ⁺]NOTA-FAPT is relatively stable in serum.

Example 6 ester Water partition coefficient measurement experiment

Adding n-octanol and 3ml of water into the test tube, and adding the solution¹⁸F]Shaking AlF-NOTA-FAPT medicine, centrifuging, collecting the upper and lower layers each at 100ul, dividing into three tubes, and measuring radioactivity value with gamma counter. Warp determination [ 2 ]¹⁸F]The distribution coefficient of AlF-NOTA-FAPT fat water is LogP-1.85 +/-0.07 (n-4), and¹⁸F]FAPI-42Log P ═ -2.43 ± 0.02(n ═ 4). Result display¹⁸F]AlF-NOTA-FAPT has a significant hydrophilicity, but is less hydrophilic than [ alpha ], [ alpha ] and [ alpha ], [ alpha ] and a ] or a¹⁸F]FAPI-42。

Example 7 in vitro cellular uptake and inhibition experiments

And (3) an A549-FAP uptake inhibition experiment with high FAP expression.

Tumor cell strain A549 of Shanghai cell bank of Chinese academy of sciences is cultured conventionally, and the A549-FAP with high FAP expression is obtained through transfection treatment. Logarithmic growthCancer cells of stage, digested with 0.25% pancreatin, washed twice with PBS; centrifuging at 1000 rpm for 5min, collecting cells, adjusting cell density to about 6 × 10⁶and/mL. Adding 0.1mL of cell suspension into each well of a 24-well plate, culturing for 24h, replacing fresh culture solution after the cells adhere to the wall, randomly dividing the culture solution into A, B, C, D, E groups, and adding¹⁸F]Continuing to culture after AlF-NOTA-FAPT, adding FAPI-04 inhibitor into the inhibition group, washing with PBS for 3 times after 5min, 15min, 30min, 60min and 120min, respectively, adding sodium dodecyl sulfate to make the cells fall off, collecting cells in each well, and measuring the count of the cells, i.e. the count of the cells taken into cytoplasm.

Results of cellular uptake and inhibition experiments: [¹⁸F]The AlF-NOTA-FAPT is quickly taken up in A549-FAP cells, reaches 13.63 +/-1.50% ID/million cells at 60 minutes, can be maintained at 16.33 +/-0.55% ID/million cells at 120 minutes, and shows lower taking values in wild A549 cells and A549-FAP cell inhibition groups, which indicates that¹⁸F]AlF-NOTA-FAPT has relatively high uptake in A549-FAP cells and has specificity (see FIG. 5).

293T-FAP uptake inhibition experiments with high FAP expression.

Tumor cell strain 293T of Shanghai cell bank of Chinese academy of sciences is cultured conventionally, and 293T-FAP with high expression of FAP is obtained through transfection treatment. Taking cancer cells in logarithmic growth phase, digesting the cancer cells with 0.25% pancreatin, and washing the cells twice with PBS; centrifuging at 1000 rpm for 5min, collecting cells, adjusting cell density to about 6 × 10⁶and/mL. Adding 0.1mL of cell suspension into each well of a 24-well plate, culturing for 24h, replacing fresh culture solution after the cells adhere to the wall, randomly dividing the culture solution into A, B, C, D, E groups, and adding¹⁸F]Continuing to culture after AlF-NOTA-FAPT, adding FAPI-04 inhibitor into the inhibition group, washing with PBS for 3 times after 5min, 15min, 30min, 60min and 120min, respectively, adding sodium dodecyl sulfate to make the cells fall off, collecting cells in each well, and measuring the count of the cells, i.e. the count of the cells taken into cytoplasm.

Results of cellular uptake experiments: [¹⁸F]AlF-NOTA-FAPT uptake rapidly in 293T-FAP cells, reaching at 60minTo 115.21. + -. 3.51% ID/million cells, in the inhibition group, showed a lower uptake value, indicating that¹⁸F]AlF-NOTA-FAPT has relatively high uptake in 293T-FAP cells and specificity (see FIG. 6).

And (4) measuring the affinity. Tumor cell plating: one 24-well plate was plated, one set of three-well cells, and 8 sets (see FIG. 7). The concentration of the competitive inhibitor (NOTA-FAPI) was (0, 10)^-5，10^-6，10^-7，10^-8，10^-9，10^-10，10^-11M). Each concentration of inhibitor was dissolved in 0.5mL of medium and added to each well separately, followed by the addition of 0.5mL of imaging agent to each well (0.5. mu. Ci/0.5 mL/well). After incubation at 37 ℃ for 1h, three washes were performed with PBS. Radioactivity was measured in a gamma counter after cells were ablated in NaOH in SDS. Representative term¹⁸F]The results of the AlF-NOTA-FAPT competitive binding experiments are shown in FIG. 8. [¹⁸F]The IC50 value of AlF-NOTA-FAPT is 1.16nM, [ alpha ], [ alpha¹⁸F]The FAPI-42 has an IC50 value of 14nM, and this result shows that¹⁸F]AlF-NOTA-FAPT has stronger affinity.

Example 8[ alpha ], [ alpha ] an¹⁸F]AlF-NOTA-FAPT in vivo biodistribution experiment

In the body of normal Kunming mouse¹⁸F]AlF-NOTA-FAPT biodistribution experiment. 6 Kunming mice were randomly divided into 2 groups of 3 mice each containing 20-40. mu. Ci [ 2 ] per injection of 0.1-0.2mL per vein¹⁸F]AlF-NOTA-FAPT solution, mice were sacrificed 30, 60, 90min post injection after removal of the eyeball for blood sampling. Tissue samples of each organ of interest (blood, brain, heart, lung, liver, gallbladder, kidney, spleen, stomach, small intestine, muscle, femur, etc.) were dissected and taken, weighed, their radioactivity counts measured, and the percentage of radioactivity injected dose (% ID/g) per gram of tissue at different time points was calculated.

Biodistribution results: [¹⁸F]The results of the in vivo biodistribution experiments of AlF-NOTA-FAPT in healthy Kunming mice are shown in FIG. 9 a. The results indicated that there was the highest radioactive uptake in the femur at 30min and no major change was seen over time; moderate radioactive uptake in blood and kidney and low radioactive uptake in liver and gall bladder, but PET imaging indicates higher radioactivity in kidneySexual uptake, which is not fully consistent with animal PET imaging results; the radioactive uptake of stomach, spleen, lung, heart, brain, muscle and intestine is low in other organs.

[¹⁸F]The in vivo biodistribution of FAPI-42 in the tumor-bearing model is shown in FIG. 9 b. The results show that the highest radioactive uptake in the gallbladder, higher radioactive uptake in bones and tumors, moderate radioactive uptake in intestinal tract, lung, kidney, muscle and blood, and lower radioactive uptake in other tissues and organs.

The result of in vivo biodistribution indicates that the value of [ alpha ], [ beta ] -is in a form of a dispersion¹⁸F]AlF-NOTA-FAPT is superior to [, ]¹⁸F]FAPI-42. Specifically, the term⁶⁸Ga]FAPI-04(⁶⁸Ga-FAPI-04)[¹⁸F]FAPI-42(¹⁸F-FAPI-42) and¹⁸F]P-FAPI(¹⁸the structural formula of F-P-FAPI) is shown as follows.

[⁶⁸Ga]The in vivo biodistribution of NOTA-FAPT in a mouse¹⁸F]AlF-NOTA-FAPT is approximately similar. However, the term⁶⁸Ga]FAPI-04 and [ alpha ], [ beta ], [ alpha ], [ beta ]¹⁸F]FAPI-42 has a high uptake of radioactivity in the hepatobiliary and intestinal systems and an unstable uptake of radioactivity in the pancreas. [¹⁸F]AlF-NOTA-FAPT and⁶⁸Ga]NOTA-FAPT exhibits the same effect as that of [ alpha ], [ alpha ]⁶⁸Ga]FAPI-04 and [ alpha ], [ beta ], [ alpha ], [ beta ]¹⁸F]FAPI-42 does not have exactly the same in vivo biodistribution characteristics, and the first two show superior pharmacokinetic properties than the latter two.

Example 9 model animal PET imaging experiment

Micro-PET/CT imaging research utilizes Siemens Inveon Micro-PET/CT (resolution about 1.4mm, aperture 12cm, axial field of view 12.7cm), an acquisition workstation is Inveon acquiriion workplace (IRW)2.0, and a new Workflow (including CT acquisition, Reconstruction, PET acquisition, PET his) is established before data acquisitiontogram, PET Recon.) to be tested, and transplanting A549FAP lung adenocarcinoma cell strain, U87 brain glioma cell strain, A549 lung adenocarcinoma cell strain and the like to the tested nude mouse to prepare the nude mouse axillary tumor model. After 10% chloral hydrate anesthesia, PET/CT scanning is carried out to collect the developer [ 2 ]¹⁸F]PET/CT images 60 minutes after 200. mu. Ci injection with AlF-NOTA-FAPT (A549-FAP; U87; A549).

After PET imaging, model animals are killed, tumor tissues are dissected and taken, and the pathological immunohistochemical results prove that: the A549FAP lung adenocarcinoma tissue and the U87 brain glioma tissue are high in FAP expression, and the A549 lung adenocarcinoma tissue is low in FAP expression or non-expression. The PET imaging result shows that: [¹⁸F]AlF-NOTA-FAPT has higher uptake in A549FAP lung adenocarcinoma model, and the inhibition of imaging can show that the uptake is obviously reduced, namely specific uptake (shown in figure 10); there was also a higher uptake in the U87 brain glioma model and a significant decrease in uptake seen by inhibition of imaging, as specific uptake (see figure 11), whereas there was little uptake in the a549 wild-type lung adenocarcinoma model (see figure 12). In the same model¹⁸F]AlF-NOTA-FAPT (NOTA-FAPT) and¹⁸F]a comparison of the FAPI-42(NOTA-FAPI-42) PET imaging results is shown in FIG. 13 a. In the same U87 tumor mouse model¹⁸F]AlF-NOTA-FAPT and¹⁸F]the comparison visualization result of FAPI-42 shows that: [¹⁸F]The tumor uptake value of AlF-NOTA-FAPT is increased to nearly 6% ID/g at 15min, and is maintained at a relatively high uptake value for 120min¹⁸F]The FAPI-42 starts to decline after reaching the highest uptake value in 10min, and the uptake value is maintained at about 3% ID/g finally.

Thus, in the aspect of tumor uptake, the term¹⁸F]AlF-NOTA-FAPT is obviously superior to [ 2 ]¹⁸F]FAPI-42. In addition, in the imaging of other organs, the term [ 2 ]¹⁸F]FAPI-42 can show obvious gallbladder intake and intestinal intake¹⁸F]Except for slightly higher uptake in the spine and joints, the AlF-NOTA-FAPT images showed low uptake in both gallbladder and intestinal tract (fig. 13 b).

Further, the term¹⁸F]AlF-NOTA-FAPT has higher uptake in both A549FAP lung adenocarcinoma (shown in FIGS. 14a, 14b and 14 c) and U87 glioma model (shown in FIGS. 15a and 15 b) from 15 minutesThe tumor maintained a relatively high uptake starting at 120 minutes.

Example 10 human PET imaging experiment

A conventional visualization and reconstruction method of the united image uEXPLORER PET/CT: the patient is allowed to rest for about 60 minutes after intravenous administration of the radioimaging agent, followed by PET/CT image scan acquisition, as required by clinical code. The tube voltage for whole body CT scanning is 120kV, and spiral scanning is carried out by adopting an automatic milliamp technology. Whole body PET was scanned using a static 3D acquisition mode with acquisition time of 5min per whole body. And (3) carrying out image reconstruction on the acquired data by adopting an ordered subset expectation maximization algorithm (OSEM) and a time of flight (TOF) after time attenuation correction and tissue attenuation correction, and finally obtaining a 3D PET image for clinical quantitative analysis and diagnosis.

Healthy volunteer [ alpha ], [ beta ] -state¹⁸F]AlF-NOTA-FAPT(¹⁸F-AlF-FAPT and¹⁸F]FAPI-42PET(¹⁸F-AlF-FAPI-42) imaging comparison (see fig. 16a, 16b and 16c) shows: [⁶⁸Ga]FAPI-04 and [ alpha ], [ beta ], [ alpha ], [ beta ]¹⁸F]FAPI-42 has high radioactive uptake in the hepatobiliary system and the intestinal system, and unstable radioactive uptake in the pancreas; and 2¹⁸F]AlF-NOTA-FAPT has very low radioactive uptake in the hepatobiliary system and the intestinal system, has high stable radioactive uptake in the pancreas, and has moderate blood radioactive uptake; other tissue and organ [ 2 ]¹⁸F]AlF-NOTA-FAPT and¹⁸F]FAPI-42 uptake is approximately similar. In sum, the term¹⁸F]AlF-NOTA-FAPT exhibits the same meaning as that of [ alpha ], [ alpha ] and [ alpha ], [ alpha ] and [ alpha ] a⁶⁸Ga]FAPI-04 and [ alpha ], [ beta ], [ alpha ], [ beta ]¹⁸F]The in vivo biodistribution characteristic of FAPI-42 is not exactly the same, and the expression "FAPI" is used for the expression of the gene¹⁸F]AlF-NOTA-FAPT exhibits superior pharmacokinetic properties.

Patient [ 2 ]¹⁸F]AlF-NOTA-FAPT PET/CT imaging is shown in FIGS. 17 a-17 c. As can be seen from fig. 17a to 17c, the tumor residues (see fig. 17 a), gastric cancer (see fig. 17 b) and peritoneal metastasis (see fig. 17 c) after treatment of appendiceal ring cell carcinoma were observed to be taken up to some extent in the PET/CT images.

[¹⁸F]AlF-NOTA-FAPT has high uptake in various solid tumors such as brain glioma and lung adenocarcinomaAnd (4) obtaining the compound, and the compound can be used for imaging with high expression of FAPPET. In the present invention [ X ]ⁿ⁺]The NOTA-FAPT analog can also exhibit the combination of¹⁸F]The similar biological functions of AlF-NOTA-FAPT can play an important clinical diagnosis role in PET imaging of tumors and other diseases. Although further improvements in their pharmacokinetic properties may be required through structural optimization, it will be apparent to those skilled in the art that modifications and variations can be made to their structures in light of the above teachings and all such modifications and variations are intended to fall within the scope of the appended claims.

Various technical features of the above embodiments may be combined arbitrarily, and for the sake of brevity, all possible combinations of the technical features of the above embodiments are not described in this specification. However, as long as there is no contradiction between combinations of these technical features, the scope of the present specification should be considered as being described. Furthermore, the above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.