CN116554146A - FAP-alpha specific radiopharmaceuticals and application thereof - Google Patents

FAP-alpha specific radiopharmaceuticals and application thereof Download PDF

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CN116554146A
CN116554146A CN202210113374.9A CN202210113374A CN116554146A CN 116554146 A CN116554146 A CN 116554146A CN 202210113374 A CN202210113374 A CN 202210113374A CN 116554146 A CN116554146 A CN 116554146A
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oncofap
hynic
peg
radionuclide
fap
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史继云
王凡
杨广杰
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Peking University
Institute of Biophysics of CAS
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Peking University
Institute of Biophysics of CAS
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Abstract

A precursor compound for forming a radionuclide complex, having the structure:wherein L is selected from: - (CH) 2 ) m-, m is an integer from 2 to 6, preferably 2 or 6; -CH 2 ‑PEG 4 ‑CH 2 -; n is selected from 0 or 1; BFC is a bifunctional chelator selected from HYNIC, MAG2, MAG3, DTPA, DOTA, NOTA, TETA. Labeling the precursor compounds with radionuclides to form radiopharmaceuticals which can be used as diagnostic imaging agents or as diagnostic imaging agents for FAP-positive tumors or FAP-positive fibrotic diseases (e.g. pulmonary fibrosis, hepatic fibrosis, etc.)A targeted therapeutic agent.

Description

FAP-alpha specific radiopharmaceuticals and application thereof
Technical Field
The invention relates to a novel oncoFAP molecule-based radiopharmaceuticals and a preparation method thereof.
Background
Cancer is the second leading cause of death worldwide, and despite the significant progress currently made in diagnosis and therapy, most of the therapies developed are directed against tumor cells, ignoring the tumor microenvironment.
The tumor entity not only comprises tumor cells, but also comprises stromal cells such as vascular cells, inflammatory cells, fibroblasts and the like. The stroma in a tumor typically represents a significant portion of the malignant tumor mass and may even represent more than 90% of the tumor mass. The existence of a complex network of interactions between stroma and tumor cells, particularly a subpopulation of cells known as tumor-associated fibroblasts (cancer associated fibroblasts, CAFs), is involved in almost all stages of tumorigenesis, which play a role in tumor initiation, progression, metastasis. CAF has therefore become one of the hot spots in the field of tumor research.
CAFs have a variety of origins, which may be derived from tumor local fibroblasts, circulating fibroblasts, vascular endothelial cells, adipocytes, bone marrow-derived stem cells, and even cancer cells, and differences in tissue type are one of the causes of heterogeneity of CAFs. Because of the source of CAFs and heterogeneity of expression patterns, it is difficult to identify CAFs of all subpopulations using one uniform marker. However, high expression of fibroblast activation protein (fibroblast activation protein, FAP) is found in the stroma CAFs of many tumors. FAP is a type II membrane-bound glycoprotein, belonging to the class II serine protease family, having dipeptidyl peptidase and endopeptidase activities. This enzyme is transiently expressed during embryonic development, is not expressed or very low expressed in normal adult tissues, and is highly expressed in more than 90% of the epithelial cancers, such as head and neck cancer, breast cancer, lung cancer, pancreatic cancer, esophageal cancer, colorectal cancer, ovarian cancer, gastric cancer, liver cancer, and the like. High expression of FAP in CAFs has been shown to be a marker of tumor invasive behavior and poor prognosis. And compared with tumors, the differential low expression of FAP in normal tissues provides excellent conditions for radionuclide-labeled FAP targeted nuclear medicine imaging, and the high expression of FAP also facilitates subsequent targeted radiotherapy or targeted drug delivery.
Recent research progress shows that the widely studied FAPI small molecules are mainly F AP-alpha inhibitors based on quinolyl-glycine- (2S) -cyano proline skeleton, and are FAPI small molecules obtained based on quinine 6-site modification. Representative are the following FAPI-02, FAPI-04, FAPI-34, FAPI-46, FAPI-74 (J Nucl Med 2021; 62:160-167):
wherein most of FAPI small molecules are positron nuclides 68 Ga、 18 F, and the like, and is applied to PET (positron emission tomography) imaging. In which only FAPI-34 is formed from a single photon species 99m Tc labeling, and SPECT imaging application. In addition, CN 111991570B discloses FAPI-04 based 99m Tc marks HFAPi and HpFApi marks medicine, optimizes 99m The in vivo pharmacokinetic profile of Tc-labeled FAPI molecules is as follows:
since the sources, numbers and distributions of FAP-expressing CAFs in human tumors and the number of FAP molecules in each cell may vary, in order to better image a tumor with a relatively low FAP expression level in clinic, it is necessary to optimize the existing FAP small molecule nuclear medicine probe to increase its tumor targeting, improve in vivo stability, make it have a higher tumor uptake and a faster systemic background clearance rate, and further obtain a significant tumor/normal tissue contrast, so as to increase the lesion detection rate.
Recently, it has been reported that oncoFAP (PNAS 2021Vol.118No.16e 2101852118), a novel FAP ligand with a binding dissociation constant in the sub-nanomolar concentration range, is obtained based on quinoline chemical modification at position 8
Current research on oncoFAPI is also limited to oncoFAP monomer-based 68 Ga and 177 labeling of Lu, preliminary PET imaging studies and in vivo mouse evaluations have been performed, but no suitable single photon nuclides have been developed 99m The molecular structure of Tc mark is used for SPECT imaging research. Compared with PET, the medicine used for SPECT (positron emission computed tomography) diagnosis is relatively simple and convenient to prepare, low in cost, moderate in clinical examination cost, high in popularity and easy to popularize and accept, so that the development of the novel nuclear medicine imaging probe based on oncofAP may have better application prospect. However, the rapid in vivo clearance of currently studied FAPI-like molecules, there is a need for researchers to continue to develop new molecular structures to modulate pharmacokinetic profiles, increase their in vivo circulation time to increase tumor uptake and retention, and further increase binding affinity to FAP to achieve higher tumor uptake and better tumor vs. non-target tissue values. The improvement of the performances is helpful to improve the detection rate and the accuracy rate of FAP positive tumors, and particularly has obviously enhanced detection efficiency for tumors and focal tissues with relatively low FAP expression, such as pulmonary fibrosis and the like.
Disclosure of Invention
The invention aims to provide a novel oncoFAP-molecule-based enhanced radiopharmaceutical. The novel enhanced oncoFAP molecule designed by the invention utilizes the SPECT imaging technology of nuclear medicine to carry out imaging diagnosis on FAP positive tumor or fibrosis diseases. Such enhanced oncoFAP compound structures may also sequester DOTA, NOTA class chelators for use in 68 Ga， 64 Cu， 177 Labeling of Lu and other metal nuclides, and PET imaging and nuclide treatment are realized.
The aim of the invention is realized by the following technical scheme:
a precursor compound for forming a radionuclide complex, having the structure:
wherein,,
l is selected from:
-(CH 2 ) m-, m is an integer from 2 to 6, preferably 2 or 6;
-CH 2 -PEG 4 -CH 2 -, wherein PEG 4 The structural formula is as follows:two ends->Represents a bond to a methylene linkage;
L 1 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 chelator selected from HYNIC, MAG2, MAG3, DTPA, DOTA, NOTA, TETA.
It is well known to those skilled in the art that the bond chain of each fragment of such compounds is in such a way that the amino group reacts with the carboxyl group to form a peptide bond, for example, wherein the BFC forms a peptide bond chain with the amino group in the main structure through its carboxyl group.
The invention also provides a complex formed by labeling the precursor compound with a radionuclide.
According to an embodiment of the invention, the radionuclide is selected from the group consisting of 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 invention, the radionuclide is selected from the group consisting of 99m Tc， 68 Ga， 64 Cu， 177 Lu。
According to an embodiment of the invention, when the radionuclide is selected from 99m At Tc, BFC is selected from HYNIC; when the radionuclide is selected from 68 Ga， 64 Cu， 177 In Lu, BFC is selected from DOTA, NOTA.
It will be appreciated by those skilled in the art that the complexes as defined above, when bifunctional chelateSynergistic ligands are also required when the agent as a ligand cannot occupy all positions of the radionuclide. Radionuclides and bifunctional chelators that require synergistic ligands in the present invention are well known to those skilled in the art. For example 99m Tc when HTNIC is used as the bifunctional chelating agent, which may be the same or different, are known in the art, wherein common synergistic ligands include water-soluble phosphines (e.g., triphenylphosphine tris-m-sulfonate sodium TPPTS), N-tris (hydroxymethyl) methylglycine (Tricine), N-bis (hydroxyethyl) glycine, glucoheptonate, ethylenediamine-N, N' -diacetate (EDDA), 3-Benzoylpyridine (BP), pyridine-2-azo-p-dimethylaniline (PADA), and the like.
As an example, the precursor compounds of the present invention are as follows:
HYNIC-[C 2 -oncoFAP] 2 the structural formula of the compound is shown as a compound 6 in the specification and the figure 2.
HYNIC-PEG 4 -[C 2 -oncoFAP] 2 The structural formula of the compound is shown as a compound 7 in the specification of figure 3.
HYNIC-[PEG 4 -oncoFAP] 2 The structural formula of the compound is shown as a compound 10 in figure 4 of the specification.
HYNIC-PEG 4 -[PEG 4 -oncoFAP] 2 The structural formula of the compound is shown as a compound 11 in figure 5 of the specification.
HYNIC-[C 6 -oncoFAP] 2 The structural formula of the compound is shown as a compound 13 in the following figure.
HYNIC-[Aoc-oncoFAP] 2 The structural formula of the compound is shown as a compound 15 in the following figure.
DOTA-[C 2 -oncoFAP] 2 The structural formula of the compound is shown as a compound A in figure 7 of the specification.
NOTA-[C 2 -oncoFAP] 2 The structural formula of the compound is shown as a compound B in the specification of figure 7.
DOTA-[PEG 4 -oncoFAP] 2 The structural formula of the compound is shown as a compound C in figure 7 of the specification.
NOTA-[PEG 4 -oncoFAP] 2 The structural formula of the compound is shown as a compound D in figure 7 of the specification.
By way of example, the complexes of the invention are as follows:
99m Tc-HYNIC-[C 2 -oncoFAP] 2 the structural formula of the compound is shown as a compound C in the specification and the figure 6.
99m Tc-HYNIC-PEG 4 -[C 2 -oncoFAP] 2 The structural formula of the compound is shown as a compound D in the specification and the figure 6.
99m Tc-HYNIC-[PEG 4 -oncoFAP] 2 The structural formula of the compound is shown as a compound E in the specification and the figure 6.
99m Tc-HYNIC-PEG 4 -[PEG 4 -oncoFAP] 2 The structural formula of the compound is shown as a compound F in the specification and the figure 6.
99m Tc-HYNIC-[C 6 -oncoFAP] 2 Structural reference 99m Tc-HYNIC-[C 2 -oncoFAP] 2 。
The invention also provides a medicine containing the complex. The medicine can be used as an imaging diagnostic agent or a radioactive targeting therapeutic agent for FAP positive tumors or FAP positive fibrosis diseases (such as pulmonary fibrosis, hepatic fibrosis and the like). The function of such radionuclides as diagnostic or therapeutic agents for cancer or tumors is well known to those skilled in the art and is primarily determined by the type of radioactivity of the nuclides. The radiopharmaceuticals obtained by the research of the invention can target FAP molecules with high expression in tumors, and are functions brought by the structures of precursor compounds, so that the precursor compounds can be respectively used as diagnostic or therapeutic agents for tumors when being matched with different nuclides. For example when the radionuclide is for 68 Ga， 64 In the case of Cu, whatThe medicine is used as PET imaging agent; when the radionuclide is 177 Lu, the drug acts as a PET therapeutic; when the radionuclide is 99m At Tc, the drug acts as a SPECT imaging agent. In general, therapeutic agents require higher uptake and longer residence time of the drug in the tumor, and as will be appreciated by those skilled in the art, the radiopharmaceuticals of the present invention have properties that meet this requirement, and thus those skilled in the art will fully appreciate that therapeutic agents formed from the precursor compounds of the present invention may be satisfactory for therapeutic use.
According to an embodiment of the invention, the medicament is an injectable formulation comprising a labeled complex as described above and an injectable carrier. Preferably, the medicament is a colorless transparent injectable formulation.
The invention also provides application of the precursor compound or the complex in preparing medicaments for diagnosing or treating FAP positive tumors or fibrosis diseases.
The beneficial effects are that:
the radiopharmaceuticals provided by the invention are brand-new FAP-targeted molecular imaging probes, and can be applied to nuclear medicine molecular imaging of various FAP-expressed tumors or FAP-positive fibrotic diseases (such as pulmonary fibrosis, hepatic fibrosis and the like), so that early diagnosis and screening of the diseases are realized. On the basis of small molecular oncoFAP, the ligand compound with larger molecular weight and volume is obtained through structural modification, and the prepared radiopharmaceuticals have remarkably excellent in-vivo stability, stronger tumor targeting and higher contrast ratio of tumor to non-target tissues. Meanwhile, the biocompatibility of the probe is improved, and the pharmacokinetic property is optimized. In addition, the ligand compound of the invention has wider application, besides SPECT imaging agent chelated with HYNIC, the ligand compound can also be chelated with DOTA and the like, and can be used for 68 Ga、 64 Cu and 177 lu markers are used for PET imaging and nuclide targeted radiotherapy of more FAP expression tumors.
The invention also researches on the simpler structural modification relative to the invention on the basis of oncoFAP to obtain the following two specific precursor compounds and corresponding compounds chelated with radionuclidesA complex: HYNIC-C 2 -oncoFAP、HYNIC-PEG 4 -C 2 -oncoFAP having the structural formula shown in compounds 3 and 4 of figure 1 of the specification. The corresponding complex is 99m Tc-HYNIC-C 2 -oncoFAP、 99m Tc-HYNIC-PEG 4 -oncoFAP, the structure of which is shown in compounds a and B of figure 6 of the description. Compared with the oncoFAP known in the prior art, the two probes have higher tumor uptake and imaging contrast, which indicates that the oncoFAP-based radioactive probe has good tumor specific targeting capability. Compared with the two probes, the molecular probe provided by the invention has the advantages that the tumor uptake is higher, and the probe is cleared from blood more quickly, so that the background of other organs is lower, and better contrast is presented in nuclear medicine imaging. The invention also researches the influence caused by the structural modification of different lengths of the same type of modified chains to obtain the following two specific precursor compounds, HYNIC 2 -oncoFAP] 2 、HYNIC-[C 6 -oncoFAP] 2 The structural formulas of the compound are shown as a compound 6 in the attached figure 2 of the specification and a compound 13 in the specification respectively. The corresponding complex is 99m Tc-HYNIC-[C 2 -oncoFAP] 2 、 99m Tc-HYNIC-[C 6 -oncoFAP] 2 . Results of in vivo biodistribution data for the mouse tumor model are shown (fig. 14C and 14G respectively), 99m Tc-HYNIC-[C 6 -oncoFAP] 2 is higher than the blood uptake of (C), and the tumor uptake is higher than that at 0.5h 99m Tc-HYNIC-[C 2 -oncoFAP] 2 Slightly lower but at 4h post injection 99m Tc-HYNIC-[C 6 -oncoFAP] 2 The tumor uptake is obviously increased 99m Tc-HYNIC-[C 2 -oncoFAP] 2 The tumor uptake of (c) decreased with time, so that the tumor uptake of both became comparable at a later time point. It can be seen that the structure-activity relationship in the field is strict, that only subtle differences in the number of different cs in the chain also significantly affect their tumor uptake, and that their trend is difficult to predict. Even with the same compound, tumor uptake increases or decreases or mutations over time are unexpected.
In addition, the present invention also investigated the fatty chain (Aoc) having a linker arm of 8-octanaminoIn the case where the precursor compound is HYNIC- [ Aoc-oncofAP] 2 As shown in compound 15 above, the corresponding complex is 99m Tc-HYNIC-[Aoc-oncoFAP] 2 The in vivo biodistribution data is shown in figure 14H. It can be seen that when the Aoc chain is exchanged, the in vivo biodistribution of the whole probe is mainly higher in the kidney (kidiny) and gall bladder (Gallbla), the tumor does not have obvious uptake, and the results of the blocking group are not significantly different from those of the non-blocking group. The different types of linker arm chains therefore have significantly different effects on the oncoFAP molecule, which severely affects the targeting effect of the compound.
Based on the above comparison, those skilled in the art will appreciate structural modifications and improvements of the present invention based on oncoFAP, particularly C 2 And PEG (polyethylene glycol) 4 The chain modified tumor targeting peptide has better tumor targeting property and in vivo metabolism property, so that the chain modified tumor targeting peptide has excellent application performance and value.
In addition, the invention also compares the typical molecular probe of CN 111991570B 99m Tc-HFAPi 99m Compared with CN 111991570B, the molecular probe has better in-vivo metabolic stability, stronger binding capacity to recombinant human FAP protein, higher absolute value of tumor uptake and quicker normal organ clearance metabolism, so that the tumor/normal organ uptake ratio of the probe is higher, and the nuclear medicine imaging of tumors is more favorable. More prominently, the molecular probe of the invention can specifically image the lung fibrosis focus area with better effect.
Drawings
(a) compound 3: HYNIC-C 2 -synthetic route to oncoFAP, (B) compound 4: HYNIC-PEG 4 -C 2 -synthetic route of oncoFAP.
Fig. 2, compound 6: HYNIC- [ C 2 -oncoFAP] 2 Is a synthetic route of (2)
Fig. 3, compound 7: HYNIC-PEG 4 -[C 2 -oncoFAP] 2 Is a synthetic route of (2)
Fig. 4, compound 10: HYNIC- [ PEG 4 -oncoFAP] 2 Is a synthetic route of (2)
Fig. 5, compound 11: HYNIC-PEG 4 -[PEG 4 -oncoFAP] 2 Is a synthetic route of (2)
FIG. 6 (A) 99m Tc-HYNIC-C 2 -oncoFAP，(B) 99m Tc-HYNIC-PEG 4 -C 2 -oncoFAP，(C) 99m Tc-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 Is a schematic structural diagram of the (c).
FIG. 7. (A) DOTA- [ C ] 2 -oncoFAP] 2 ，(B)NOTA-[C 2 -oncoFAP] 2 (C) DOTA- [ PEG 4 -oncoFAP] 2 ，(D)NOTA-[PEG 4 -oncoFAP] 2 。
FIG. 8 shows the radioactive HPLC profile of the probe in urine samples at different times after BALB/c mice injection. (A) 99m Tc-HYNIC-C 2 -oncoFAP，(B) 99m Tc-HYNIC-PEG 4 -C 2 -oncoFAP，(C) 99m Tc-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 。
Fig. 9 (A) 99m Radioactive HPLC profile of probes in urine samples at different times after BALB/c mice injection. (B) 99m Radioactivity HPLC profile of probes in urine samples at different times after BALB/c mice injection.
FIG. 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 experiments with recombinant human FAP-alpha protein (rhFAP-alpha). (B) 99m Tc-HYNIC-FAPI-04( 99m Tc-HFAPi)， 99m Tc-HYNIC-PEG 4 -FAPI-04( 99m Tc-HpFApi) and recombinant human FAP-alpha protein (rhFAP-alpha). Percent binding of each probe in the (C) protein binding assay.
FIG. 11 (A) injection in U87MG glioblastoma model 99m Tc-HYNIC-C 2 SPECT/CT images after 0.5, 1, 2 and 4h after oncoFAP; (B) SPECT/CT images of the 0.5h unlabeled oncoFAP blocking group; (C) Injection in the U87MG glioblastoma model 99m Tc-HYNIC-PEG 4 -C 2 SPECT/CT images after 0.5, 1, 2 and 4h after oncoFAP; (D) SPECT/CT image of the 0.5h unlabeled oncoFAP blocking group.
FIG. 12 (A) injection in U87MG glioblastoma model 99m Tc-HYNIC-[C 2 -oncoFAP] 2 SPECT/CT images after 0.5, 1, 2 and 4 hours; (B) SPECT/CT images of the 0.5h unlabeled oncoFAP blocking group; (C) Injection in the U87MG glioblastoma model 99m Tc-HYNIC-PEG 4 -[C 2 -oncoFAP] 2 SPECT/CT images after 0.5, 1, 2 and 4 hours; (D) SPECT/CT image of the 0.5h unlabeled oncoFAP blocking group.
FIG. 13 (A) injection in U87MG glioblastoma model 99m Tc-HYNIC-[PEG 4 -oncoFAP] 2 SPECT/CT images after 0.5, 1, 2 and 4 hours; (B) SPECT/CT images of the 0.5h unlabeled oncoFAP blocking group; (C) Injection in the U87MG glioblastoma model 99m Tc-HYNIC-PEG 4 -[PEG 4 -oncoFAP] 2 SPECT/CT images after 0.5, 1, 2 and 4 hours; (D) SPECT/CT image of the 0.5h unlabeled oncoFAP blocking group.
Fig. 14. In vivo biological profile of radioactive probe in the U87MG glioblastoma model. (A) 99m Tc-HYNIC-C 2 -oncoFAP，(B) 99m Tc-HYNIC-PEG 4 -C 2 -oncoFAP，(C) 99m Tc-HYNIC-[C 2 -oncoFAP] 2 ，(D) 99m Tc-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 。
Fig. 15 (A) 99m Tc-HYNIC-FAPI-04( 99m Tc-HFAPi) in vivo biodistribution in the U87MG glioblastoma model. (B) Quantitative uptake values of the radioactive probe in U87MG glioblastoma were compared. * Indicating significant differences (p < 0.05), ns indicating no significant differences.
FIG. 16 (A) M1-M2 are shown 99m SPECT/CT imaging results of Tc-HFAPI-04 in bleomycin-induced mouse pulmonary fibrosis model, M3-M4 are shown 99m SPECT/CT imaging results in normal control mice with Tc-HFAPI-04. (B) M5-M7 are shown as 99m Tc-HYNIC-[C 2 -oncoFAP] 2 SPECT/CT imaging results in bleomycin-induced mouse lung fibrosis model, M8-M9 are shown 99m Tc-HYNIC-[C 2 -oncoFAP] 2 SPECT/CT imaging results in normal control mice.
FIG. 17 (A) M1-M2 shows 99m Tc-HYNIC-PEG 4 SPECT/CT imaging of FAPI-04 in bleomycin-induced mouse lung fibrosis model, M3 is shown 99m Tc-HYNIC-PEG 4 SPECT/CT imaging results of FAPI-04 in normal control mice. (B) M4-M5 are shown as 99m Tc-HYNIC-PEG 4 -[C 2 -oncoFAP] 2 SPECT/CT imaging results in bleomycin-induced mouse lung fibrosis model, M6 is shown 99m Tc-HYNIC-PEG 4 -[C 2 -oncoFAP] 2 SPECT/CT imaging results in normal control mice.
FIG. 18 (A) M1-M2 shows 99m Tc-HYNIC-[PEG 4 -oncoFAP] 2 Bleomycin (Boletia) BotrytisSPECT/CT imaging results in a plain-induced mouse lung fibrosis model, M3 is shown as 99m Tc-HYNIC-[PEG 4 -oncoFAP] 2 SPECT/CT imaging results in normal control mice. (B) M4-M5 are shown as 99m Tc-HYNIC-PEG 4 -[PEG 4 -oncoFAP] 2 SPECT/CT imaging results in bleomycin-induced mouse lung fibrosis model, M6 is shown 99m Tc-HYNIC-PEG 4 -[PEG 4 -oncoFAP] 2 SPECT/CT imaging results in normal control mice.
FIG. 19 is a graphical representation of HE staining, masson staining and immunohistochemical staining of FAP protein in normal mouse lung tissue, lung fibrosis model mouse lung tissue.
Detailed Description
The materials adopted in the embodiment of the invention are as follows:
HYNIC-NHS (dihydrazinamide) was purchased from Noca-biochem, usa. 1-amino-3,6,9, 12-tetraoxaptantectan-15-oic acid (NH) 2 -PEG 4 -COOH) from the company xianruixi biotechnology limited. Dichloromethane (DCM), 4-Dimethylaminopyridine (DMAP), tetrahydrofuran (THF) were all purchased from the general fine chemicals company of beijing. succinic acid, disodium succinate hexahydrate (disodium succinate), trisodium triphenylphosphine-3,3',3 "-trisulfinate (TPPTS, sodium triphenylphosphine trisulphonate), N, N-Dimethylform amide (DMF, N, N-dimethylformamide), tricine (trimethylglycine), trifluoroacetic acid (TFA, trifluoroacetic acid), N-ethylideopropylamine (DIPEA, N, N-diisopropylethylamine) were all purchased from Sigma-Aldrich, USA. Na (Na) 99m TcO 4 The eluate was purchased from Beijing atomic Gaokang Co.
Preparation example
1、HYNIC-C 2 -oncoFAP and HYNIC-PEG 4 -C 2 Preparation of oncoFAP (fig. 1)
a、HYNIC-PEG 4 Preparation of-COOH
Weighing NH 2 -PEG 4 -COOH (1.2 eq) was added to an EP tube, dissolved in 50. Mu.L DMF, and DIEA was added to adjust the pH to 7.8-8.0; the HYNIC-NHS (1 eq) was further weighed into an EP tube and dissolved in 50mu.L of DMF. Adding HYNIC-NHS into NH 2 -PEG 4 Mixing with COOH solution, adding DIEA to regulate pH value to 7.8-8.0, and reacting at room temperature overnight. The reaction was monitored by high performance liquid chromatography and the target product was isolated and purified (method one). The elution peak was collected for 18.2 minutes and the eluate was lyophilized using a vacuum freeze-drying method to give a solid product. After a small amount of product is dissolved, HPLC is used for identifying the purity>98%. M/z= 569.6 ([ m+h) by TOF MS (es+) mass spectrometry] + ) Confirmed to be the expected product HYNIC-PEG 4 -COOH。
b. Preparation of oncoFAP
8-aminoquinoline-4-carboxylic acid (1 eq), (S) -1- (2-aminoacetyl) -4.4-difluoropyrrolidine-2-carbonitrile hydrochloride (1 eq) and HATU (1 eq) were added to a 25mL round bottom flask and dissolved with 900. Mu.L DMF and 4mL DCM followed by dropwise addition of DIEA (4 eq) and stirring. The crude product was diluted with DCM, washed with water, na 2 SO 4 Drying, filtering, and finally removing the solvent by a rotary evaporator to obtain a crude product (S) -8-amino-N- (2- (2-cyano-4, 4-difluoro-pyrrolidin-1-yl) -2-carbonyl-ethyl) -quinoline-4-carboxamide. The above crude product (1 eq), succinic anhydride (50 eq) and DAMP (0.5 eq) were added to a 25mL round bottom flask and dissolved with 3mL THF and reacted for 6 hours at 60 ℃. The solvent was removed from the reaction mixture by rotary evaporation, diluted with water, extracted with DCM, and then Na 2 SO 4 After drying, filtration, drying, analysis by TOF MS (es+) mass spectrometry, M/z=460.1 ([ m+h)] + ) The expected product oncoFAP was identified.
c、C 2 Preparation of oncoFAP
oncoFAP (1 eq) and HATU (1.5 eq) were weighed into an EP tube, dissolved in 200 μl DMF, DIEA (2 eq) was added and reacted at room temperature for about 30 minutes. Then, N-tert-butoxycarbonyl ethylenediamine (N-Boc-ethylenediamine, CAS: 57260-73-8) (1.5 eq) was weighed and added to the reaction solution, DIEA was added to adjust pH to 8.5-9.0, and the reaction was carried out at room temperature overnight, and the reaction was monitored by high performance liquid chromatography to isolate and purify the target product (method II). The elution peak was collected for 19.8 minutes and the eluate was lyophilized using a vacuum freeze-drying method to give the desired product Boc-C 2 -oncoFAP. The lyophilized product was dissolved in 1mL TFA and reacted at room temperature for 10min, and reversedThe reaction solution was dried with nitrogen and the product obtained was analyzed by TOF MS (ES+) mass spectrometry, M/z=502.2 ([ M+H)] + ) Identified as the expected product C 2 -oncoFAP。
d、HYNIC-C 2 Preparation of oncoFAP
C is C 2 -oncoFAP (1 eq) and HYNIC-NHS (1 eq) were dissolved in DMF, DIEA was added to adjust pH to 8.5-9.0, reacted overnight at room temperature, monitored for reflection using high performance liquid chromatography, and the target product was isolated and purified (method two). The elution peak was collected for 14.0 minutes and the eluate was lyophilized using a vacuum freeze-drying method to give a solid product. M/z= 805.2 ([ m+h) by MALDI-TOF mass spectrometry] + ) Confirmed to be the expected product HYNIC-C 2 -oncoFAP。
e、HYNIC-PEG 4 -C 2 Preparation of oncoFAP
Weighing HYNIC-PEG 4 -COOH (1 eq), HATU (1.5 eq) in EP tube in 200. Mu.L DMF and DIEA (2 eq) added and reacted at room temperature for about 30 minutes. Weighing NH 2 -oncoFAP (1 eq) was added to the reaction solution, DIEA was added to adjust pH to 8.5-9.0, and the reaction was carried out overnight at room temperature, and the reaction was monitored by hplc, and the target product was isolated and purified (method two). The elution peak was collected for 15.1 minutes and the eluate was lyophilized using a vacuum freeze-drying method to give a solid product. M/z= 1052.3 ([ m+h) by MALDI-TOF mass spectrometry] + ) Confirmed to be the expected product HYNIC-PEG 4 -C 2 -oncoFAP。
2、HYNIC-[C 2 -oncoFAP] 2 And HYNIC-PEG 4 -[C 2 -oncoFAP] 2 Is shown in figures 2 and 3
a、Glu-(C 2 -oncoFAP) 2 Is prepared from
C is C 2 -oncoFAP (1 eq) and Boc-Glu-OSu 2 (0.5 eq) was dissolved in DMF, DIEA was added to adjust pH to 8.5-9.0, stirred overnight at room temperature, and the reaction was monitored by HPLC to isolate and purify the target product (method II). The elution peak was collected for 19.9 minutes, and the eluate was lyophilized by vacuum freeze-drying to give the compound Boc-Glu- (C) 2 -oncoFAP) 2 The method comprises the steps of carrying out a first treatment on the surface of the The lyophilized product was dissolved in 1mL TFA, reaction at room temperature for 10min, drying the reaction solution with nitrogen, and subjecting the obtained product to TOF MS (ES+) mass spectrometry analysis, M/z= 557.7 ([ M+H)] 2+ ) Confirmed to be the expected product Glu- (C) 2 -oncoFAP) 2 。
b、HYNIC-oncoFAP 2 Is prepared from
Glu- (oncofAP) 2 (1 eq) and HYNIC-NHS (1 eq) were dissolved in DMF, DIEA was added to adjust pH to 8.5-9.0, reacted overnight at room temperature, and the reaction was monitored by HPLC and the target product was isolated and purified (method two). The elution peak was collected for 16.9 minutes and the eluate was lyophilized using a vacuum freeze-drying method to give a solid product. M/z=709.2 ([ m+2h) by TOF MS (es+) mass spectrometry] 2+ ) Confirmed to be the expected product HYNIC-C 2 -oncoFAP 2 。
c、HYNIC-PEG 4 -[C 2 -oncoFAP] 2 Is prepared from
Weighing HYNIC-PEG 4 -COOH (1 eq), HATU (1.5 eq) in EP tube in 200. Mu.L DMF and DIEA (2 eq) added and reacted at room temperature for about 30 minutes. Weighing Glu- (C) 2 -oncoFAP) 2 (1 eq) was added to the reaction mixture, DIEA was added to adjust pH to 8.5-9.0, and the mixture was allowed to react overnight at room temperature, followed by monitoring the reaction and separation and purification of the target product by HPLC (method II). The elution peak was collected for 17.5 minutes and the eluate was lyophilized using a vacuum freeze-drying method to give a solid product. M/z= 832.8 ([ m+2h) by TOF MS (es+) mass spectrometry] 2+ ) Confirmed to be the expected product HYNIC-PEG 4 -[C 2 -oncoFAP] 2 。
3、HYNIC-[PEG 4 -oncoFAP] 2 And HYNIC-PEG 4 -[PEG 4 -oncoFAP] 2 Is shown in FIGS. 4 and 5
a、Boc-PEG 4 Preparation of oncoFAP
oncoFAP (1 eq) and HATU (1.5 eq) were weighed into an EP tube, dissolved in 200 μl DMF, DIEA (2 eq) was added and reacted at room temperature for about 30 minutes. Thereafter, tert-butyl (14-amino-3, 6,9, 12-tetraoxatetradecyl) carbamate (Boc-NH-PEG) was weighed 4 -NH 2 CAS 811442-84-9) (1.5 eq) was added to the reaction solution and DIEA was added thereto to adjust the reaction solutionThe pH value is regulated to 8.5-9.0, the reaction is carried out overnight at room temperature, the high performance liquid chromatography is used for monitoring the reflection, and the target product is separated and purified (method II). The elution peak was collected for 20.4 minutes and the eluate was lyophilized using a vacuum freeze-drying method to give a solid product. M/z= 339.6 ([ m+2h) by TOF MS (es+) mass spectrometry] 2+ ) Identified as the desired product Boc-PEG 4 -oncoFAP。
b、Glu-[PEG 4 -oncoFAP] 2 Is prepared from
Boc-PEG 4 -oncoFAP (1 eq) was dissolved in 1mL TFA and reacted at room temperature for 10 minutes, the reaction mixture was dried with nitrogen and then dissolved in DMF, DIEA was added to adjust pH to 8.5-9.0; weighing Boc-Glu-OSu 2 (0.5 eq) was added to the reaction mixture, DIEA was added to adjust the pH to 8.5-9.0, and the mixture was stirred overnight at room temperature, and the reaction was monitored by high performance liquid chromatography to isolate and purify the target product (method II). The elution peak was collected for 19.9 minutes and the eluate was lyophilized using a vacuum freeze-drying method to obtain the desired product Boc-Glu- [ PEG 4 -oncoFAP] 2 The method comprises the steps of carrying out a first treatment on the surface of the The lyophilized product was dissolved in 1mL of TFA and reacted at room temperature for 10min, the reaction solution was dried with nitrogen, and the obtained product was analyzed by MALDI-TOF-MS mass spectrometry, M/z= 1466.5 ([ M+H)] + ) Glu- [ PEG confirmed as the expected product 4 -oncoFAP] 2 。
c、HYNIC-[PEG 4 -oncoFAP] 2 Is prepared from
Glu- [ PEG 4 -oncoFAP] 2 (1 eq) and HYNIC-NHS (1 eq) were dissolved in DMF, DIEA was added to adjust pH to 8.5-9.0, reacted overnight at room temperature, and the reaction was monitored by HPLC and the target product was isolated and purified (method two). The elution peak was collected for 17.6 minutes and the eluate was lyophilized using a vacuum freeze-drying method to give a solid product. M/z= 885.3 ([ m+2h) by TOF MS (es+) mass spectrometry] 2+ ) Confirmed to be the expected product HYNIC- [ PEG 4 -oncoFAP] 2 。
d、HYNIC-PEG 4 -[PEG 4 -oncoFAP] 2 Is prepared from
Weighing HYNIC-PEG 4 -COOH (1 eq), HATU (1.5 eq) in EP tube, dissolved in 200. Mu.L DMF, added DIEA (2 eq), and inverted at room temperatureShould be about 30 minutes. Weighing Glu- [ PEG 4 -oncoFAP] 2 (1 eq) was added to the reaction mixture, DIEA was added to adjust pH to 8.5-9.0, and the mixture was allowed to react overnight at room temperature, followed by monitoring the reaction and separation and purification of the target product by HPLC (method II). The elution peak was collected for 17.5 minutes and the eluate was lyophilized using a vacuum freeze-drying method to give a solid product. M/z= 1009.4 ([ m+2h) by TOF MS (es+) mass spectrometry] 2+ ) Confirmed to be the expected product HYNIC-PEG 4 -[PEG 4 -oncoFAP] 2 。
4、HYNIC-[C 6 -oncoFAP] 2 And HYNIC- [ Aoc-oncofAP] 2 Is prepared from
a、HYNIC-[C 6 -oncoFAP] 2 Is prepared from
Glu- [ C 6 -oncoFAP] 2 (1 eq) and HYNIC-NHS (1 eq) were dissolved in DMF, DIEA was added to adjust pH to 8.5-9.0, reacted overnight at room temperature, and the reaction was monitored by HPLC and the target product was isolated and purified (method two). The elution peak was collected for 20.8 minutes and the eluate was lyophilized using a vacuum freeze-drying method to give a solid product. M/z=765.3 ([ m+2h) by TOF MS (es+) mass spectrometry] 2+ ) Confirmed to be the expected product HYNIC 6 -oncoFAP] 2 。
b、HYNIC-[Aoc-oncoFAP] 2 Is prepared from
Glu- [ Aoc-oncoFAP] 2 (1 eq) and HYNIC-NHS (1 eq) were dissolved in DMF, DIEA was added to adjust pH to 8.5-9.0, reacted overnight at room temperature, and the reaction was monitored by HPLC and the target product was isolated and purified (method two). The elution peak was collected for 23.8 minutes and the eluate was lyophilized using a vacuum freeze-drying method to give a solid product. M/z=708.3 ([ m+2h) by TOF MS (es+) mass spectrometry] 2+ ) Confirmed to be the expected product HYNIC- [ Aoc-oncoFAP] 2 。
5、DOTA-[PEG 4 -oncoFAP] 2 Is shown in FIG. 7
By DOTA- [ PEG 4 -oncoFAP] 2 For example, glu- [ PEG 4 -oncoFAP] 2 (1 eq) and DOTA-NHS (1 eq) were dissolved in DMF and adjusted by the addition of DIEAThe pH value is 8.5-9.0, the reaction is carried out overnight at room temperature, the high performance liquid chromatography is used for monitoring the reflection, and the target product is separated and purified (method II). The elution peak was collected for 18.3 minutes and the eluate was lyophilized using a vacuum freeze-drying method to give a solid product. M/z= 926.9 ([ m+2h) by TOF MS (es+) mass spectrometry] 2+ ) DOTA- [ PEG confirmed as the expected product 4 -oncoFAP] 2 。
6、 99m Tc-HYNIC-PKM-L-oncoFAP and 99m Tc-HYNIC-PKM-[L-oncoFAP] 2 is prepared from
Preparation of 20. Mu.g of HYNIC-PKM-L-oncoFAP or HYNIC-PKM- [ L-oncoFAP] 2 5.0mg of triphenylphosphine trisulphonate (TPPTS) and 6.5mg of trimethylol glycine (Tricine) were dissolved in 1mL of 0.5M succinic acid buffer solution (pH 4.8), the mixed solution was placed in a 10mL penicillin bottle, and the mixed solution was lyophilized to obtain a labeled kit. Adding 1.0-1.5mL Na into lyophilized powder of labeled kit 99m TcO 4 Placing the solution in a heating reactor such as 75-110deg.C water bath, air bath or metal bath, heating for 20-30 min, cooling at room temperature for 5min after the reaction is completed, and making into the final product 99m Tc-HYNIC-PKM-L-oncofAP and HYNIC-PKM- [ L-oncofAP] 2 . Analysis by radioactive HPLC was performed. The HPLC method is as follows:
the method for separating and purifying the target product by high performance liquid chromatography comprises the following steps: the Agilent 1260HPLC system was equipped with a YMC-Pack ODS-A C18 semi-preparative column (250X 10mml. D.S. -5 μm,12 nm). Gradient elution was set to 80% A and 20% B at the beginning, 80% A and 20% B at 5min, 40% A and 60% B at 25min, and 2.0mL/min at a flow rate of 2.0mL/min, where the mobile A phase was deionized water (0.05% TFA) and the mobile B phase was acetonitrile (0.05% TFA).
And a second method for separating and purifying the target product by high performance liquid chromatography: the Agilent 1260HPLC system was equipped with a Venusil MP C18 semi-preparative column (250X 10mml. D.S. -5 μm). Gradient elution was set to 90% A and 10% B at the beginning, 40% A and 60% B at 20min, and 90% A and 10% B at 25min, with a flow rate of 3.2mL/min, where the mobile A phase was deionized water (0.05% TFA) and the mobile B phase was acetonitrile (0.05% TFA).
Example 1 oncoFAP-based 99m In vivo stability of Tc-labeled radioactive probes in Normal BALB/c mice
(1) oncoFAP-based 99m Verification of Tc-labeled radioactive probe stability in mice
BALB/c normal mice were divided into 6 groups of 2. 100 μL (37 MBq) of each group is injected through tail vein 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 -conFAP] 2 、 99m Tc-HYNIC-[PEG 4 -oncoFAP] 2 And 99m Tc-HYNIC-PEG 4 -[PEG 4 -oncoFAP] 2 the urine from the mice was taken 30 and 120 minutes after injection, mixed with 50% acetonitrile/water, and analyzed by HPLC with radioactivity, and the results are shown in FIG. 8. 99m Tc-HYNIC-C 2 -oncoFAP 99m Tc-HYNIC-PEG 4 -C 2 -oncoFAP is metabolized in mice and undergoes a certain decomposition but still retains a substantial portion of the proto-drug; 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 stronger stability and do not decompose with the prolongation of metabolic time.
(2) 99m Tc-HFAPi 99m Verification of Tc-HpFApi stability in mice
The experimental method was the same as (1), and the results are shown in FIG. 9. 99m Tc-HFAPi 99m After Tc-HpFApi is metabolized in mice, most of the drug in urine is broken down, with only a small amount of proto-drug.
In vivo stability results analysis and comparison:
in previous studies 99m Tc-HFAPi 99m Tc-HpFApi is rapidly decomposed in mice with very littlePart of the drug is still intact; the radioactive probe is very stable in the body, most of radiopharmaceuticals can still be kept complete after 2 hours, only a small amount of radiopharmaceuticals are metabolized and decomposed, so that the radioactive probe has better in-vivo metabolic stability, and the introduction of a novel coupling mode and a pharmacokinetics connecting molecule is helpful for improving the stability of the probe.
Example 2 in vitro rhFAP-alpha protein binding assay
(1) oncoFAP-based 99m Binding experiments of Tc-labeled radioactive probes and recombinant human FAP-alpha protein (rhFAP-alpha)
rhFAP-alpha protein was dissolved in ELISA coating buffer (1X) (concentration 2. Mu.g/mL), coated in 96-well plates at 0.2. Mu.g/100. Mu.L per well, overnight at 4 ℃. After the coating is finished, the coating liquid is discarded, and the 96-well plate is repeatedly washed 3-5 times by using PBS. Blocking solution (5% calf serum/PBS buffer, pH 7.4) was added to the 96-well plate and incubated at 37℃for 2 hours. After the end of the blocking, the 96-well plate was repeatedly washed 3 to 5 times with PBS. Will prepare 99m Tc-labeled probes were added to the rhFAP-alpha coated sample wells, each well was filled with 0.3. Mu. Ci/100. Mu.L of radiolabel, and 4 parallel wells were set. Adding the same amount to another 4 sample wells 99m Tc marks the probe and then add 1000 times molar amount of oncoFAP and mix well. 96-well plates were incubated at 37℃for 1 hour. Four put-free tubes were prepared, and equal amounts of radiolabel were added 99m Tc marks the probe, leaving it as a standard. After incubation was completed, the incubation was decanted, then washed five times with PBS, after which the liquid was discarded, each sample well was cut with scissors, placed in a disposable tube, and the radioactivity count in each well was measured with a radioactivity gamma-full automatic counter. Then calculate by formula 99m Percent binding of Tc-labeled probes to rhFAP-alpha. The experimental results are shown in FIGS. 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 ) The% AD values for binding to rhFAP-a 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%), respectively; the% AD values of the oncofaP block experimental groups under the same conditions 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%), and the binding groups were significantly different (p)<0.0001). Experimental results demonstrate that oncoFAP-based 99m Tc-labeled radioactive probes can specifically bind to rhFAP-alpha protein.
(2) 99m Tc-HFAPi 99m Binding experiments of Tc-HpFApi and recombinant human FAP-alpha protein (rhFAP-alpha)
The steps are the same as (1), and the results are shown in FIGS. 10 (B) and (C), 99m Tc-HFAPi 99m The% AD values of Tc-HpFApi binding to rhFAP-alpha were 4.4% (+ -0.2%), 4.3% (+ -0.2%), respectively, whereas the% AD values of the FAPI block experimental groups under the same conditions were 0.95% (+ -0.02%), 1.1% (+ -0.02%), respectively.
In vitro rhFAP-alpha protein binding experimental result comparison analysis:
under the same experimental conditions, and 99m Tc-HFAPi 99m Compared with Tc-HpFApi, the radioactive probe of the invention has stronger binding capacity to recombinant human FAP protein in protein binding experiments and better property.
Example 3 oncoFAP-based 99m SPECT/CT imaging of Tc-labeled radioactive probes in tumor-bearing mice
(1) 99m Tc-HYNIC-C 2 -oncoFAP 99m Tc-HYNIC-PEG 4 -C 2 -oncoFAP, imaging in a U87MG tumor-bearing mouse model will prepare 99m Tc-HYNIC-C 2 -oncoFAP 99m Tc-HYNIC-PEG 4 After preparation of 37 MBq/100. Mu.L of oncoFAP with physiological saline, each mouse was injected via tail vein100 μl (37 MBq) was subjected to SPECT/CT imaging at 0.5, 1, 2 and 4 hours after injection. The mice in the closed group were injected with 100. Mu.L (500. Mu.g) of oncoFAP at the same time as the imaging drug and were imaged 0.5 hours after administration. Mice were anesthetized during imaging with 1.5% isoflurane-oxygen. After the imaging, reconstructing the SPECT image and fusing the SPECT image with the CT image to obtain a 3D imaging image, wherein a post image (Posterior view) is used for displaying and the tumor position is marked by an arrow. The result of the development is shown in FIG. 11.
(2) 99m Tc-HYNIC-[C 2 -oncoFAP] 2 And 99m Tc-HYNIC-PEG 4 -[C 2 -oncoFAP] 2 imaging in the U87MG tumor-bearing mouse model will prepare 99m Tc-HYNIC-[C 2 -oncoFAP] 2 And 99m Tc-HYNIC-PEG 4 -[C 2 -oncoFAP] 2 after preparation of 37 MBq/100. Mu.L with physiological saline, each mouse was injected with 100. Mu.L (37 MBq) via the tail vein, and 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. Mu.L (500. Mu.g) of oncoFAP at the same time as the imaging drug and were imaged 0.5 hours after administration. Mice were anesthetized during imaging with 1.5% isoflurane-oxygen. After the imaging, reconstructing the SPECT image and fusing the SPECT image with the CT image to obtain a 3D imaging image, wherein a post image (Posterior view) is used for displaying and the tumor position is marked by an arrow. The result of the development is shown in FIG. 12.
(3) 99m Tc-HYNIC-[PEG 4 -oncoFAP] 2 And 99m Tc-HYNIC-PEG 4 -[PEG 4 -oncoFAP] 2 imaging in a U87MG tumor-bearing mouse model
To be prepared 99m Tc-HYNIC-[PEG 4 -oncoFAP] 2 And 99m Tc-HYNIC-PEG 4 -[PEG 4 -oncoFAP] 2 after preparation of 37 MBq/100. Mu.L with physiological saline, each mouse was injected with 100. Mu.L (37 MBq) via the tail vein, and 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. Mu.L (500. Mu.g) of oncoFAP at the same time as the imaging drug and were imaged 0.5 hours after administration. Mice were anesthetized with 1.5% isoflurane-oxygen during imagingAnd (3) drunk. After the imaging, reconstructing the SPECT image and fusing the SPECT image with the CT image to obtain a 3D imaging image, wherein a post image (Posterior view) is used for displaying and the tumor position is marked by an arrow. The imaging results are shown in FIG. 13.
And (3) 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 obvious tumor uptake and higher imaging contrast can be seen, which indicates that the radioactive probe of the invention has good tumor specific targeting capability. In the blocking group experiments, tumor uptake was significantly reduced, demonstrating specific binding of the probe to FAP sites at the tumor site. Further analysis and 99m Tc-HYNIC-C 2 -oncoFAP、 99m Tc-HYNIC-PEG 4 -C 2 -oncoFAP compared to probe 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 the tumor uptake is higher, and the probe is cleared from the blood faster, so that the background of other organs is lower, thereby presenting better contrast in the nuclear medicine imaging, being beneficial to the diagnosis of the tumor and being capable of providing clearer imaging diagnosis for some FAP low-expression tumors and fibrosis focuses (such as pulmonary fibrosis and hepatic fibrosis).
Example 4 oncoFAP-based 99m Biodistribution of Tc-labeled radioactive probes in tumor-bearing mice
BALB/c Nude mice were divided into 6 groups of 6 tumors each. Each group of mice was injected with 100. Mu.L (-74 KBq) via the tail vein, respectively 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 、 99m Tc-HYNIC-PEG 4 -[PEG 4 -oncoFAP] 2 、 99m Tc-HYNIC-[C 6 -oncoFAP] 2 A kind of electronic device 99m Tc-HYNIC-[Aoc-oncoFAP] 2 And sacrificed 0.5 and 4 hours after injection; blood and major viscera were taken, weighed and radioactivity counts were measured, and after decay correction, percent injection dose rate per gram of tissue (% ID/g) was calculated. The biodistribution results are expressed as mean ± standard deviation (means ± SD, n=3), and the biodistribution experimental results are shown in fig. 14.
Biodistribution results analysis and comparison:
CN 111991570B is concerned with 99m Tc-HFAPi 99m Tc-HpFApi, both of which are similar in biodistribution in the U87MG tumor model, wherein 99m The biological distribution of Tc-HFAPi is shown in FIG. 15 (A), which shows the partial probe and the partial probe according to the invention 99m The tumor uptake value pair of Tc-HFAPi is shown in FIG. 15 (B). And (3) with 99m Compared with Tc-HFAPi, the probe has higher tumor uptake absolute value and higher normal organ clearance metabolism, so that the tumor/normal organ uptake ratio of the probe is higher, the nuclear medicine imaging of the tumor is more facilitated, particularly the SPECT imaging diagnosis is very sensitive to background signal noise, and the low background is more beneficial to accurately detecting tiny focus.
Example 5 oncoFAP-based 2 A kind of electronic device 99m SPECT/CT imaging of Tc-labeled radioactive probes in Bleomycin (Bleomycin) -induced C57BL/6 mouse model
The pulmonary fibrosis mouse model is formed by slowly instilling a physiological saline solution (7 mg/kg, 100. Mu.L) of bleomycin into the trachea, followed by conventional 2 weeks of feeding. When the pulmonary fibrosis model and normal control mice were visualized, 100. Mu.L (. About.18 MBq) of each mouse was injected via the tail vein 99m Tc-HFAPI、 99m Tc-HpFAPI、 99m Tc-HYNIC-[C 2 -oncoFAP] 2 、 99m Tc-HYNIC-PEG 4 -[C 2 -oncoFAP] 2 、 99m Tc-HYNIC-[PEG 4 -oncoFAP] 2 Or (b) 99m Tc-HYNIC-PEG 4 -[PEG 4 -oncoFAP] 2 And SPECT/CT imaging was performed 1 hour after injection. Mice were anesthetized with 0.5-1.5% isoflurane-oxygen during imaging. The imaging results are shown in FIGS. 16-18.
Analysis of pulmonary fibrosis imaging results:
as in FIG. 16 (A), in 99m In the Tc-HFAPi imaging group, M1-M3 is a bleomycin-induced pulmonary fibrosis model mouse, and the CT confirms that the lung of the mouse has obvious fibrosis focus, and no obvious probe uptake signal appears in a fibrosis area in a corresponding SPECT image; in normal control mice, neither CT nor SPECT seen pulmonary fibrosis signals. As shown in FIG. 16 (B), in 99m Tc-HYNIC-[C 2 -oncoFAP] 2 In the imaging group, M1-M3 are model mice with bleomycin-induced pulmonary fibrosis, obvious substantive areas are visible in the lung from CT signals, the model mice are fibrosis focuses, and aggregation of radioactive signals is visible in SPECT/CT images corresponding to the areas, so that the areas concentrated by the probes can be analyzed to be pulmonary fibrosis areas; in normal control mice, no significant fibrotic signal was seen in both CT and SPECT images. It can thus be considered that, 99m Tc-HYNIC-[C 2 -oncoFAP] 2 can specifically image focus of pulmonary fibrosis region, and has better probe concentration intensity and contrast with surrounding organs 99m Tc-HFAPi. The white dashed boxes in the figure select lung tissue contours.
As in FIG. 17 (A), in 99m In the Tc-HpFAPI imaging group, M1-M2 is a bleomycin-induced pulmonary fibrosis mouse, and the lung of the mouse is confirmed to have obvious fibrosis focus by CT, and no obvious probe uptake signal appears in a fibrosis area in a corresponding SPECT image; it should be noted that significant radioactive signal uptake can be seen in adjacent sites, such as the spine, sternum sites, where the high intensity signal affects the uptake of the probe in the lungs and reconstruction of SPECT signals; in normal control mice (M3), neither CT nor SPECT seen pulmonary fibrosis signals. As shown in FIG. 17 (B)In the following 99m Tc-HYNIC-PEG 4 -[C 2 -oncoFAP] 2 In the imaging group, M4-M5 is a bleomycin-induced pulmonary fibrosis model mouse, a significant substantive region in the lung is confirmed by CT signals, the region is a fibrosis focus, and aggregation of radioactive signals can be seen in SPECT/CT images corresponding to the regions, so that the region concentrated by the probe can be analyzed to be a pulmonary fibrosis region; in the normal control mice (M6), no significant materialized fibrosis signal and probe uptake signal were seen in both CT and SPECT images. It can thus be considered that, 99m Tc-HYNIC-PEG 4 -[C 2 -oncoFAP] 2 can specifically image focus of pulmonary fibrosis region, and has better probe concentration intensity and contrast with surrounding organs 99m Tc-HpFApi. In the figure, the white dotted boxes are all selected from the lung, and the red dotted boxes are all selected from the joint.
In FIG. 18, and 99m Tc-HYNIC-[C 2 -oncoFAP] 2 and 99m Tc-HYNIC-PEG 4 -[C 2 -oncoFAP] 2 similarly to this, the process is carried out, 99m Tc-HYNIC-[PEG 4 -oncoFAP] 2 and 99m Tc-HYNIC-PEG 4 -[PEG 4 -oncoFAP] 2 can specifically image the lung fibrosis focus area.
In conclusion, the method comprises the steps of, 99m Tc-HFAPi 99m Tc-HpFApi does not work well in the imaging of pulmonary fibrosis, and does not image the areas of fibrosis well; whereas the oncoFAP 2-based radioactive probe involved in the present invention 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 can specifically image the focus area of pulmonary fibrosis, and has better effect than that of the lung fibrosis 99m Tc-HFAPi 99m Tc-HpFApi makes it have wider clinical application value.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A precursor compound for forming a radionuclide complex, having the structure:
wherein,,
l is selected from: - (CH) 2 ) m-, m is an integer from 2 to 6, preferably 2 or 6; -CH 2 -PEG 4 -CH 2 -；
Wherein PEG is 4 The structural formula is as follows:two ends->Represents a bond to a methylene linkage;
L 1 is-C (O) -L-NH-wherein L is as defined above;
n is selected from 0 or 1;
BFC is a bifunctional chelator selected from HYNIC, MAG2, MAG3, DTPA, DOTA, NOTA, TETA.
2. The precursor compound of claim 1, HYNIC- [ C 2 -oncoFAP] 2 Which is selected from the following specific compounds:
HYNIC-PEG 4 -[C 2 -oncoFAP] 2 ，
HYNIC-[PEG 4 -oncoFAP] 2 ，
HYNIC-PEG 4 -[PEG 4 -oncoFAP] 2 ，
HYNIC-[C 6 -oncoFAP] 2 ，
DOTA-[C 2 -oncoFAP] 2 ，
NOTA-[C 2 -oncoFAP] 2 ，
DOTA-[PEG 4 -oncoFAP] 2 ，
NOTA-[PEG 4 -oncoFAP] 2 。
3. a complex formed by labeling a precursor compound of claim 1 with a radionuclide.
4. The complex of claim 2, said radionuclide being selected from the group consisting of 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 of claim 2, said radionuclide being selected from the group consisting of 99m Tc， 68 Ga， 64 Cu， 177 Lu。
6. The complex of claim 2, when the radionuclide is selected from the group consisting of 99m At Tc, BFC is selected from HYNIC; when the radionuclide is selected from 68 Ga， 64 Cu， 177 In Lu, BFC is selected from DOTA, NOTA.
7. A complex according to claim 3 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 。
8. a pharmaceutical composition comprising the complex of any one of claims 3-7. Preferably, the medicament may be used as an imaging diagnostic or as a radiotargeted therapeutic for FAP-positive tumors or FAP-positive fibrotic diseases (e.g. pulmonary fibrosis, liver fibrosis, etc.). For example when the radionuclide is for 68 Ga， 64 In the case of Cu, the drug acts as a PET imaging agent; when the radionuclide is 177 Lu, the drug acts as a PET therapeutic; when the radionuclide is 99m At Tc, the drug acts as a SPECT imaging agent.
9. The pharmaceutical composition of claim 8, which is an injectable formulation comprising the labeled complex and an injectable carrier. Preferably, the medicament is a colorless transparent injectable formulation.
10. Use of a precursor compound according to any one of claims 1-2 or a complex according to any one of claims 3-7 for the preparation of a medicament for the diagnosis or treatment of FAP-positive tumors or fibrotic diseases.
CN202210113374.9A 2022-01-29 2022-01-29 FAP-alpha specific radiopharmaceuticals and application thereof Pending CN116554146A (en)

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