WO2023143502A1 - FAP-α SPECIFIC RADIOPHARMACEUTICAL AND APPLICATION THEREOF - Google Patents

Abstract

A precursor compound for forming a radionuclide complex, having the structure of formula I or formula II: wherein L is selected from -(CH2)m- and -CH2-PEG4-CH2-; L1 is -C(O)-L-NH-; BFC is a bifunctional chelating agent. A radiopharmaceutical is formed by the radionuclide-labeled precursor compound, and the pharmaceutical can be used as a diagnostic imaging agent or a radioactive targeted therapeutic agent for FAP-positive tumors or FAP-positive fibrotic diseases (such as pulmonary fibrosis and liver fibrosis).

Description

A kind of FAP-α specific radiopharmaceutical and its application

This application claims the priority of the previous application with the patent application number 202210113374.9 and the title of the invention "A FAP-α-specific radiopharmaceutical and its application" submitted to the State Intellectual Property Office of China on January 29, 2022. The entirety of the application is incorporated by reference into this application.

technical field

The invention relates to a novel class of radiopharmaceuticals based on oncoFAP molecules and a preparation method thereof.

Background technique

Cancer is the second leading cause of death in the world, and despite significant advances in diagnosis and treatment, most developed therapies target tumor cells while ignoring the tumor microenvironment.

Tumor entities contain not only tumor cells, but also stromal cells such as vascular cells, inflammatory cells, and fibroblasts. The stroma in tumors usually accounts for a large part of the malignant tumor entity, and can even account for more than 90% of the tumor mass. There is a complex interaction network between stroma and tumor cells, especially the cell subset called cancer associated fibroblasts (CAFs), which is involved in almost all stages of tumorigenesis. , Transfer plays a role. Therefore, CAF has become one of the hotspots in the field of tumor research.

CAFs have multiple origins, and they may originate from local tumor fibroblasts, circulating fibroblasts, vascular endothelial cells, adipocytes, bone marrow-derived stem cells, and even cancer cells, and the difference in tissue types is one of the reasons for the heterogeneity of CAFs . Due to the heterogeneity of origin and expression patterns of CAFs, it is difficult to use a unified marker to identify CAFs of all subpopulations. However, high expression of fibroblast activation protein (FAP) has been found in stromal CAFs of many tumors. FAP is a type II membrane-bound glycoprotein belonging to the type II serine protease family with dipeptidyl peptidase and endopeptidase activities. This enzyme is transiently expressed during embryonic development, has no or very low expression in normal adult tissues, and is highly expressed in more than 90% of epithelial cancers, such as head and neck cancer, breast cancer, lung cancer, pancreatic cancer, esophageal cancer, colorectal cancer Cancer, ovarian cancer, gastric cancer, liver cancer, etc. High expression of FAP in CAFs has been shown to be a marker of tumor aggressive behavior and poor prognosis. Compared with tumors, the differentially low expression of FAP in normal tissues provides excellent conditions for radionuclide-labeled FAP-targeted nuclear medicine imaging, and its high expression also provides a basis for subsequent radiation-targeted therapy or targeted drugs. Delivery comes with convenience.

The latest research progress shows that the widely studied FAPI small molecules are mainly FAP-α inhibitors based on the quinolinyl-glycine-(2S)-cyanoproline skeleton, all of which are FAPI obtained by modifying the 6-position of quinine Small molecule. Representative ones are the following FAPI-02, FAPI-04, FAPI-34, FAPI-46, FAPI-74 (J Nucl Med 2021; 62:160–167):

Among them, most of the above-mentioned FAPI small molecules are labeled with positron nuclides ⁶⁸ Ga, ¹⁸ F, etc., and applied to PET (Positron Emission Tomography) imaging. Among them, only FAPI-34 is labeled with single-photon nuclide ^99m Tc for SPECT imaging applications. In addition, CN 111991570 B announced ^99m Tc-labeled HFAPi and HpFAPi-labeled drugs based on FAPI-04, and optimized the in vivo pharmacokinetic characteristics of ^99m Tc-labeled FAPI molecules. The specific structure is as follows:

Since in human tumors, the origin, number and distribution of FAP-expressing CAFs and the number of FAP molecules per cell may vary, in order to better perform nuclear medicine on tumors with relatively low FAP expression levels in clinical Explore Needle imaging requires optimization of existing FAP small molecule nuclear medicine probes to enhance their tumor targeting, improve in vivo stability, enable higher tumor uptake and faster systemic background clearance, and then Obtain remarkable tumor/normal tissue contrast to improve lesion detection rate.

OncoFAP (PNAS 2021 Vol.118 No.16 e2101852118) was recently reported based on the 8-position chemical modification of quinoline, which is a novel FAP ligand with a binding dissociation constant in the subnanomolar concentration range. The structural formula of oncoFAP is

At present, the research on oncoFAPI is limited to the labeling of ⁶⁸ Ga and ¹⁷⁷ Lu based on oncoFAP monomers. Preliminary PET imaging studies and in vivo evaluations in mice have been carried out, but no molecular structure suitable for single-photon nuclide ^99m Tc labeling has been developed yet. , for SPECT imaging research. Compared with PET, SPECT (Positron Emission Computed Tomography) diagnosis uses relatively simple and low-cost drug preparation, moderate clinical examination costs, high penetration rate, and easy promotion and acceptance. Like probes may have better application prospects. However, the FAPI molecules currently studied are fast in vivo, and researchers still need to continue to develop new molecular structures to adjust pharmacokinetic characteristics, increase their circulation time in vivo, improve tumor uptake and retention, and further improve the combination with FAP affinity for higher tumor uptake and better tumor to non-target tissue contrast. The improvement of these performances will help to improve the detection rate and accuracy of FAP-positive tumors, especially for tumors and lesion tissues with relatively low FAP expression, such as pulmonary fibrosis, etc., which have significantly enhanced detection efficiency.

Contents of the invention

The purpose of the present invention is to provide a new type of radiopharmaceutical based on oncoFAP molecular enhancement. The novel enhanced oncoFAP molecule designed in the present invention utilizes the SPECT imaging technology of nuclear medicine to perform imaging diagnosis on FAP-positive tumors or fibrotic diseases. This enhanced oncoFAP compound structure can also chelate DOTA and NOTA chelating agents for labeling metal nuclides such as ⁶⁸ Ga, ⁶⁴ Cu, ¹⁷⁷ Lu, etc., to realize PET imaging and nuclide therapy.

The purpose of the present invention is achieved through the following technical solutions:

A precursor compound for forming a radionuclide complex, which has a structure shown in the following formula I or formula II:

in,

L is selected from:

-(CH ₂ )m-, m is an integer of 2-6, preferably 2 or 6;

-CH ₂ -PEG ₄ -CH ₂ -, wherein the structural formula of PEG ₄ is: both ends Represents a bond to a methylene bond chain;

_L is -C(O)-L-NH-, wherein L is as defined above;

n is selected from 0 or 1;

BFC is selected from BFC is a bifunctional chelating agent, selected from HYNIC, MAG2, MAG3, DTPA, DOTA, NOTA, TETA.

It is well known to those skilled in the art that the bond chains of each fragment of such compounds are formed by the reaction of amino groups and carboxyl groups to form peptide bonds. For example, BFC forms peptide bond chains through its carboxyl groups and amino groups in the main structure.

The present invention also provides a complex formed after the above-mentioned precursor compound is labeled with a radionuclide.

According to an embodiment of the present invention, the radionuclide is selected from ¹¹¹ In, ⁶⁴ Cu, ^99m Tc, ⁶⁸ Ga, ¹²³ I, ¹⁸ F, ⁹⁰ Y, ¹⁷⁷ Lu, ¹³¹ I, ¹²⁵ I, ⁸⁹ Sr, ¹⁵³ Sm.

According to an embodiment of the present invention, the radionuclide is selected ^from99mTc , ^68Ga , ^64Cu , ^177Lu .

According to an embodiment of the present invention, when the radionuclide is selected from ^99m Tc, BFC is selected from HYNIC; when the radionuclide is selected from ⁶⁸ Ga, ⁶⁴ Cu, ¹⁷⁷ Lu, BFC is selected from DOTA, NOTA.

Those skilled in the art can understand that for the complex defined above, when the bifunctional chelating agent as a ligand cannot occupy all positions of the radionuclide, a coordinating ligand is also required. The radionuclides and bifunctional chelators for which cooperating ligands are required in the present invention are well known to those skilled in the art. For example, when ^99m Tc uses HTNIC as a bifunctional chelating agent, as a synergistic ligand, it can be the same or different, and is all those known in the prior art, wherein common synergistic ligands include water-soluble phosphine (such as triphenylphosphine Trimesansulfonic acid sodium salt TPPTS), N-tris(hydroxymethyl)methylglycine (Tricine), N-bis(hydroxyethyl)glycine, glucoheptonate, ethylenediamine-N,N'-di Acetate (EDDA), 3-benzoylpyridine (BP), pyridine-2-azo-p-xylidine (PADA), etc.

As examples, precursor compounds of the invention are described below:

HYNIC-[C ₂ -oncoFAP] ₂ , its structural formula is shown in compound 6 in the accompanying drawing 2 of the description.

HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ , the structural formula of which is shown in Compound 7 in Figure 3 of the specification.

HYNIC-[PEG ₄ -oncoFAP] ₂ , the structural formula of which is shown in compound 10 in Figure 4 of the description.

HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ , the structural formula of which is shown in Compound 11 in Figure 5 of the specification.

HYNIC-[C ₆ -oncoFAP] ₂ , its structural formula is shown as compound 13 in the figure below.

HYNIC-[Aoc-oncoFAP] ₂ , its structural formula is shown as compound 15 in the figure below.

DOTA-[C ₂ -oncoFAP] ₂ , its structural formula is shown in compound A in the accompanying drawing 7 of the description.

NOTA-[C ₂ -oncoFAP] ₂ , the structural formula of which is shown in compound B in Figure 7 of the specification.

DOTA-[PEG ₄ -oncoFAP] ₂ , its structural formula is shown in Compound C in Figure 7 of the specification.

NOTA-[PEG ₄ -oncoFAP] ₂ , the structural formula of which is shown in Compound D in Figure 7 of the specification.

The structural formulas of HYNIC-C ₂ -oncoFAP and HYNIC-PEG ₄ -C ₂ -oncoFAP are as shown in compounds 3 and 4 in Figure 1 of the specification.

As examples, complexes of the invention are described below:

^99m Tc-HYNIC-[C ₂ -oncoFAP] ₂ , the structural formula of which is shown in Compound C in Figure 6 of the specification.

^99m Tc-HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ , the structural formula of which is shown in Compound D in Figure 6 of the specification.

^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ , the structural formula of which is shown in Compound E in Figure 6 of the specification.

^99m Tc-HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ , the structural formula of which is shown in Compound F in Figure 6 of the specification.

^99m Tc-HYNIC-[C ₆ -oncoFAP] ₂ , the structure refers to ^99m Tc-HYNIC-[C ₂ -oncoFAP] ₂ .

The structural formulas of ^99m Tc-HYNIC-C ₂ -oncoFAP and ^99m Tc-HYNIC-PEG ₄ -oncoFAP are shown in Compounds A and B in Figure 6 of the specification.

The present invention also provides a medicine containing the complex. The medicament can be used as an imaging diagnostic agent or a radiation-targeted therapeutic agent for FAP-positive tumors or FAP-positive fibrotic diseases (such as pulmonary fibrosis, liver fibrosis, etc.). Those skilled in the art are well aware that the functions of the above radionuclides as diagnostic or therapeutic agents for cancer or tumors are mainly determined by the type of radioactive rays of the nuclides. The radiopharmaceuticals obtained in the present invention can target the highly expressed FAP molecule in the tumor, which is the function brought by the structure of the precursor compound. Therefore, when the precursor compound of the present invention is combined with different nuclides, it can be used as the target of the tumor respectively. diagnostic or therapeutic agents. For example, when the radionuclide is ⁶⁸ Ga, ⁶⁴ Cu, the drug is used as a PET imaging agent; when the radionuclide is ¹⁷⁷ Lu, the drug is used as a PET therapeutic agent; when the radionuclide is ^99m Tc, The drug acts as a SPECT imaging agent. Usually, the therapeutic agent needs the drug to have higher uptake and longer residence time in the tumor. It can be known from the examples of the present invention that the radiopharmaceutical of the present invention has the performance to meet this requirement, so those skilled in the art can fully predict Therapeutic agents formed from the inventive precursor compounds are suitable for therapeutic use.

According to an embodiment of the present invention, the drug is an injectable preparation comprising the above-mentioned labeled complex and an injectable carrier. Preferably, the drug is a colorless and transparent injectable preparation.

The present invention also provides the application of the above precursor compound or complex in the preparation of medicaments for diagnosing or treating FAP-positive tumors or fibrotic diseases.

Beneficial effects: the radiopharmaceutical of the present invention is a brand-new FAP-targeted molecular imaging probe, which can be applied to Nuclear medicine molecular imaging of various FAP-expressing tumors or FAP-positive fibrotic diseases (such as pulmonary fibrosis, liver fibrosis, etc.), so as to realize early diagnosis and screening of diseases. On the basis of the small molecule oncoFAP, the present invention obtains ligand compounds with larger molecular weight and volume through structural modification, and the prepared radiopharmaceuticals have significantly excellent in vivo stability, stronger tumor targeting, and higher tumor and tumor resistance. Contrast of non-target tissue. At the same time, the biocompatibility of the probe is improved, and the pharmacokinetic properties are optimized. In addition, the ligand compound of the present invention has wider applicability. In addition to the SPECT imaging agent chelated with HYNIC, it can also be chelated with DOTA, etc., and can be used for ⁶⁸ Ga, ⁶⁴ Cu and ¹⁷⁷ Lu labeling, and can be applied to more PET imaging and nuclide-targeted radiation therapy for FAP-expressing tumors.

Based on oncoFAP, the present invention firstly obtains the following two specific precursor compounds and corresponding complexes chelated with radionuclides through structure modification: HYNIC-C ₂ -oncoFAP, HYNIC-PEG ₄ -C ₂ -oncoFAP , and its structural formula is shown in compound 3 and 4 of accompanying drawing 1 of the description. The corresponding complexes are ^99m Tc-HYNIC-C ₂ -oncoFAP and ^99m Tc-HYNIC-PEG ₄ -oncoFAP, the structures of which are shown in compounds A and B in Figure 6 of the specification. Compared with oncoFAP known in the prior art, the two probes have higher tumor uptake and imaging contrast, indicating that the oncoFAP-based radioactive probe has good tumor-specific targeting ability. On this basis, the present invention also obtains a molecular probe with higher tumor uptake and faster blood clearance, which makes the background of other organs lower and thus presents better contrast in nuclear medicine imaging. The present invention also studies the impact of structural modifications of the same type of modified chains with different lengths, and obtains the following two specific precursor compounds, HYNIC-[C ₂ -oncoFAP] ₂ and HYNIC-[C ₆ -oncoFAP] ₂ , whose structural formula They are respectively shown in compound 6 in the accompanying drawing 2 of the description and compound 13 in the description. The corresponding complexes are ^99m Tc-HYNIC-[C ₂ -oncoFAP] ₂ , ^99m Tc-HYNIC-[C ₆ -oncoFAP] ₂ . The results of the in vivo biodistribution data of the mouse tumor model (shown in Figure 14C and Figure 14G, respectively) showed that the blood uptake of ^99m Tc-HYNIC-[C ₆ -oncoFAP] ₂ was higher, and the tumor uptake was higher at 0.5 h than ^99m Tc-HYNIC-[C ₂ -oncoFAP] ₂ was slightly lower, but ^99m Tc-HYNIC-[C ₆ -oncoFAP] ₂ tumor uptake was significantly increased at 4 h after injection, while ^99m Tc-HYNIC-[C ₂ - Tumor uptake of oncoFAP] ₂ decreased over time, so tumor uptake of the two became comparable at later time points. It can be seen that the structure-activity relationship in this field is tight, and the slight difference in the number of C in the chain also obviously affects its tumor uptake, and its trend is difficult to predict. Even for the same compound, tumor uptake increases or decreases or mutations over time are unpredictable.

In addition, the present invention also studies the case where the connecting arm is an aliphatic chain (Aoc) of 8-octylamino, the precursor compound is HYNIC-[Aoc-oncoFAP] ₂ , as shown in the above compound 15, and the corresponding complex is ^99m The in vivo biodistribution data of Tc-HYNIC-[Aoc-oncoFAP] ₂ are shown in Figure 14H. It can be seen that when the Aoc chain is replaced, the in vivo biodistribution of the entire probe is mainly absorbed in the kidney (kideny) and gallbladder (Gallbla), and there is no obvious uptake in the tumor. The results of the blocking group are similar to those of the non-blocking group Tumor uptake was not significantly different. Therefore different types of tether chains are The impact brought by the oncoFAP molecule is obviously different, which seriously affects the targeting effect of the compound.

Based on the above comparison, those skilled in the art can understand that the structural modifications and improvements made on the basis of oncoFAP in the present invention, especially those modified by the _C2 and _PEG4 chains, have better tumor targeting properties and in vivo metabolic properties, so Has excellent application performance and value.

In addition, the present invention also compares the typical molecular probes ^99m Tc-HFAPi and ^99m Tc-HpFAPi of CN 111991570 B with the molecular probe of the present invention. Compared with CN 111991570 B, the molecular probe of the present invention has better Stability in body metabolism, stronger binding ability to recombinant human FAP protein, higher absolute value of tumor uptake, faster clearance metabolism of normal organs, higher tumor/normal organ uptake ratio of probes, more conducive to nuclear medicine of tumors imaging. More prominently, the molecular probe of the present invention can specifically image the lesion area of pulmonary fibrosis, and the effect is better.

Description of drawings

Figure 1. (A) Compound 3: Synthetic route of HYNIC-C ₂ -oncoFAP, (B) Compound 4: Synthetic route of HYNIC-PEG ₄ -C ₂ -oncoFAP.

Figure 2. Synthetic route of compound 6: HYNIC-[C ₂ -oncoFAP] ₂ .

Figure 3. Synthetic route of compound 7: HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ .

Figure 4. Synthetic route of compound 10: HYNIC-[PEG ₄ -oncoFAP] ₂ .

Figure 5. Synthetic route of compound 11: HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ .

Figure 6. (A) ^99mTc -HYNIC-C ₂ -oncoFAP, (B) ^99mTc -HYNIC-PEG ₄ -C ₂ -oncoFAP, (C) ^99mTc -HYNIC-[C ₂ -oncoFAP] ₂ , (D ) ^99m Tc-HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ , (E) ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ , (F) ^99m Tc-HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ Schematic diagram of the structure.

Figure 7. Schematic structure of (A) DOTA-[C ₂ -oncoFAP] ₂ , (B) NOTA-[C ₂ -oncoFAP] ₂ , (C) DOTA-[PEG ₄ -oncoFAP] ₂ , (D) NOTA- [PEG ₄ -oncoFAP] ₂ .

Figure 8. Radioactive HPLC profiles of probes in urine samples of BALB/c mice at different times after injection. (A) ^99mTc -HYNIC-C ₂ -oncoFAP, (B) ^99mTc -HYNIC-PEG ₄ -C ₂ -oncoFAP, (C) ^99mTc -HYNIC-[C ₂ -oncoFAP] ₂ , (D) ^99mTc -HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ , (E) ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ , (F) ^99m Tc-HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ .

Figure 9. (A) Radioactive HPLC spectra of ^99m Tc-HFAPi in urine samples of BALB/c mice at different times after injection. (B) Radioactive HPLC spectra of ^99m Tc-HpFAPi probe in urine samples of BALB/c mice at different times after injection.

Figure 10. (A) ^99m Tc-HYNIC-C ₂ -oncoFAP ( ^99m Tc-HC-oFP), ^99m Tc-HYNIC-PEG ₄ -C ₂ -oncoFAP ( ^99m Tc-HP-oFP), ^99m Tc-HYNIC- [C ₂ -oncoFAP] ₂ ( ^99m Tc-H-CoFP ₂ ), ^99m Tc-HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ ( ^99m Tc-HP-CoFP ₂ ), ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ ( ^99m Tc-H-PoFP ₂ ), ^99m Tc-HYNIC-PEG ₄ -(PEG ₄ -oncoFAP) ₂ ( ^99m Tc-HP-PoFP ₂ ) binding experiment to recombinant human FAP-α protein (rhFAP-α). (B) Binding experiment of ^99m Tc-HYNIC-FAPI-04 ( ^99m Tc-HFAPi), ^99m Tc-HYNIC-PEG ₄ -FAPI-04 ( ^99m Tc-HpFAPi) and recombinant human FAP-α protein (rhFAP-α) . (C) Binding percentage of each probe in protein binding assay.

Fig. 11. (A) SPECT/CT images after 0.5, 1, 2 and 4 h after injection of ^99m Tc-HYNIC-C ₂ -oncoFAP in U87MG glioblastoma model; (B) 0.5 h without SPECT/CT images of labeled oncoFAP blocking group; (C) 0.5, 1, 2 and 4 h after injection of ^99m Tc-HYNIC-PEG ₄ -C ₂ -oncoFAP in U87MG glioblastoma model SPECT/CT imaging images; (D) SPECT/CT imaging images of unlabeled oncoFAP blocking group at 0.5 h.

Figure 12. (A) SPECT/CT images of ^99m Tc-HYNIC-[C ₂ -oncoFAP] ₂ injected in U87MG glioblastoma model after 0.5, 1, 2 and 4 h; (B) SPECT/CT images _of unlabeled oncoFAP blocking group at ^0.5 h; (C) 0.5 _{, 1} , SPECT/CT imaging images after 2 and 4 hours; (D) SPECT/CT imaging images of unlabeled oncoFAP blocking group at 0.5 h.

Figure 13. (A) SPECT/CT images of 0.5, 1, 2 and 4 h after injection of ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ in U87MG glioblastoma model; (B) SPECT/CT image of unlabeled oncoFAP blocking group at 0.5 h; (C) ^0.5, _{1 ,} 0.5, ₁ , SPECT/CT images after 2 and 4 h; (D) SPECT/CT images of unlabeled oncoFAP blocking group at 0.5 h.

Figure 14. In vivo biodistribution of radioactive probe in U87MG glioblastoma model. (A) ^99mTc -HYNIC-C ₂ -oncoFAP, (B) ^99mTc -HYNIC-PEG ₄ -C ₂ -oncoFAP, (C) ^99mTc -HYNIC-[C ₂ -oncoFAP] ₂ , (D) ^99mTc -HYNIC-PEG ₄ -[C ₂ -oncoFAPI] ₂ , (E) ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ , (F) ^99m Tc-HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ , ( G) ^99m Tc-HYNIC-[C ₆ -oncoFAP] ₂ , (H) ^99m Tc-HYNIC-[Aoc-oncoFAP] ₂ .

Figure 15. (A) In vivo biodistribution of ^99m Tc-HYNIC-FAPI-04 ( ^99m Tc-HFAPi) in U87MG glioblastoma model. (B) Comparison of quantitative uptake values of radioactive probes in U87MG glioblastoma. * indicates significant difference (p<0.05), ns indicates no significant difference.

Figure 16. (A) M1-M2 shows the SPECT/CT imaging results of ^99m Tc-HFAPI-04 in the bleomycin-induced mouse lung fibrosis model, and M3-M4 shows ^99m Tc-HFAPI -04 SPECT/CT imaging results in normal control mice. (B) M5-M7 shows the SPECT/CT imaging results of ^99m Tc-HYNIC-[C ₂ -oncoFAP] ₂ in the bleomycin-induced mouse lung fibrosis model, and M8-M9 shows ^{the 99m} SPECT/CT imaging results of Tc-HYNIC-[C ₂ -oncoFAP] ₂ in normal control mice.

Figure 17. (A) M1-M2 shows the role of ^99m Tc-HYNIC-PEG ₄ -FAPI-04 in the bleomycin-induced mouse lung fibrosis model M3 shows the SPECT/CT imaging results of ^99m Tc-HYNIC-PEG ₄ -FAPI-04 in normal control mice. (B) M4-M5 shows the SPECT/CT imaging results of ^99m Tc-HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ in the bleomycin-induced mouse lung fibrosis model, and M6 shows SPECT/CT imaging results of ^99m Tc-HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ in normal control mice.

Figure 18. (A) M1-M2 shows the SPECT/CT imaging results of ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ in the bleomycin-induced mouse lung fibrosis model, and M3 shows SPECT/CT imaging results of ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ in normal control mice. (B) M4-M5 shows the SPECT/CT imaging results of ^99m Tc-HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ in the bleomycin-induced mouse lung fibrosis model, M6 shows SPECT/CT imaging results of ^99m Tc-HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ in normal control mice.

Fig. 19 shows HE staining, Masson staining and immunohistochemical staining of FAP protein in normal mouse lung tissue and pulmonary fibrosis model mouse lung tissue.

Detailed ways

The material adopted in the embodiment of the present invention:

HYNIC-NHS (nicotinamide hydrazide) was purchased from Noca-biochem, USA. 1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid (NH ₂ -PEG ₄ -COOH) was purchased from Xi'an Ruixi Biotechnology Co., Ltd. Dichloromethane (DCM), 4-dimethylaminopyridine (DMAP) and tetrahydrofuran (THF) were purchased from Beijing Tongguang Fine Chemical Company. succinic acid (succinic acid), disodium succinate hexahydrate (disodium succinate), trisodium triphenylphosphine-3,3',3"-trisulfonate (TPPTS, sodium triphenylphosphine trisulfonate), N,N-Dimethylform amide (DMF , N,N-dimethylformamide), tricine (trimethylolglycine), trifluoroacetic acid (TFA, trifluoroacetic acid), N-Ethyldiisopropylamine (DIPEA, N,N-diisopropylethylamine) are all purchased From Sigma-Aldrich Company of the United States. ^{Na 99m} TcO ₄ eluent was purchased from Beijing Atomic High-Tech Co., Ltd.

Preparation example

1. Preparation of HYNIC-C ₂ -oncoFAP and HYNIC-PEG ₄ -C ₂ -oncoFAP (Figure 1)

a. Preparation of HYNIC-PEG ₄ -COOH

Weigh NH ₂ -PEG ₄ -COOH (1.2eq) into EP tube, dissolve in 50μL DMF, add DIEA to adjust pH to 7.8-8.0; weigh HYNIC-NHS (1eq) in EP tube, dissolve in 50μL DMF middle. Add HYNIC-NHS to the NH ₂ -PEG ₄ -COOH solution, mix thoroughly, then add DIEA to adjust the pH to 7.8-8.0, and react overnight at room temperature. The reaction was monitored by high performance liquid chromatography, and the target product was separated and purified (method 1). The eluted peak at 18.2 minutes was collected, and the eluate was freeze-dried by vacuum freeze-drying method to obtain a solid product. After taking a small amount of product to dissolve, HPLC identification Determined to be >98% pure. Through TOF MS (ES+) mass spectrometry analysis, m/z=569.6 ([M+H] ⁺ ), confirmed to be the expected product HYNIC-PEG ₄ -COOH.

b. Preparation of oncoFAP

8-Aminoquinoline-4-carboxylic acid (1eq), (S)-1-(2-aminoacetyl)-4.4-difluoropyrrolidine-2-carbonitrile hydrochloride (1eq) and HATU (1eq) were added 25mL round bottom flask, and dissolved with 900μL DMF and 4mL DCM, then added dropwise DIEA (4eq) and stirred. The crude product was diluted _with DCM, washed with water, dried over _Na2SO4 , filtered, and finally the solvent was removed with a rotary evaporator to obtain the crude product (S)-8-amino-N-(2-(2-cyano-4,4- Difluoropyrrolidin-1-yl)-2-carbonylethyl)quinoline-4-carboxamide. The above crude product (1eq), succinic anhydride (50eq) and DAMP (0.5eq) were added into a 25mL round bottom flask, dissolved in 3mL THF, and reacted at 60°C for 6 hours. The reaction solution was removed from the solvent by a rotary evaporator, diluted with water, extracted with DCM, dried with Na ₂ SO ₄ , filtered and dried, and analyzed by TOF MS (ES+) mass spectrometry, m/z=460.1 ([M+H] ⁺ ), Confirmed as expected product oncoFAP.

c. Preparation of C ₂ -oncoFAP

Weigh oncoFAP (1eq) and HATU (1.5eq) into EP tubes, dissolve in 200μL DMF, add DIEA (2eq), and react at room temperature for about 30 minutes. Then weigh N-tert-butoxycarbonylethylenediamine (N-Boc-ethylenediamine, CAS: 57260-73-8) (1.5eq) into the above reaction solution, add DIEA to adjust the pH value to 8.5-9.0, room temperature After reacting overnight, the reaction was monitored by high performance liquid chromatography, and the target product was separated and purified (method 2). The eluted peak at 19.8 minutes was collected, and the eluate was freeze-dried by vacuum freeze-drying method to obtain the expected product Boc-C ₂ -oncoFAP. The lyophilized product was dissolved in 1 mL TFA, reacted at room temperature for 10 min, and the reaction liquid was blown dry with nitrogen gas. The obtained product was analyzed by TOF MS (ES+), m/z=502.2 ([M+H] ⁺ ), confirmed to be the expected product C ₂ -oncoFAP.

d. Preparation of HYNIC-C ₂ -oncoFAP

Dissolve C ₂ -oncoFAP (1eq) and HYNIC-NHS (1eq) in DMF, add DIEA to adjust the pH value to 8.5-9.0, react overnight at room temperature, use high performance liquid chromatography to monitor the reaction, and separate and purify the target product (Method Two). The eluted peak at 14.0 minutes was collected, and the eluate was freeze-dried by vacuum freeze-drying method to obtain a solid product. After MALDI-TOF mass spectrometry analysis, m/z=805.2 ([M+H] ⁺ ), confirmed to be the expected product HYNIC-C ₂ -oncoFAP.

e. Preparation of HYNIC-PEG ₄ -C ₂ -oncoFAP

Weigh HYNIC-PEG ₄ -COOH (1eq) and HATU (1.5eq) into EP tubes, dissolve in 200 μL DMF, add DIEA (2eq), and react at room temperature for about 30 minutes. Weigh NH ₂ -oncoFAP (1eq) into the above reaction solution, add DIEA to adjust the pH value to 8.5-9.0, react overnight at room temperature, use high performance liquid chromatography to monitor the reaction, and separate and purify the target product (method 2) . The eluted peak at 15.1 minutes was collected, and the eluate was freeze-dried by vacuum freeze-drying method to obtain a solid product. After MALDI-TOF mass spectrometry analysis, m/z=1052.3 ([M+H] ⁺ ), confirmed to be the expected product HYNIC- _PEG4 - _C2 -oncoFAP.

2. Preparation of HYNIC-[C ₂ -oncoFAP] ₂ and HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ (Figures 2 and 3)

a. Preparation of Glu-(C ₂ -oncoFAP) ₂

Dissolve C ₂ -oncoFAP (1eq) and Boc-Glu-OSu ₂ (0.5eq) in DMF, add DIEA to adjust the pH value to 8.5-9.0, stir at room temperature overnight, use high performance liquid chromatography to monitor the reaction, the target product Carry out separation and purification (method 2). The eluted peak was collected at 19.9 minutes, and the eluate was lyophilized by vacuum freeze-drying method to obtain the compound Boc-Glu-(C ₂ -oncoFAP) ₂ ; the lyophilized product was dissolved in 1mL TFA, and reacted at room temperature for 10min, and the reaction solution was blown with nitrogen Blow dry, and the obtained product was analyzed by TOF MS (ES+), m/z=557.7 ([M+H] ²⁺ ), which was confirmed to be the expected product Glu-(C ₂ -oncoFAP) ₂ .

b. Preparation of HYNIC-oncoFAP ₂

Dissolve Glu-(oncoFAP) ₂ (1eq) and HYNIC-NHS (1eq) in DMF, add DIEA to adjust the pH value to 8.5-9.0, react overnight at room temperature, use high performance liquid chromatography to monitor the reaction and separate the target product and purification (method 2). The eluted peak at 16.9 minutes was collected, and the eluate was freeze-dried by vacuum freeze-drying method to obtain a solid product. After TOFMS (ES+) mass spectrometry analysis, m/z=709.2 ([M+2H] ²⁺ ), confirmed to be the expected product HYNIC-C ₂ -oncoFAP ₂ .

c. Preparation of HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂

Weigh HYNIC-PEG ₄ -COOH (1eq) and HATU (1.5eq) into EP tubes, dissolve in 200 μL DMF, add DIEA (2eq), and react at room temperature for about 30 minutes. Weigh Glu-(C ₂ -oncoFAP) ₂ (1eq) into the above reaction solution, add DIEA to adjust the pH value to 8.5-9.0, react overnight at room temperature, use high performance liquid chromatography to monitor the reaction, and separate and analyze the target product. Purification (method two). The eluted peak at 17.5 minutes was collected, and the eluate was freeze-dried by vacuum freeze-drying method to obtain a solid product. After TOF MS (ES+) mass spectrometry analysis, m/z=832.8 ([M+2H] ²⁺ ), confirmed to be the expected product HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ .

3. Preparation of HYNIC-[PEG ₄ -oncoFAP] ₂ and HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ (Figure 4 and Figure 5)

a. Preparation of Boc-PEG ₄ -oncoFAP

Weigh oncoFAP (1eq) and HATU (1.5eq) into EP tubes, dissolve in 200μL DMF, add DIEA (2eq), and react at room temperature for about 30 minutes. Then weigh (14-amino-3,6,9,12-tetraoxatetradecyl) tert-butyl carbamate (Boc-NH-PEG ₄ -NH ₂ , CAS: 811442-84-9) (1.5 eq) adding to the above reaction solution, adding DIEA to adjust the pH value to 8.5-9.0, reacting at room temperature overnight, using high performance liquid chromatography to monitor the reaction, and separating and purifying the target product (method 2). The eluted peak at 20.4 minutes was collected, and the eluate was freeze-dried by vacuum freeze-drying method to obtain a solid product. After TOF MS (ES+) mass spectrometry analysis, m/z=339.6 ([M+2H] ²⁺ ), it was confirmed to be the expected product Boc-PEG ₄ -oncoFAP.

b. Preparation of Glu-[PEG ₄ -oncoFAP] ₂

Dissolve Boc-PEG ₄ -oncoFAP (1eq) in 1mL TFA and react at room temperature for 10 minutes. The reaction solution is blown dry with nitrogen and dissolved in DMF. Add DIEA to adjust the pH value to 8.5-9.0; weigh Boc-Glu-OSu ₂ ( 0.5eq) was added to the above reaction solution, DIEA was added to adjust the pH value to 8.5-9.0, stirred overnight at room temperature, the reaction was monitored by high performance liquid chromatography, and the target product was separated and purified (method 2). The elution peak at 19.9 minutes was collected, and the eluate was lyophilized by vacuum freeze-drying method to obtain the expected product Boc-Glu-[PEG ₄ -oncoFAP] ₂ ; Drying with nitrogen gas, the obtained product was analyzed by MALDI-TOF-MS mass spectrometry, m/z=1466.5 ([M+H] ⁺ ), confirmed to be the expected product Glu-[PEG ₄ -oncoFAP] ₂ .

c. Preparation of HYNIC-[PEG ₄ -oncoFAP] ₂

Dissolve Glu-[PEG ₄ -oncoFAP] ₂ (1eq) and HYNIC-NHS (1eq) in DMF, add DIEA to adjust the pH value to 8.5-9.0, react at room temperature overnight, use high performance liquid chromatography to monitor the reaction, target The product was isolated and purified (method 2). The eluted peak at 17.6 minutes was collected, and the eluate was freeze-dried by vacuum freeze-drying method to obtain a solid product. After TOF MS (ES+) mass spectrometry analysis, m/z=885.3 ([M+2H] ²⁺ ), confirmed to be the expected product HYNIC-[PEG ₄ -oncoFAP] ₂ .

d. Preparation of HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂

Weigh HYNIC-PEG ₄ -COOH (1eq) and HATU (1.5eq) into EP tubes, dissolve in 200 μL DMF, add DIEA (2eq), and react at room temperature for about 30 minutes. Weigh Glu-[PEG ₄ -oncoFAP] ₂ (1eq) and add it to the above reaction solution, add DIEA to adjust the pH value to 8.5-9.0, react at room temperature overnight, use high performance liquid chromatography to monitor the reaction, and separate and analyze the target product Purification (method two). The eluted peak at 17.5 minutes was collected, and the eluate was freeze-dried by vacuum freeze-drying method to obtain a solid product. After TOF MS (ES+) mass spectrometry analysis, m/z=1009.4 ([M+2H] ²⁺ ), it was confirmed to be the expected product HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ .

4. Preparation of HYNIC-[C ₆ -oncoFAP] ₂ and HYNIC-[Aoc-oncoFAP] ₂

a. Preparation of HYNIC-[C ₆ -oncoFAP] ₂

Dissolve Glu-[C ₆ -oncoFAP] ₂ (1eq) and HYNIC-NHS (1eq) in DMF, add DIEA to adjust the pH value to 8.5-9.0, react overnight at room temperature, use high performance liquid chromatography to monitor the reaction, target The product was isolated and purified (method 2). The eluted peak at 20.8 minutes was collected, and the eluate was freeze-dried by vacuum freeze-drying method to obtain a solid product. After TOF MS (ES+) mass spectrometry analysis, m/z=765.3 ([M+2H] ²⁺ ), confirmed to be the expected product HYNIC-[C ₆ -oncoFAP] ₂ .

b. Preparation of HYNIC-[Aoc-oncoFAP] ₂

Dissolve Glu-[Aoc-oncoFAP] ₂ (1eq) and HYNIC-NHS (1eq) in DMF, add DIEA to adjust the pH value to 8.5-9.0, react overnight at room temperature, use high performance liquid chromatography to monitor the reaction, and the target product Carry out separation and purification (method 2). The eluted peak at 23.8 minutes was collected, and the eluate was freeze-dried by vacuum freeze-drying method to obtain a solid product. After TOF MS (ES+) mass spectrometry analysis, m/z=708.3 ([M+2H] ²⁺ ), confirmed to be the expected product HYNIC-[Aoc-oncoFAP] ₂ .

5. Preparation of DOTA-[PEG ₄ -oncoFAP] ₂ (Figure 7)

Taking the preparation of DOTA-[PEG ₄ -oncoFAP] ₂ as an example, dissolve Glu-[PEG ₄ -oncoFAP] ₂ (1eq) and DOTA-NHS (1eq) in DMF, add DIEA to adjust the pH value to 8.5-9.0, room temperature After reacting overnight, the reaction was monitored by high performance liquid chromatography, and the target product was separated and purified (method 2). The eluted peak at 18.3 minutes was collected, and the eluate was freeze-dried by vacuum freeze-drying method to obtain a solid product. After TOF MS (ES+) mass spectrometry analysis, m/z=926.9 ([M+2H] ²⁺ ), confirmed to be the expected product DOTA-[PEG ₄ -oncoFAP] ₂ .

6. Preparation of ^99m Tc-HYNIC-PKM-L-oncoFAP and ^99m Tc-HYNIC-PKM-[L-oncoFAP] ₂

Prepare 20 μg of HYNIC-PKM-L-oncoFAP or HYNIC-PKM-[L-oncoFAP] ₂ , 5.0 mg of sodium triphenylphosphinetrisulfonate (TPPTS), 6.5 mg of trihydroxymethylglycine (Tricine) in 1 mL 0.5 M succinic acid buffer solution (pH 4.8), the mixed solution was placed in a 10 mL vial, and the mixed solution was lyophilized to obtain a labeled kit. Add 1.0-1.5mL Na ^99m TcO ₄ solution to the freeze-dried powder in the marked kit, place it in a heating reactor such as a water bath, air bath or metal bath at 75-110°C, heat the reaction for 20-30 minutes, and wait for the reaction to complete After cooling at room temperature for 5 minutes, ^99m Tc-HYNIC-PKM-L-oncoFAP and HYNIC-PKM-[L-oncoFAP] ₂ were prepared. It was analyzed by radioactive HPLC for later use.

Described HPLC method is as follows:

Method 1 for separation and purification of the target product by high performance liquid chromatography: Agilent 1260 HPLC system is equipped with YMC-Pack ODS-A C18 semi-preparative column (250×10mml.D.S-5μm, 12nm). Gradient elution for 25min, flow rate 2.0mL/min, wherein the mobile phase A is deionized water (containing 0.05% TFA), the mobile phase B is acetonitrile (containing 0.05% TFA), and the elution gradient is set to 80% A and 20% B, 80% A and 20% B at 5 min, 40% A and 60% B at 25 min.

Method 2 for separating and purifying the target product by high performance liquid chromatography: Agilent 1260 HPLC system is equipped with Venusil MP C18 semi-preparative column (250×10 mml.DS-5 μm). Gradient elution for 25min with a flow rate of 3.2mL/min, wherein the mobile phase A is deionized water (containing 0.05% TFA), the mobile phase B is acetonitrile (containing 0.05% TFA), and the elution gradient is set to 90% A and 10% B, 40% A and 60% B at 20 min, 90% A and 10% B at 25 min.

Example 1 In vivo stability of oncoFAP-based ^99m Tc-labeled radioactive probes in normal BALB/c mice

(1) Validation of the stability of oncoFAP-based ^99m Tc-labeled radioactive probes in mice

BALB/c normal mice were divided into 6 groups with 2 mice in each group. Each group was injected with 100μL (37MBq) ^99mTc -HYNIC-C ₂ -oncoFAP, ^99mTc -HYNIC-PEG ₄ -C ₂ -oncoFAP, ^99mTc -HYNIC-[C ₂ -oncoFAP] ₂ , ^99mTc- HYNIC-PEG ₄ -[C ₂ -conFAP] ₂ , ^99mTc -HYNIC-[PEG ₄ -oncoFAP] ₂ and ^99mTc -HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ , and at 30 and 120 minutes after injection The mouse urine was taken, mixed with 50% acetonitrile/water, and analyzed by radioactive high-performance liquid chromatography. The results are shown in FIG. 8 . After ^99m Tc-HYNIC-C ₂ -oncoFAP and ^99m Tc-HYNIC-PEG ₄ -C ₂ -oncoFAP are metabolized in mice, there is a certain decomposition phenomenon, but most of the original drug can still be retained; ^99m Tc- HYNIC-[C ₂ -oncoFAP] ₂ , ^99m Tc-HYNIC-PEG ₄ -[C ₂ -conFAP] ₂ , ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ and ^99m Tc-HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ These dimer molecules have strong stability and will not decompose with the prolongation of metabolic time.

(2) Verification of the stability of ^99m Tc-HFAPi and ^99m Tc-HpFAPi in mice

The experimental method is the same as (1), and the results are shown in Figure 9. After ^99m Tc-HFAPi and ^99m Tc-HpFAPi were metabolized in mice, most of the drugs in the urine were decomposed, and only a small amount of the original drug was present.

Analysis and comparison of in vivo stability results:

^{The 99m} Tc-HFAPi and ^99m Tc-HpFAPi in the previous study were quickly decomposed in the mouse body, and only a very small part was still a complete drug; while the radioactive probe of the present invention was very stable in vivo, after 2 hours, most of the The radiopharmaceutical can still remain intact, and only a small amount is metabolized, indicating that it has better metabolic stability in vivo. The introduction of new coupling methods and pharmacokinetic linker molecules is helpful to improve the stability of the probe .

Example 2 In vitro rhFAP-α protein binding experiment

(1) Binding experiment of oncoFAP-based ^99m Tc-labeled radioactive probe to recombinant human FAP-α protein (rhFAP-α)

The rhFAP-α protein was dissolved in ELISA coating buffer (1×) (concentration: 2 μg/mL), 0.2 μg/100 μL per well was coated in a 96-well plate, and left overnight at 4°C. After coating, the coating solution was discarded, and the 96-well plate was repeatedly washed 3 to 5 times with PBS. Add blocking solution (5% calf serum/PBS buffer, pH 7.4) to a 96-well plate, place it at 37° C. and incubate for 2 hours. After blocking, the 96-well plate was repeatedly washed 3-5 times with PBS. The prepared ^99m Tc-labeled probes were respectively added to the sample wells coated with rhFAP-α, and 0.3 μCi/100 μL of radioactive label was added to each well, and 4 parallel wells were set up. Prepare another 4 sample wells and add an equal amount of ^99m Tc-labeled probe, then add 1000-fold molar amount of oncoFAP, and mix well. Incubate the 96-well plate at 37°C for 1 hour. Prepare another four immunoprecipitation tubes, add an equal amount of radiolabeled ^99m Tc-labeled probes, and reserve as standard Sample. After the incubation is completed, pour off the incubation solution, and then wash five times with PBS, then discard the liquid, cut out each sample hole with scissors, place it in a radioactive tube, and measure the concentration in each hole with a radioactive γ-automatic counter. radioactive counts. Afterwards, the binding percentage of the ^99m Tc-labeled probe to rhFAP-α was calculated by the formula. The experimental results are shown in Figure 10(A) and (C), ^99m Tc-HYNIC-C ₂ -oncoFAP( ^99m Tc-HC-oFP), ^99m Tc-HYNIC-PEG ₄ -C ₂ -oncoFAP( ^99m Tc-HP-oFP ), ^99m Tc-HYNIC-[C ₂ -oncoFAP] ₂ ( ^99m Tc-H-CoFP ₂ ), ^99m Tc-HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ ( ^99m Tc-HP-CoFP ₂ ), ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ ( ^99m Tc-H-PoFP ₂ ) and ^99m Tc-HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ ( ^99m Tc-HP-PoFP ₂ ) bind to rhFAP-α The %AD values were 15.47% (±1.53%), 22.36% (±2.65%), 27.59% (±3.49%), 20.53% (±0.82%), 25.65% (±1.88%), 36.87% (± 1.90%); while in the oncoFAP block experimental group under the same conditions, the %AD values were 0.098% (±0.096%), 0.025% (±0.002%), 0.033% (±0.003%), 0.027% (±0.004%) %), 0.029% (±0.004%), 0.056% (±0.041%), which were significantly different from the binding group (p<0.0001). The experimental results indicated that the oncoFAP-based ^99m Tc-labeled radioactive probe could specifically bind to rhFAP-α protein.

(2) Binding experiment of ^99m Tc-HFAPi and ^99m Tc-HpFAPi to recombinant human FAP-α protein (rhFAP-α)

The steps are the same as (1), and the results are shown in Figure 10 (B) and (C). The %AD values of ^99m Tc-HFAPi and ^99m Tc-HpFAPi combined with rhFAP-α are 4.4% (±0.2%) and 4.3% (±0.2%) respectively. 0.2%), while in the FAPI block experimental group under the same conditions, the %AD values were 0.95% (±0.02%) and 1.1% (±0.02%) respectively.

Comparative analysis of the results of in vitro rhFAP-α protein binding experiments:

Under the same experimental conditions, compared with ^99m Tc-HFAPi and ^99m Tc-HpFAPi, the radioactive probe of the present invention shows stronger binding ability to recombinant human FAP protein in the protein binding experiment, and its properties are better.

Example 3 SPECT/CT imaging of oncoFAP-based ^99m Tc-labeled radioactive probes in tumor-bearing mice

(1) Imaging of ^99m Tc-HYNIC-C ₂ -oncoFAP and ^99m Tc-HYNIC-PEG ₄ -C ₂ -oncoFAP in U87MG tumor-bearing mouse model

After the prepared ^99m Tc-HYNIC-C ₂ -oncoFAP and ^99m Tc-HYNIC-PEG ₄ -oncoFAP were formulated with physiological saline to 37MBq/100μL, each mouse was injected with 100μL (37MBq) through the tail vein. After injection, 0.5 , 1, 2 and 4 hours for SPECT/CT imaging. The mice in the closed group were injected with 100 μL (500 μg) of oncoFAP at the same time as the imaging drug, and imaging was performed 0.5 hours after administration. Mice were anesthetized with 1.5% isoflurane-oxygen during imaging. After imaging, the SPECT image was reconstructed and fused with the CT image to obtain a 3D imaging image. The posterior view was used for display and the tumor location was marked with an arrow. The imaging results are shown in Figure 11.

(2) Imaging of ^99m Tc-HYNIC-[C ₂ -oncoFAP] ₂ and ^99m Tc-HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ in U87MG tumor-bearing mouse model

The prepared ^99m Tc-HYNIC-[C ₂ -oncoFAP] ₂ and ^99m Tc-HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ were respectively prepared with normal saline to 37MBq/100μL, and each mouse was injected through the tail vein 100 μL (37 MBq), SPECT/CT imaging was performed at 0.5, 1, 2 and 4 hours after injection. The mice in the closed group were injected with 100 μL (500 μg) of oncoFAP at the same time as the imaging drug, and imaging was performed 0.5 hours after administration. Mice were anesthetized with 1.5% isoflurane-oxygen during imaging. After imaging, the SPECT image was reconstructed and fused with the CT image to obtain a 3D imaging image. The posterior view was used for display and the tumor location was marked with an arrow. The imaging results are shown in Figure 12.

(3) Imaging of ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ and ^99m Tc-HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ in U87MG tumor-bearing mouse model

The prepared ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ and ^99m Tc-HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ were formulated with normal saline to 37MBq/100μL, and each mouse was injected via the tail vein 100 μL (37 MBq), SPECT/CT imaging was performed at 0.5, 1, 2 and 4 hours after injection. The mice in the closed group were injected with 100 μL (500 μg) of oncoFAP at the same time as the imaging drug, and imaging was performed 0.5 hours after administration. Mice were anesthetized with 1.5% isoflurane-oxygen during imaging. After imaging, the SPECT image was reconstructed and fused with the CT image to obtain a 3D imaging image. The posterior view was used for display and the tumor location was marked with an arrow. The imaging results are shown in Figure 13.

Analysis of imaging results:

In the U87MG tumor-bearing mouse model, ^99m Tc-HYNIC-C ₂ -oncoFAP, ^99m Tc-HYNIC-PEG ₄ -C ₂ -oncoFAP, ^99m Tc-HYNIC-[C ₂ -oncoFAP] ₂ , ^99m Tc-HYNIC- PEG ₄ -[C ₂ -oncoFAP] ₂ , ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ and ^99m Tc-HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ all showed obvious tumor uptake, and the imaging contrasts were both Higher, indicating that the radioactive probe of the present invention has good tumor-specific targeting ability. In the blocking group experiment, the tumor uptake was significantly reduced, indicating the specific binding of the probe to the FAP site at the tumor site. Further analysis, compared with ^99mTc -HYNIC-C ₂ -oncoFAP, ^99mTc -HYNIC-PEG ₄ -C ₂ -oncoFAP, the probes ^99mTc -HYNIC-[C ₂ -oncoFAP] ₂ , ^99mTc -HYNIC-PEG _4- [C ₂ -oncoFAP] ₂ , ^99mTc -HYNIC-[PEG ₄ -oncoFAP] ₂ and ^99mTc -HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ tumor uptake was higher and probe was cleared from blood Faster, so that the background of other organs is lower, so that it shows better contrast in nuclear medicine imaging, which is beneficial to the diagnosis of tumors, and can detect some FAP low-expressing tumors and fibrotic lesions (such as pulmonary fibrosis and liver fibrosis) ) provide a clearer imaging diagnosis.

Example 4 Biodistribution of oncoFAP-based ^99m Tc-labeled radioactive probes in tumor-bearing mice

BALB/c Nude mice bearing U87MG tumors were divided into 6 groups, 6 rats in each group. Each group of mice was injected with 100μL (~74kBq) ^{of 99mTc} -HYNIC-C ₂ -oncoFAP, ^99mTc -HYNIC-PEG ₄ -C ₂ -oncoFAP, ^99mTc -HYNIC-[C ₂ -oncoFAP] _2, ^99m Tc-HYNIC-PEG ₄ -[C ₂ -oncoFAP] _2, ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ , ^99m Tc-HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ , ^99m Tc-HYNIC- [C ₆ -oncoFAP] ₂ , and ^99m Tc-HYNIC-[Aoc-oncoFAP] ₂ , and sacrificed at 0.5 and 4 hours after injection; blood and major organs were collected, weighed and radioactive counts were measured, calculated after decay correction Percent injected dose rate per gram of tissue (%ID/g). The biodistribution results are expressed as mean ± standard deviation (means ± SD, n = 3), and the experimental results of biodistribution are shown in FIG. 14 .

Analysis and comparison of biodistribution results:

^99m Tc-HFAPi and ^99m Tc-HpFAPi are involved in CN 111991570 B, and the biodistribution of the two in the U87MG tumor model is similar, and the biodistribution results of ^99m Tc-HFAPi are shown in Figure 15 (A), part of the present invention The comparison of the tumor uptake values of the probe and ^99m Tc-HFAPi is shown in Fig. 15(B). Compared with ^99m Tc-HFAPi, the probe of the present invention has a higher absolute value of tumor uptake, faster clearance and metabolism of normal organs, so that the ratio of tumor/normal organ uptake of the probe is higher, which is more conducive to nuclear medicine imaging of tumors, especially Because SPECT imaging diagnosis is very sensitive to background signal noise, low background is more conducive to accurate detection of tiny lesions.

Example 5 SPECT/CT imaging of ^99m Tc-labeled radioactive probes based on oncoFAP ₂ in the C57BL/6 mouse model induced by Bleomycin

The mouse model of pulmonary fibrosis was formed by slowly instilling bleomycin saline solution (7 mg/kg, 100 μL) into the trachea, and then routinely fed for 2 weeks. When imaging the pulmonary fibrosis model and normal control mice, each mouse was injected with 100 μL (~18MBq) of ^99m Tc-HFAPI, ^99m Tc-HpFAPI, ^99m Tc-HYNIC-[C ₂ -oncoFAP] through the tail vein _2, ^99m Tc-HYNIC-PEG ₄ -[C ₂ -oncoFAP] _2, ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ or ^99m Tc-HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ , and inject SPECT/CT imaging was performed 1 hour later. Mice were anesthetized with 0.5-1.5% isoflurane-oxygen during imaging. The imaging results are shown in Figure 16-18.

Analysis of pulmonary fibrosis imaging results:

As shown in Figure 16(A), in ^{the 99m} Tc-HFAPi imaging group, M1-M3 were bleomycin-induced pulmonary fibrosis model mice, and it was confirmed by CT that obvious fibrosis lesions appeared in the lungs of the mice, while the corresponding In the SPECT images, there was no obvious probe uptake signal in the fibrosis area; in the normal control mice, neither CT nor SPECT showed pulmonary fibrosis signals. As shown in Figure 16(B), In ^{the 99m} Tc-HYNIC-[C ₂ -oncoFAP] ₂ imaging group, M1-M3 were bleomycin-induced pulmonary fibrosis model mice, and there were obvious solidified areas in the lungs from the CT signal, which was for Fibrosis lesions, in the SPECT/CT images corresponding to these areas, there is also the accumulation of radioactive signals, so it can be analyzed that the area where the probe is concentrated is the area of pulmonary fibrosis; while in normal control mice, CT There was no obvious fibrosis signal in both SPECT and SPECT images. Therefore, it can be considered that ^99m Tc-HYNIC-[C ₂ -oncoFAP] ₂ can specifically image the regional lesions of pulmonary fibrosis, and its concentration intensity of the probe in the lesions and the contrast with the surrounding organs are obviously better than those of ^99mTc -HFAPi. The outlines of lung tissue are selected in the white dotted line box in the figure.

As shown in Figure 17(A), in ^{the 99m} Tc-HpFAPI imaging group, M1-M2 were mice with pulmonary fibrosis in the bleomycin-induced group. It was confirmed by CT that obvious fibrosis lesions appeared in the lungs of the mice, while In the corresponding SPECT image, there is no obvious probe uptake signal in the fibrosis area; it should be noted that obvious radioactive signal uptake can be seen in adjacent parts such as the spine and sternum, and the high-intensity signal in these parts affects The uptake of the probe in the lungs and the reconstruction of the SPECT signal were confirmed; in the normal control group mice (M3), no pulmonary fibrosis signal was seen in CT and SPECT. As shown in Figure 17(B), in ^{the 99m} Tc-HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ imaging group, M4-M5 were bleomycin-induced pulmonary fibrosis model mice, which were confirmed by CT signal in the lung There are obvious solidified areas in the center, which are fibrosis lesions. In the SPECT/CT images corresponding to these areas, radioactive signal accumulation can also be seen, so it can be analyzed that the areas where the probe is concentrated are areas of pulmonary fibrosis ; while in the normal control mice (M6), there were no obvious parenchymal fibrosis signals and probe uptake signals in both CT and SPECT images. Therefore, it can be considered that ^99m Tc-HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ can specifically image the regional lesions of pulmonary fibrosis, and the concentration intensity of the probe in the lesions and the contrast with the surrounding organs should be higher than that of the surrounding organs. Obviously better than ^99m Tc-HpFAPi. In the figure, the white dotted line boxes selected are lungs, and the red dotted line boxes selected are joints.

In Fig. 18, similar to ^99m Tc-HYNIC-[C ₂ -oncoFAP] ₂ and ^99m Tc-HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ , ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ and ^99m Tc -HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ can perform specific imaging on pulmonary fibrosis lesions.

In summary, ^99m Tc-HFAPi and ^99m Tc-HpFAPi are not effective in the imaging of pulmonary fibrosis, and cannot image the fibrotic area well; while the radioactive probe ^99m based on oncoFAP2 involved in the present invention Tc-HYNIC-[C ₂ -oncoFAP] ₂ , ^99m Tc-HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ , ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ and ^99m Tc-HYNIC-PEG ₄ -[ PEG ₄ -oncoFAP] ₂ can specifically image the area of pulmonary fibrosis, and the effect is obviously better than ^99m Tc-HFAPi and ^99m Tc-HpFAPi, so it has a wider clinical application value.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above-mentioned embodiments. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. A precursor compound for forming a radionuclide complex, which has a structure shown in the following formula I or formula II:
   

   in,

   L is selected from: -(CH ₂ )m-, m is an integer of 2-6, preferably 2 or 6; -CH ₂ -PEG ₄ -CH ₂ -;

   Wherein the PEG ₄ structural formula is: both ends Represents a bond to a methylene bond chain;

   _L is -C(O)-L-NH-, wherein L is as defined above;

   n is selected from 0 or 1;

   BFC is a bifunctional chelating agent selected from HYNIC, MAG2, MAG3, DTPA, DOTA, NOTA, TETA.
2. The precursor compound according to claim 1, which is selected from the following specific compounds:

   HYNIC-[C ₂ -oncoFAP] ₂ ,

   HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ ,

   HYNIC-[PEG ₄ -oncoFAP] ₂ ,

   HYNIC- _PEG4- [ _PEG4 -oncoFAP] ₂ ,

   HYNIC-[ _C6 -oncoFAP] ₂ ,

   DOTA-[C ₂ -oncoFAP] ₂ ,

   NOTA-[C ₂ -oncoFAP] ₂ ,

   DOTA-[PEG ₄ -oncoFAP] ₂ ,

   NOTA-[PEG ₄ -oncoFAP] ₂ ,

   HYNIC-C ₂ -oncoFAP,

   HYNIC- _PEG4 - _C2 -oncoFAP.
3. A radionuclide-labeled complex formed from the precursor compound of claim 1.
4. The complex according to claim 2, wherein the radionuclide is selected from ¹¹¹ In, ⁶⁴ Cu, ^99m Tc, ⁶⁸ Ga, ¹²³ I, ¹⁸ F, ⁹⁰ Y, ¹⁷⁷ Lu, ¹³¹ I, ¹²⁵ I, ⁸⁹ Sr, ¹⁵³ Sm.
5. The complex according to claim 2, wherein the radionuclide is selected from ^99m Tc, ⁶⁸ Ga, ⁶⁴ Cu, ¹⁷⁷ Lu.
6. According to the complex described in claim 2, when the radionuclide is selected from ^99m Tc, BFC is selected from HYNIC; when the radionuclide is selected from ⁶⁸ Ga, ⁶⁴ Cu, ¹⁷⁷ Lu, BFC is selected from DOTA, NOTA.
7. The complex according to claim 3, which is selected from the following specific complexes:

   ^99m Tc-HYNIC-[C ₂ -oncoFAP] ₂ ,

   ^99m Tc-HYNIC-PEG ₄ -[C ₂ -oncoFAP] ₂ ,

   ^99m Tc-HYNIC-[PEG ₄ -oncoFAP] ₂ ,

   ^99m Tc-HYNIC-PEG ₄ -[PEG ₄ -oncoFAP] ₂ ,

   ^99m Tc-HYNIC-[C ₆ -oncoFAP] _2,

   ^99m Tc-HYNIC-C ₂ -oncoFAP,

   ^99mTc -HYNIC- _PEG4 - _C2 -oncoFAP.
8. A pharmaceutical composition, which contains the complex described in any one of claims 3-7. Preferably, the drug can be used as an imaging diagnostic agent or radioactive targeted therapeutic agent for FAP-positive tumors or FAP-positive fibrotic diseases (such as pulmonary fibrosis, liver fibrosis, etc.). For example, when the radionuclide is ⁶⁸ Ga, ⁶⁴ Cu, the drug is used as a PET imaging agent; when the radionuclide is ¹⁷⁷ Lu, the drug is used as a PET therapeutic agent; when the radionuclide is ^99m Tc, The drug acts as a SPECT imaging agent.
9. The pharmaceutical composition according to claim 8, which is an injectable preparation, comprising the above-mentioned labeled complex and an injectable carrier. Preferably, the drug is a colorless and transparent injectable preparation.
10. Use of the precursor compound according to any one of claims 1-2 or the complex compound according to any one of claims 3-7 in the preparation of medicaments for diagnosing or treating FAP-positive tumors or fibrotic diseases.