CN118356515A - Conjugates with radionuclides targeting FAP and their applications - Google Patents

Abstract

The present disclosure discloses a conjugate labeled with a radionuclide targeting fibroblast activation protein (FAP) and its application. The conjugate of the present disclosure comprises: a polypeptide targeting FAP; and a chelating group bound to the radionuclide, wherein the radionuclide comprises one or more of ¹⁸ F, ⁶⁸ Ga, ⁶⁴ Cu and ¹⁷⁷ Lu. The conjugate of the present disclosure ensures diagnostic specificity and can also significantly improve the overall quality of the image.

Description

Conjugates with radionuclides targeting FAP and their applications

Technical Field

The invention belongs to the field of biotechnology, and in particular relates to a conjugate with radionuclides targeting FAP and an application thereof.

Background technique

Fibroblast activation protein (FAP) is specifically upregulated on the cell surface of cancer-associated fibroblasts (CAFs), but is rarely expressed in fibroblasts in normal adult tissues. Therefore, FAP is used as a target in clinical practice, and PET/CT scanning imaging is used to show FAP expression, thereby diagnosing tumors.

At present, quinoline small molecule inhibitors (FAPIs) targeting FAP, including FAPI-04, FAPI-46, FAPI-74, FAP-2286, etc., which are labeled with ⁶⁸ Ga, have been widely used in the clinic to diagnose various types of cancer. However, the production capacity of ⁶⁸ Ge/ ⁶⁸ Ga generators is limited, and they can only be produced once every 4 to 6 hours on average, and the availability of radioactive nuclides ⁶⁸ Ga is limited (T _1/2 ≈ 68 minutes), which makes it difficult for ⁶⁸ Ga-labeled radioactive tracers to meet the needs when they are clinically used in medical centers with a large number of patients. On the other hand, ⁶⁸ Ga has a long positron range and a low positron yield, and has low spatial resolution and image contrast. Therefore, it is still necessary to develop radioactive probes that can show high affinity and specificity for FAP, and have good tumor uptake and long tumor retention time.

Summary of the invention

In order to solve the problems of the short half-life and low yield of ⁶⁸ Ga in the prior art, which are difficult to meet the needs of high-throughput medical institutions, the present invention provides a new FAP-targeted cyclic peptide, which specifically targets FAP and tumor cells, can be quickly taken up by tumor cells, and has a low excretion rate, thereby prolonging the retention time in the body, ensuring the diagnosis and its specificity, and significantly improving the overall quality of the image.

According to one aspect of the present disclosure, a conjugate targeting fibroblast activation protein (FAP) is provided, the conjugate comprising: a binding peptide that specifically binds to FAP; a chelating group that binds to a radionuclide; and a linker that connects the polypeptide to the chelating group.

In some embodiments, the chelating group is selected from one or more of DOTA, DOTAGA, NOTA, NODAGA, NODA-MPAA, HBED, TETA, CB-TE2A, DTPA, DFO, Macropa, HOPO, TRAP, THP, DATA, NOTP, sarcophagine, FSC, NETA, H4octapa, Pycup, N _x S _4-x (N4, N2S2, N3S), Hynic, ^99m Tc (CO) _3- groups. In a preferred embodiment, the chelating group can be selected from DOTA and/or NOTA.

In some embodiments, the binding peptide can be a cyclic peptide. In some embodiments, the binding peptide can have, for example, 3 to 40 amino acid residues. In some embodiments, the binding peptide can have 3 to 20 amino acid residues. In some embodiments, the binding peptide can have, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues.

In some embodiments, the binding peptide can be a single-ring peptide.

In some embodiments, the binding peptide can be cyclized through a peptide bond, an alkyl bond, an alkenyl bond, an ester bond, a thioester bond, an ether bond, a thioether bond, a phosphate ether bond, an azo bond, a C-N-C bond, a C=N-C bond, a C=N-O bond, an amide bond, a lactam bridge, a carbamoyl bond, a urea bond, a thiourea bond, an amine bond and/or a thioamide bond. In a specific embodiment, the binding peptide can be cyclized through a thioether bond.

Except for the amino acid residues at the N-terminus and the C-terminus which are cyclized in the above-mentioned manner, the other amino acid residues in the binding peptide are connected by peptide bonds.

In some embodiments, the binding peptide may have a (hetero) aromatic or (hetero) alicyclic moiety. In some embodiments, the binding peptide may have a trisubstituted (hetero) aromatic or (hetero) alicyclic moiety. In a specific embodiment, the binding peptide has a 1,3,5-trimethylbenzene moiety. The binding peptide can be obtained by reacting and coupling the amino acid residues (e.g., Cys) at the N-terminus and C-terminus of a linear peptide with 1,3,5-tris(bromomethyl)benzene.

In some embodiments, the cyclic peptide may have a structure of Formula I,

CX ₁ -X ₂ -X ₃ -X ₄ -X ₅ -C(I),

Wherein X ₁ -X ₅ can be selected from any amino acid residues.

In some embodiments, the amino acid residues of _Xi to _X5 are each independently selected from phenylalanine (Phe), proline (Pro), glutamine (Gln), threonine (Thr), cysteine (Cys), glutamic acid (Glu), alanine (Ala), glycine (Gly), valine (Val), leucine (Leu), isoleucine (Ile), tryptophan (Trp), tyrosine (Tyr), aspartic acid (Asp), asparagine (Asn), lysine (Lys), methionine (Met), serine (Ser), histidine (His), or derivatives thereof.

In some embodiments, _X1 can be an aromatic amino acid residue, for example, can be selected from Phe, Tyr or Trp, preferably Phe. In some embodiments, _X2 and _X3 can each independently be an uncharged polar amino acid residue, for example, can be selected from Ser, Thr, Gln, Asn, Met or Cys. In a preferred embodiment, _X2 can be Gln. In a preferred embodiment, _X3 can be Thr. In some embodiments, _X4 and _X5 can each independently be an uncharged non-polar amino acid residue, for example, can be selected from Gly, Ala, Pro, Val, Leu or Ile. In a preferred embodiment, _X4 can be Pro. In a preferred embodiment, _X5 can be Pro.

In a specific embodiment, the cyclic peptide may have an amino acid sequence of Cys-Phe-Gln-Thr-Pro-Pro-Cys.

In some embodiments, any amino acid residue of the cyclic peptide may have one or more modifications. In some embodiments, the N-terminal and/or C-terminal residues of the cyclic peptide may have one or more modifications. In some embodiments, the N-terminal and/or C-terminal residues of the cyclic peptide may have a modification of (C ₁ -C ₆ alkyl)-NH-(C＝O) _n -, wherein n is an integer selected from 1 to 5. In some embodiments, the amino acid residue at the N-terminal of the cyclic peptide has modification.

In specific embodiments, the cyclic peptide may have one or more of the following structures (the attachment point to the chelating group is not shown):

In some embodiments, the cyclic peptide is linked to the chelating group via a thioether bond.

In some embodiments, the radionuclide may be one or more of ¹⁸ F, ⁶⁸ Ga, ⁶⁴ Cu, and ¹⁷⁷ Lu.

In specific embodiments, the conjugate may have one or more of the following structures (nuclides not shown):

In some embodiments, the conjugate may be one or more selected from ¹⁸ F-(NOTA)-FAP-NUR, ¹⁸ F-(NOTA)-FAP-NOX, ⁶⁴ Cu-(NOTA)-FAP-NOX, ¹⁷⁷ Lu-(DOTA)-FAP-DOX, ⁶⁸ Ga-(DOTA)-FAP-DOX.

According to yet another aspect of the present disclosure, a pharmaceutical composition is provided, which includes the above-mentioned conjugate of the present disclosure and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition may further include additional therapeutic agents and/or imaging agents.

According to yet another aspect of the present disclosure, a kit for targeted medical imaging is provided, which comprises the conjugate labeled with a radionuclide of the present disclosure.

According to yet another aspect of the present disclosure, a method for imaging and/or treating a diseased area using the conjugate labeled with a radionuclide of the present disclosure is provided.

According to another aspect of the present disclosure, provided is a use of the conjugate labeled with a radionuclide of the present disclosure in preparing a lesion imaging agent.

According to another aspect of the present disclosure, provided is a use of the conjugate labeled with a radionuclide of the present disclosure in preparing a method for treating or preventing a disease.

In some embodiments, the lesions include diseases or conditions associated with fibroblast activation protein, such as tumors and/or inflammation. In some embodiments, the tumors may include, but are not limited to, breast cancer, ovarian cancer, lung cancer, colorectal cancer, prostate cancer, lung cancer, fibrosarcoma, bone and connective tissue sarcoma, renal cell carcinoma, gastric cancer, liver cancer, brain tumor, bladder cancer, colorectal cancer, pancreatic cancer, or melanoma. In some embodiments, the inflammation may include, but is not limited to, osteoarthritis, rheumatoid arthritis, granulation tissue, liver fibrosis, pulmonary fibrosis, or cirrhosis.

In some embodiments, the imaging is performed in vivo. In some embodiments, the imaging can be positron emission tomography (PET) and/or X-ray computed tomography (CT) imaging.

In some embodiments, the lesion can be diagnosed by imaging the lesion site using the radionuclide-labeled conjugate of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 schematically shows the chemical structures of NOTA-FAP-NUR, NOTA-FAP-NOX, DOTA-FAP-DOX, DOTA-FAPI-04, and DOTA-FAP-2286.

FIG. 2 shows the cell binding affinity of NOTA-FAP-NUR and its in vitro stability after radionuclide ¹⁸ F labeling.

FIG3 shows the cell binding affinity of NOTA-FAP-NOX and its in vitro stability after radionuclide ⁶⁴ Cu labeling.

FIG. 4 shows the uptake and excretion results of Al ¹⁸ F-FAP-NUR and ⁶⁸ Ga-FAP-2286 by HT1080-FAP cells.

FIG. 5 shows the results of uptake and excretion of Al ¹⁸ F-FAP-NOX by HT1080-FAP cells.

FIG. 6 shows the results of uptake and excretion of ⁶⁴ Cu-FAP-NOX and ⁶⁴ Cu-FAPI-04 by A549-FAP cells.

FIG. 7 shows the results of uptake and excretion of ¹⁷⁷ Lu-FAP-DOX by HT1080-FAP cells.

FIG. 8 shows the biodistribution results of A1 ¹⁸ F-FAP-NUR in normal KM mice and 293T-FAP tumor-bearing mice.

FIG. 9 shows the biodistribution results of Al ¹⁸ F-FAP-NOX in 293T-FAP tumor-bearing mice.

FIG. 10 shows the biodistribution results of ⁶⁴ Cu-FAP-NOX and ⁶⁴ Cu-FAPI-04 in 293T-FAP tumor-bearing mice and normal KM mice.

FIG. 11 shows the tumor-to-non-target organ uptake ratios of ⁶⁴ Cu-FAP-NOX and ⁶⁴ Cu-FAPI-04 in 293T-FAP tumor-bearing mice.

FIG. 12 shows the biodistribution results of ¹⁷⁷ Lu-FAP-DOX in 293T-FAP tumor-bearing mice.

FIG. 13 shows small animal PET/CT results of A1 ¹⁸ F-FAP-NUR and ⁶⁸ Ga-FAP-2286 in 293T-FAP tumor-bearing mice.

FIG. 14 shows the small animal PET/CT results of A1 ¹⁸ F-FAP-NOX in 293T-FAP tumor-bearing mice.

FIG. 15 shows the small animal PET/CT results of ⁶⁴ Cu-FAP-NOX, ⁶⁴ Cu-FAPI-04, and ⁶⁸ Ga-FAP-2286 in 293T-FAP tumor-bearing mice.

FIG. 16 shows the small animal PET/CT results of ⁶⁸ Ga-FAP-DOX in 293T-FAP tumor-bearing mice.

Detailed ways

It is reported that the retention time of ⁶⁸ Ga-FAP-2286, a small molecule inhibitor labeled with ⁶⁸ Ga, in tumors is greatly prolonged, but its liver uptake is higher than that of ⁶⁸ Ga-FAPI-46 in clinical imaging. Therefore, the pharmacokinetic properties need to be further optimized. The present disclosure has found a radionuclide-labeled cyclic peptide with significantly improved properties such as targeting and uptake through screening, which further optimizes the pharmacokinetic properties while retaining the advantage of long-term tumor retention.

The cyclic peptide provided by the present disclosure is used as a probe, and the specific targeting of FAP is significantly improved. Compared with the uptake of normal cells, the uptake of the cyclic peptide provided by the present disclosure by tumor cells is significantly increased, and the excretion rate is reduced, thereby prolonging the retention time of the cyclic peptide labeled with radionuclide in the body or in tumor cells. Therefore, the labeled cyclic peptide of the present disclosure is suitable for delayed imaging. Moreover, the conjugate of the present disclosure significantly improves the overall quality of the image.

definition

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

The term "positron emission tomography (PET)," as used herein, is a nuclear imaging technique (also called molecular imaging) that can visualize metabolic processes in the body. The basis of PET imaging is that the technique detects pairs of gamma rays emitted indirectly by positron-emitting radionuclides (also called radiopharmaceuticals, radionuclides, or radiotracers). The tracer is injected into a vein with a biologically active molecule, usually a sugar used for cellular energy. The PET system's sensitive detectors capture the gamma-ray radiation inside the body and use software to map and triangulate the emission sources, creating a three-dimensional computed tomography image of the tracer concentration in the body.

The term "X-ray computed tomography (CT)" used in this article can be used to perform tomographic imaging of biological tissue and engineering material samples without destroying the samples, using the sample's absorption characteristics of radiation energy, thereby obtaining structural information inside the samples. CT images can reflect the fine structure inside the human body, and the grayscale distribution on the CT image reflects the distribution of the attenuation coefficient of the X-rays in the energy range of various materials inside the object to be tested.

In a specific embodiment, examples of chelating groups used to construct the conjugates of the present disclosure include DOTA and NOTA. DOTA, the structures of which are shown below:

In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

Unless otherwise noted, all starting materials and reagents are of standard commercial grade and are used without further purification or are readily prepared from such materials by conventional methods. Based on this disclosure, one skilled in the art of organic synthesis and solid phase synthesis will recognize that the starting materials and reaction conditions may be varied, including additional steps for producing the compounds encompassed by the present invention.

Abbreviations used in this article:

Fmoc-amino acid-OH: Fmoc-AA-OH;

Fmoc-Cys(Trt)-OH;

Fmoc-Phe-OH: Fmoc-Phe-OH;

Fmoc-N-trityl-L-glutamine: Fmoc-Gln(Trt)-OH;

Fmoc-O-tert-butyl-L-threonine: Fmoc-Thr(tBu)-OH;

Fmoc-L-proline: Fmoc-Pro-OH;

Fmoc-Pro(4,4-F2)-OH: 4,4-difluoro-L-proline;

2-Chlorotrityl chloride: CTC;

N,N-diisopropylethylamine: DIPEA;

1-Hydroxybenzotriazole: HOBT;

1,3-Diisopropylcarbodiimide: DIC;

Trifluoroacetic acid: TFA;

Piperidine: Pip;

N,N-dimethylformamide:DMF;

Dichloromethane: DCM.

Example 1. Preparation of NOTA-FAP-NUR

The cyclic peptide was synthesized on solid phase according to the route shown below.

1. Linear peptide solid phase synthesis: Weigh 0.445 g of 2-chlorotrityl chloride (CTC) resin (loaded at 0.45 μmol/g) with weighing paper into a peptide solid phase synthesis reaction tube, add DCM (5 ml) and DMF (5 ml) to swell the resin, and drain after 20 minutes. Rinse with DMF and DCM 5 times, add Fmoc-Cys(Trt)-OH (0.585 g, 1 mmol) and DIPEA (333 μl) in DMF solution into the reaction tube, and fix the reaction tube in a constant temperature oscillator for 4 hours at room temperature. Wash with DCM and DMF 5 times each, drain and add 20% piperidine in DMF solution (10 ml), repeat 2 times, 10 minutes each time, so as to remove the Fmoc protecting group on the amino acid, and then wash with DMF and DCM 5 times each. Then, the prepared DMF solution of Fmoc-amino acid-OH (1 mmol, 5 equivalents), HOBT (1 mmol, 5 equivalents), and DIC (1 mmol, 5 equivalents) was added to the reaction tube, and the reaction tube was fixed in a constant temperature oscillator and oscillated at room temperature for 2 hours. After the reaction was completed, it was rinsed with DMF and DCM 5 times. Repeat the above steps to couple amino acids Fmoc-Phe-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, and Fmoc-Cys(Trt)-OH in sequence.

2. Modification of the nitrogen terminus of the linear peptide: After all amino acids are coupled, a linear peptide-resin complex is obtained, and a 10 ml DMF solution of prepared butylcarbamic acid (1 mmol, 5 equivalents), HOBT (1 mmol, 5 equivalents), and DIC (1 mmol, 5 equivalents) is added to the reaction tube. The reaction tube is fixed in a constant temperature oscillator and shaken at room temperature for 2 hours. After the reaction is completed, it is rinsed with DMF and DCM 5 times.

3. Cutting and purification of linear peptides: After the modification of the nitrogen end of the linear peptide is completed, the modified linear peptide-resin complex is obtained, which is evaporated with anhydrous ether, and 10 ml of a mixed solution of TFA/water (95:5, volume/volume) is added, and placed on an orbital shaker for 4 hours at room temperature. The resin is filtered, and the resin is washed with new TFA 2-3 times, the filtrate is combined, the filtrate is precipitated with ice anhydrous ether, and the precipitate is washed with ice anhydrous ether 3 times, and finally the precipitate is placed in a vacuum drying kettle and dried at room temperature for 24 hours to obtain a crude linear peptide.

4. Cyclization of linear peptide: The crude linear peptide was dissolved in 20 ml of a 1:1 mixture of ethanol and acetonitrile. 140 μl of N,N-diisopropylethylamine and 1,3,5-tri(bromoethyl)benzene (0.26 mmol, 1.3 equivalents compared to the initial resin load) were added to this mixture. The solution was stirred for 1 hour, and then 171.2 mg of 2-aminoethanethiol (2.2 mmol, 11 equivalents compared to the initial resin load) was added. After 1 hour, the solvent was evaporated and the remaining solvent was dissolved in acetonitrile and water (20 ml, 1:1 mixture, containing 100 μl of TFA). The lyophilized crude product was purified by reverse phase high performance liquid chromatography (RP-HPLC) to obtain the cyclic peptide intermediate.

5. Cyclic peptide coupling with bifunctional chelating group: To the intermediate solution in 450 μl DMSO, 8 μl DIPEA was added to adjust the pH to about 7.5-8, and then 30.75 mg NOTA-NHS (40.5 μmol, 1.5 equivalents compared to the cyclic peptide intermediate) in 200 μl DMSO was added. During the reaction monitored by LC-MS, 8 μl DIPEA was added three times to readjust the pH to the starting value. After the amino acid coupling was completed, the reaction solution was purified by HPLC to obtain the target product NOTA-FAP-NUR. LC-MS: accurate mass 1383.570 (calculated value 1383.800). c (MW = 1384.650).

Example 2. Preparation of NOTA-FAP-NOX

The cyclic peptide was synthesized on solid phase according to the route shown below.

1. Linear peptide solid phase synthesis: Weigh 0.445 g of 2-chlorotrityl chloride (CTC) resin (loaded at 0.45 μmol/g) with weighing paper into a peptide solid phase synthesis reaction tube, add DCM (5 ml) and DMF (5 ml) to swell the resin, and drain after 20 minutes. Rinse with DMF and DCM 5 times, add Fmoc-Cys(Trt)-OH (0.585 g, 1 mmol) and DIPEA (333 μl) in DMF solution into the reaction tube, and fix the reaction tube in a constant temperature oscillator for 4 hours at room temperature. Wash with DCM and DMF 5 times each, drain and add 20% piperidine in DMF solution (10 ml), repeat 2 times, 10 minutes each time, so as to remove the Fmoc protecting group on the amino acid, and then wash with DMF and DCM 5 times each. Then, the prepared DMF solution of Fmoc-amino acid-OH (1 mmol, 5 equivalents), HOBT (1 mmol, 5 equivalents), and DIC (1 mmol, 5 equivalents) was added to the reaction tube, and the reaction tube was fixed in a constant temperature oscillator and oscillated at room temperature for 2 hours. After the reaction was completed, it was rinsed with DMF and DCM 5 times. Repeat the above steps to couple the amino acids Fmoc-Phe-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, and Fmoc-Cys(Trt)-OH in sequence.

2. Modification of the nitrogen terminus of the linear peptide: After all amino acids are coupled, a linear peptide-resin complex is obtained, and the prepared 2-oxo-2-propylamino-acetic acid (1 mmol, 5 equivalents), HOBT (1 mmol, 5 equivalents), and DIC (1 mmol, 5 equivalents) in a volume of 10 ml of DMF solution are added to the reaction tube. The reaction tube is fixed in a constant temperature oscillator and shaken at room temperature for 2 hours. After the reaction is completed, it is rinsed with DMF and DCM 5 times.

3. Cutting and purification of linear peptides: After the modification of the nitrogen end of the linear peptide is completed, the modified linear peptide-resin complex is obtained, which is evaporated with anhydrous ether, and 10 ml of a mixed solution of TFA/water (95:5, volume/volume) is added, and placed on an orbital shaker for 4 hours at room temperature. The resin is filtered, and the resin is washed with new TFA 2-3 times, the filtrate is combined, the filtrate is precipitated with ice anhydrous ether, and the precipitate is washed with ice anhydrous ether 3 times, and finally the precipitate is placed in a vacuum drying kettle and dried at room temperature for 24 hours to obtain a crude linear peptide.

4. Cyclization of linear peptide: The crude linear peptide was dissolved in 20 ml of a 1:1 mixture of ethanol and acetonitrile. 140 μl of N,N-diisopropylethylamine and 1,3,5-tri(bromoethyl)benzene (0.26 mmol, 1.3 equivalents compared to the initial resin load) were added to this mixture. The solution was stirred for 1 hour, and then 171.2 mg of 2-aminoethanethiol (2.2 mmol, 11 equivalents compared to the initial resin load) was added. After 1 hour, the solvent was evaporated and the remaining solvent was dissolved in acetonitrile and water (20 ml, 1:1 mixture, containing 100 μl of TFA). The lyophilized crude product was purified by reverse phase high performance liquid chromatography (RP-HPLC) to obtain the cyclic peptide intermediate.

5. Cyclic peptide coupling with bifunctional chelating group: To the intermediate solution in 450 μL DMSO, 8 μL DIPEA was added to adjust the pH to about 7.5-8, and then 30.75 mg NOTA-NHS (40.5 μmol, 1.5 equivalents compared to the cyclic peptide intermediate) in 200 μL DMSO was added. During the reaction monitored by LC-MS, 8 μL DIPEA was added three times to readjust the pH to the starting value. After the amino acid coupling was completed, the reaction solution was purified by HPLC to produce the target compound NOTA-FAP-NOX. LC-MS: accurate mass 1369.590 (calculated value 1370.200). c (MW = 1370.670).

Example 3. Preparation of DOTA-FAP-DOX

The cyclic peptide was synthesized on solid phase according to the route shown below.

1. Linear peptide solid phase synthesis: Weigh 0.445 g of 2-chlorotrityl chloride (CTC) resin (loaded at 0.45 μmol/g) with weighing paper into a peptide solid phase synthesis reaction tube, add DCM (5 ml) and DMF (5 ml) to swell the resin, and drain after 20 minutes. Rinse with DMF and DCM 5 times, add Fmoc-Cys(Trt)-OH (0.585 g, 1 mmol) and DIPEA (333 μl) in DMF solution into the reaction tube, and fix the reaction tube in a constant temperature oscillator for 4 hours at room temperature. Wash with DCM and DMF 5 times each, drain and add 20% piperidine in DMF solution (10 ml), repeat 2 times, 10 minutes each time, so as to remove the Fmoc protecting group on the amino acid, and then wash with DMF and DCM 5 times each. Then, the prepared DMF solution of Fmoc-amino acid-OH (1 mmol, 5 equivalents), HOBT (1 mmol, 5 equivalents), and DIC (1 mmol, 5 equivalents) was added to the reaction tube, and the reaction tube was fixed in a constant temperature oscillator and oscillated at room temperature for 2 hours. After the reaction was completed, it was rinsed with DMF and DCM 5 times. Repeat the above steps to couple amino acids Fmoc-Phe-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, and Fmoc-Cys(Trt)-OH in sequence.

2. Modification of the nitrogen terminus of the linear peptide: After all amino acids are coupled, a linear peptide-resin complex is obtained, and the prepared 2-oxo-2-propylamino-acetic acid (1 mmol, 5 equivalents), HOBT (1 mmol, 5 equivalents), and DIC (1 mmol, 5 equivalents) in a volume of 10 ml of DMF solution are added to the reaction tube. The reaction tube is fixed in a constant temperature oscillator and shaken at room temperature for 2 hours. After the reaction is completed, it is rinsed with DMF and DCM 5 times.

3. Cutting and purification of linear peptides: After the modification of the nitrogen end of the linear peptide is completed, the modified linear peptide-resin complex is obtained, which is evaporated with anhydrous ether, and 10 ml of a mixed solution of TFA/water (95:5, volume/volume) is added, and placed on an orbital shaker for 4 hours at room temperature. The resin is filtered, and the resin is washed with new TFA 2-3 times, the filtrate is combined, the filtrate is precipitated with ice anhydrous ether, and the precipitate is washed with ice anhydrous ether 3 times, and finally the precipitate is placed in a vacuum drying kettle and dried at room temperature for 24 hours to obtain a crude linear peptide.

4. Cyclization of linear peptide: The crude linear peptide was dissolved in 20 ml of a 1:1 mixture of ethanol and acetonitrile. 140 μl of N,N-diisopropylethylamine and 1,3,5-tri(bromoethyl)benzene (0.26 mmol, 1.3 equivalents compared to the initial resin load) were added to this mixture. The solution was stirred for 1 hour, and then 171.2 mg of 2-aminoethanethiol (2.2 mmol, 11 equivalents compared to the initial resin load) was added. After 1 hour, the solvent was evaporated and the remaining solvent was dissolved in acetonitrile and water (20 ml, 1:1 mixture, containing 100 μl of TFA). The lyophilized crude product was purified by reverse phase high performance liquid chromatography (RP-HPLC) to obtain the cyclic peptide intermediate.

5. Cyclic peptide coupling with bifunctional chelating group: To the intermediate solution in 450 μL DMSO, 8 μL DIPEA was added to adjust the pH to about 7.5-8, and then 30.75 mg DOTA-NHS (40.5 μmol, 1.5 equivalents compared to the cyclic peptide intermediate) in 200 μL DMSO was added. During the reaction monitored by LC-MS, 8 μL DIPEA was added three times to readjust the pH to the starting value. After the reaction was completed, the reaction solution was purified by HPLC to produce the target compound DOTA-FAP-DOX. LC-MS: accurate mass 1484.610 (calculated value 1485.100). c (MW = 1485.750).

Example 4. Synthesis of radionuclide-labeled FAP-NUR, FAP-NOX, FAP-DOX and FAP-2286

1. ¹⁸ F-FAP-NUR:

The nuclear reaction ¹⁸ O(p,n) ¹⁸ F was carried out using a GE PET trace 860 cyclotron to generate [ ¹⁸ F]F ^- . The ¹⁸ F fluorine source was transferred to a Sep-Park Light QMA anion exchange column in the form of ¹⁸ F/[ ¹⁸ O]H ₂ O and captured by QMA. Subsequently, ¹⁸ F was eluted into the reaction vessel with 0.5 mL of 0.9% NaCl aqueous solution to obtain 600 mCi ¹⁸ F. Drying was completed by azeotropic distillation using 1 mL of acetonitrile. After the drying process is completed, a mixed solution containing 350 μL acetonitrile, 250 μL 0.2M NaOAc buffer (pH 4.0), 7.0 μL 10.0mM AlCl ₃ solution in 0.2M NaOAc buffer and 100 μL NOTA-FAP-NUR (chemical structure as shown in Figure 1) (1mg/mL) dissolved in 0.125M L-ascorbic acid is added to the reaction vessel. After heating at 100°C for 15 minutes, the mixture is cooled to 70°C and diluted with 10mL water. Next, the diluted solution is transferred to a C ₁₈ column (pre-treated with 10mL ethanol and 10mL water). Then, the filter cartridge is washed with 10mL water and 5mL water in turn. The product is eluted into a collection bottle with 1.5mL50% EtOH solution (pre-added with 6.5mL 0.9% sterile NaCl for injection, containing 10mg/mL L-ascorbic acid at the same time). 8 mL of the final product solution was transferred to a sterile bottle by sterilization filtration using a Millex GV 33 mm 0.22 μm filter, and the radioactivity was 130 mCi. Al ¹⁸ F-labeled FAP-NUR (denoted as Al ¹⁸ F-FAP-NUR) was successfully prepared.

2. ¹⁸ F-FAP-NOX:

(1) A GE PET trace 860 cyclotron was used to perform a nuclear reaction ¹⁸ O(p,n) ¹⁸ F reaction to generate [ ¹⁸ F]F ^- . The ¹⁸ F fluorine source was transferred to a Sep-Park Light QMA anion exchange column in the form of ¹⁸ F/[ ¹⁸ O]H ₂ O via a channel and captured by QMA. Subsequently, ¹⁸ F was eluted into a reaction vessel with 0.5 mL of 0.9% NaCl aqueous solution to obtain 560 mCi ¹⁸ F. Drying was accomplished by azeotropic distillation using 1 mL of acetonitrile. After the drying process is completed, a mixed solution containing 350 μL acetonitrile, 250 μL 0.2M NaOAc buffer (pH 4.0), 7.0 μL 10.0mM AlCl ₃ solution in 0.2M NaOAc buffer and 100 μL NOTA-FAP-NOX (1mg/mL, chemical structure as shown in Figure 1) dissolved in 0.125M L-ascorbic acid is added to the reaction vessel. After heating at 100°C for 15 minutes, the mixture is cooled to 70°C and diluted with 10mL water. Next, the diluted solution is transferred to a C ₁₈ column (pre-treated with 10mL ethanol and 10mL water). Then, the filter cartridge is washed with 10mL water and 5mL water in turn. The product is eluted into a collection bottle with 1.5mL 50% EtOH solution (pre-added with 6.5mL 0.9% sterile NaCl for injection, containing 10mg/mL L-ascorbic acid at the same time). 8 mL of the final product solution was transferred to a sterile bottle by sterile filtration using a Millex GV 33 mm 0.22 μm filter, and the radioactivity was 115 mCi. Al ¹⁸ F-labeled FAP-NOX (denoted as Al ¹⁸ F-FAP-NOX) was successfully prepared.

(2) ⁶⁴ CuCl ₂ was stored in 0.05 M HCl solution. Take 50 μL of NOTA-FAP-NOX (1 mg/mL) dissolved in 0.125 M L-ascorbic acid and mix thoroughly with 1 mL of sodium L-ascorbate (0.8 M). Add 20 mCi ⁶⁴ CuCl ₂ to the above mixed solution at pH 4. Incubate the reaction mixture at 50 °C for 10 min. The final ⁶⁴ Cu-labeled FAP-NOX (denoted as ⁶⁴ Cu-FAP-NOX) was obtained, and the final radioactivity was determined to be 19.5 mCi. Quality control of the radioligand was performed using HPLC. The radioligand was used for in vitro and in vivo experiments without further purification.

3. ⁶⁸ Ga-FAP-DOX:

(1) ^{The 68} Ga required for labeling was obtained using the ⁶⁸ Ge/ ⁶⁸ Ga generator of iThemba LABS in South Africa. 100 μg of the precursor DOTA-FAP-DOX (chemical structure shown in Figure 1) was dissolved in 100 μL of L-ascorbic acid (0.125 M) in advance to prepare a precursor solution with a concentration of 1 μg/μL. 6.0 mL of HCl (0.6 M) was used to elute the ⁶⁸ Ge/ ⁶⁸ Ga generator. 1.5 mL of the highest intermediate activity GaCl ₂ solution (~740 MBq) was collected and added to a 10 mL reaction tube containing 50 μL of DOTA-FAP-DOX (50 μg, 36 nmol) precursor solution. 0.9 mL of sodium acetate buffer (1 M, pH 5.2) was added to the mixed solution to adjust the pH value of the mixed solution to 3-3.5. The reaction tube was then placed in a metal thermostat and incubated at 95°C for 15 minutes, the obtained product was diluted and cooled, and then the product solution in the reaction tube was subjected to solid phase extraction through a _C18 column, the product was adsorbed by the _C18 column, 10 mL of injection water was taken to rinse the _C18 column, and the residual organic solvent and other impurities on the _C18 column were removed. The final product adsorbed by the _C18 column was eluted into the product bottle with 1.5 mL of 50% ethanol, and diluted with physiological saline until the ethanol content in the product was less than 10%, and the final radioactivity was determined to be 560 MBq. The final ⁶⁸ Ga-labeled FAP-DOX (referred to as ⁶⁸ Ga-FAP-DOX) was obtained.

(2) Take 100 μg of precursor DOTA-FAP-DOX and dissolve it in 100 μL L-ascorbic acid (0.125 M) to prepare a precursor solution with a concentration of 1 μg/μL. Add 20 μL DOTA-FAP-DOX (20 μg, 13.5 nmol), 20 μL sodium acetate buffer (2 M, pH 5.5) and 200 μL deionized water to a 10 mL reaction tube. Take 10 mCi of ¹⁷⁷ LuCl3 solution and mix it evenly with the solution in the reaction tube. Place the reaction tube in a metal constant temperature oscillator and incubate at 70 ° C for 30 minutes. The final ¹⁷⁷ Lu labeled FAP-DOX (referred to as ¹⁷⁷ Lu-FAP-DOX) is obtained, and the final radioactivity is measured to be 9.8 mCi. Then use HPLC for quality control, and the radiochemical purity should be greater than 95%. The radioligand can be used for in vitro and in vivo experiments without further purification.

4. ⁶⁸ Ga-FAP-2286:

(1) ^{The 68} Ga required for labeling was obtained using the ⁶⁸ Ge/ ⁶⁸ Ga generator of iThemba LABS in South Africa. 50 μg of the precursor DOTA-FAP-2286 (chemical structure shown in Figure 1) was dissolved in 50 μL of L-ascorbic acid (0.125 M) in advance to prepare a precursor solution with a concentration of 1 μg/μL. 6.0 mL of HCl (0.6 M) was used to elute the ⁶⁸ Ge/ ⁶⁸ Ga generator. 1.5 mL of the highest intermediate activity GaCl ₂ solution (~740 MBq) was collected and added to a 10 mL reaction tube containing 50 μL of DOTA-FAP-2286 (50 μg, 36 nmol) precursor solution. 0.9 mL of sodium acetate buffer (1 M, pH 5.2) was added to the mixed solution, and the pH value of the mixed solution was adjusted to 3-3.5. The reaction tube was then placed in a metal thermostat and incubated at 95°C for 15 minutes, the obtained product was diluted and cooled, and then the product solution in the reaction tube was subjected to solid phase extraction through a _C18 column, the product was adsorbed by the _C18 column, 10 mL of injection water was taken to rinse the _C18 column, and the organic solvent and other impurities remaining on the _C18 column were removed. The final product adsorbed by the _C18 column was eluted into the product bottle with 1.5 mL of 50% ethanol, and diluted with physiological saline until the ethanol content in the product was less than 10%, and the final radioactivity was determined to be 540 MBq. The final ⁶⁸ Ga-labeled FAP-2286 (referred to as ⁶⁸ Ga-FAP-2286) was obtained.

(2) 100 μg of precursor DOTA-FAP-2286 was dissolved in 100 μL L-ascorbic acid (0.125 M) to prepare a precursor solution with a concentration of 1 μg/μL. 20 μL DOTA-FAP-2286 (20 μg, 13.5 nmol, chemical structure shown in Figure 1), 20 μL sodium acetate buffer (2 M, pH 5.5) and 200 μL deionized water were added to a 10 mL reaction tube. 10 mCi of ¹⁷⁷ LuCl3 solution was mixed evenly with the solution in the reaction tube, and the reaction tube was placed in a metal constant temperature oscillator and incubated at 70 ° C for 30 minutes. ¹⁷⁷ Lu-labeled FAP-2286 (referred to as ¹⁷⁷ Lu-FAP-2286) was successfully prepared, and the final radioactivity was determined to be 9.6 mCi. HPLC was then used for quality control, and the radiochemical purity should be greater than 95%. The radioligand can be used for in vitro and in vivo experiments without further purification.

5. ⁶⁴ Cu-FAPI-04

⁶⁴ CuCl ₂ was stored in 0.05 M HCl solution. Take 50 μL of DOTA-FAPI-04 (1 mg/mL) dissolved in 0.125 M L-ascorbic acid, mix thoroughly with 1 mL of L-sodium ascorbate (0.8 M), and add 21 mCi ⁶⁴ CuCl ₂ to the above mixed solution at a pH of 4. Incubate the reaction mixture at 50 °C for 10 minutes. The final ⁶⁴ Cu-labeled FAPI-04 (denoted as ⁶⁴ Cu-FAPI-04) was obtained, and the final radioactivity was determined to be 20.4 mCi. Quality control of the radioligand was performed using HPLC. The radioligand was used for in vitro and in vivo experiments without further purification.

Example 5. Radiolabeling, in vitro stability and partition coefficient

Steps:

(1) In vitro stability assay: The corresponding radioactive drug (100 μCi, 10 μL) was added to 90 μL of phosphate buffered saline (PBS, 0.01 M, pH = 7.4), and the mixture was incubated at 37°C for 4 hours. The mixture was directly placed in a radio-HPLC (Thermo Fisher Scientific, USA) for analysis. The radiochemical purity was automatically analyzed by radio-HPLC.

(2) Determination of distribution coefficient: 100 μL of the corresponding radioactive drug was added to a mixed solution of equal volumes of n-octanol and PBS (0.01 M, pH = 7.4) with a total volume of 5 mL, mixed evenly, and centrifuged at 12000×rpm for 5 minutes (n = 3). Then, the mixture was centrifuged, and equal volumes of the supernatant (n-octanol containing the radioactive drug) and the bottom liquid (PBS solution containing the radioactive drug) were taken, counted using an automatic gamma counter (Hidex AMG, Hidex Oy, Finland), and the average LogP value was calculated.

result:

Al ¹⁸ F-FAP cyclic peptide:

(1) In Example 1, Al and ¹⁸ F were chelated to successfully prepare Al ¹⁸ F labeled FAP-NUR and FAP-NOX. The yields without attenuation correction were determined by HPLC and were 21.67% and 20.54%, respectively, with radiochemical purities exceeding 95%. The total synthesis time from ¹⁸ F transfer to obtaining the purified product was approximately 44 minutes.

(2) The lipid-water distribution coefficients (LogP) of Al ¹⁸ F-FAP-NUR and ⁶⁸ Ga-FAP-2286 were -2.45±0.07 (n=3) and -2.61±0.01 (n=3), respectively.

(3) As shown in FIG. 2 b , the stability of Al ¹⁸ F-FAP-NUR remained above 90% after 4 hours in vitro.

⁶⁴ Cu-FAP-NOX:

(1) As shown in FIG3 b , no obvious detachment of the chelating agent was observed after ⁶⁴ Cu-FAP-NOX was incubated in PBS and serum for 24 hours, and the radiochemical purity was maintained at more than 95%.

(2) The n-octanol/PBS partition coefficients (LogD _7.4 ) of ⁶⁴ Cu-FAP-NOX, ⁶⁴ Cu-FAPI-04 and ⁶⁸ Ga-FAP-2286 are -3.07±0.04, -2.87±0.02 and -2.68±0.05, respectively.

(3) As shown in FIG. 3 b , the stability of ⁶⁴ Cu-FAP-NOX remained above 90% after 4 hours in vitro.

⁶⁸ Ga-FAP cyclic peptide:

In Example 1, ⁶⁸ Ga-labeled FAP-DOX and FAP-NOX were successfully prepared. The yields without attenuation correction were determined by HPLC and were 75.68% and 70.54%, respectively, with a radiochemical purity of over 95%.

¹⁷⁷ Lu-FAP cyclic peptide:

In Example 1, ¹⁷⁷ Lu-labeled FAP-DOX and FAP-2286 were successfully prepared. The yields without attenuation correction were determined by HPLC and were 98% and 96%, respectively, and the radiochemical purity exceeded 95%.

Example 6. Cell experiment

Steps:

(1) Cell competition binding assay

1) Cell competition binding experiment of NOTA-FAP-NUR

¹⁷⁷ Lu-FAP-2286 (74 kBq/tube, 180 μL/tube), different amounts of competitive non-radioactive ligand (NOTA-FAP-NUR or DOTA-FAP-2286) and DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) were mixed, and the mixture was added to a glass test tube (3×10 ⁴ cells/tube, 180 μL/tube) inoculated with 293T cells (293T-FAP cells) overexpressing human FAP (Gene ID: 2191), so that the concentration of the competitive non-radioactive ligand in the final solution was 7 different concentrations (1×10 ^-5 to 1×10 ^-11 M, with a concentration gradient of 10 times between each concentration). The glass test tube was placed in a 37°C water bath and incubated for 1 hour. The cells were then harvested using a Brandel M-48T cell harvester (Biomedical Research and Development Laboratories, Inc., Gaithersburg, MD, USA), and the radioactivity of the cells was measured using a gamma counter.

2) Cell competition binding experiment of NOTA-FAP-NOX

A549 cells (A549-FAP cells) overexpressing human FAP (Gene ID: 2191) were seeded in 24-well plates 24 hours in advance (8×10 ⁴ cells/well), and the medium was removed and the cells were washed twice with PBS before adding non-radioactive competitive ligands (NOTA-FAP-NOX/DOTA-FAP-DOX/DOTA-FAPI-04/DOTA-FAP-2286). Eight non-radioactive competitive ligands of different concentrations (ranging from 2×10 ^-5 to 2×10 ^-12 M, with a concentration gradient of 10 times between each concentration) dissolved in DMEM medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin) were mixed with ¹⁷⁷ Lu-FAP-2286, so that the final solution concentrations were 8 different concentrations (1×10 ^-5 to 1×10 ^-12 M, with a concentration gradient of 10 times between each concentration). The mixed solution was added to the well plate inoculated with A549-FAP cells, each well containing 74KBq ¹⁷⁷ Lu-FAP-2286, and incubated for 1 hour at 37°C. The cells were then washed twice with cold PBS, and finally lysed with 1M NaOH solution, the solution containing cell fragments was collected in a plastic tube, and the radioactivity of the cells was measured with a γ counter.

(2) Cell uptake experiment

1) Cellular uptake experiments of Al ¹⁸ F-FAP-NUR, Al ¹⁸ F-FAP-NOX and ⁶⁸ Ga-FAP-2286

HT1080 cells overexpressing human FAP (Gene ID: 2191) (HT1080-FAP cells) (5×10 ⁴ cells/well) or HT1080 cells (5×10 ⁴ cells/well) were seeded in 24-well plates 24 hours in advance. Before the experiment, the culture medium was removed and the cells were washed twice with PBS. Subsequently, serum-free DMEM basal medium (74 kBq/well, 1 mL/well) containing Al ¹⁸ F-FAP-NUR/ ⁶⁸ Ga-FAP-2286/Al ¹⁸ F-FAP-NOX was added to the well plate and incubated at 37°C for 5, 15, 30, 60 and 120 minutes. After reaching the designated time point, the culture medium containing the radioactive drug was immediately removed, the cells were washed twice with PBS, and finally the cells were lysed with 1M NaOH solution. The solution containing cell debris was collected in a plastic tube, and the radioactivity of the cells was measured with a γ counter.

2) Cellular uptake experiments of ⁶⁴ Cu-FAP-NOX and ⁶⁴ Cu-FAPI-04

A549-FAP cells (8×10 ⁴ cells/well) or A549 cells (8×10 ⁴ cells/well) were seeded in 24-well plates 24 hours in advance. Before the experiment, the culture medium was removed and the cells were washed twice with PBS. Subsequently, serum-free DMEM basal medium containing ⁶⁴ Cu-FAP-NOX/ ⁶⁴ Cu-FAPI-04 (74 kBq/well, 1 mL/well) was added to the well plate and incubated at 37°C for 0.5, 1, 2, 4, 6, 12, 24 and 48 hours. After reaching the designated time point, the culture medium containing the radioactive drug was immediately removed, the cells were washed twice with PBS, and finally the cells were lysed with 1M NaOH solution. The solution containing cell fragments was collected in a plastic tube, and the radioactivity of the cells was measured with a γ counter.

3) Cellular uptake experiment of ^177Lu -FAP-DOX

HT1080-FAP cells (5×10 ⁴ cells/well) were seeded in 24-well plates 24 hours in advance. Before the experiment, the culture medium was removed and the cells were washed twice with PBS. Subsequently, serum-free DMEM basal medium containing ¹⁷⁷ Lu-FAP-DOX (74 kBq/well, 1 mL/well) was added to the well plate and incubated at 37°C for 0.5, 1, 2, 4, 8, 24, 48 and 72 hours. After reaching the designated time point, the culture medium containing the radioactive drug was immediately removed, the cells were washed twice with PBS, and finally the cells were lysed with 1M NaOH solution, the solution containing cell fragments was collected in a plastic tube, and the radioactivity of the cells was measured with a γ counter.

(3) Cell inhibition experiment

1) Cell inhibition experiments of Al ¹⁸ F-FAP-NUR, Al ¹⁸ F-FAP-NOX, and ⁶⁸ Ga-FAP-2286

HT1080-FAP cells (5×10 ⁴ cells/well) were seeded in 24-well plates 24 hours in advance. Before the experiment, the culture medium was removed and the cells were washed twice with PBS. Subsequently, serum-free DMEM basal medium (74 kBq/well, 1 mL/well) containing Al ¹⁸ F-FAP-NUR/ ⁶⁸ Ga-FAP-2286/Al ¹⁸ F-FAP-NOX was added to the well plate. The radioactive medium contained non-radioactive ligand DOTA-FAP-2286 (3 μM/mL) and was incubated with the cells at 37°C for 120 minutes. After reaching the designated time point, the culture medium containing the radioactive drug was immediately removed, the cells were washed twice with PBS, and finally the cells were lysed with 1 M NaOH solution. The solution containing cell fragments was collected in a plastic tube, and the radioactivity of the cells was measured with a γ counter.

2) Cell inhibition experiments of ⁶⁴ Cu-FAP-NOX and ⁶⁴ Cu-FAPI-04

A549-FAP cells (8×10 ⁴ cells/well) were seeded in 24-well plates 24 hours in advance. Before the experiment, the culture medium was removed and the cells were washed twice with PBS. Subsequently, serum-free DMEM basal medium containing ⁶⁴ Cu-FAP-NOX/ ⁶⁴ Cu-FAPI-04 (74 kBq/well, 1 mL/well) was added to the well plate. The radioactive medium contained non-radioactive ligand DOTA-FAP-2286 (3 μM/mL) and was incubated with the cells at 37°C for 4 hours. After reaching the designated time point, the culture medium containing the radioactive drug was immediately removed, the cells were washed twice with PBS, and finally the cells were lysed with 1 M NaOH solution. The solution containing cell fragments was collected in a plastic tube, and the radioactivity of the cells was measured with a γ counter.

3) Cell inhibition experiment of ¹⁷⁷ Lu-FAP-DOX

HT1080-FAP cells (8×10 ⁴ cells/well) were seeded in 24-well plates 24 hours in advance. Before the experiment, the culture medium was removed and the cells were washed twice with PBS. Subsequently, serum-free DMEM basal medium containing ¹⁷⁷ Lu-FAP-DOX (74 kBq/well, 1 mL/well) was added to the well plate. The radioactive medium contained non-radioactive ligand DOTA-FAP-2286 (3 μM/mL) and was incubated with the cells at 37°C for 4 hours. After reaching the designated time point, the culture medium containing the radioactive drug was immediately removed, the cells were washed twice with PBS, and finally the cells were lysed with 1 M NaOH solution. The solution containing cell fragments was collected in a plastic tube, and the radioactivity of the cells was measured with a γ counter.

(4) Cell internalization experiment

1) Cellular internalization experiments of Al ¹⁸ F-FAP-NUR, Al ¹⁸ F-FAP-NOX, and ⁶⁸ Ga-FAP-2286

HT1080-FAP cells (5×10 ⁴ cells/well) were seeded in 24-well plates 24 hours in advance. Before the experiment, the culture medium was removed and the cells were washed twice with PBS. Subsequently, serum-free DMEM basal medium (74 kBq/well, 1 mL/well) containing Al ¹⁸ F-FAP-NUR/ ⁶⁸ Ga-FAP-2286/Al ¹⁸ F-FAP-NOX was added to the well plate and incubated with the cells at 37°C for 5, 15, 30, 60 and 120 minutes. After reaching the designated time point, the culture medium containing the radioactive drug was immediately removed, the cells were washed twice with PBS, and 1 mL of pre-cooled glycine-HCl (0.05 M, pH=2.8) solution was added to each well and incubated with the cells for 10 minutes. The incubated solution in each well was collected, the cells were washed twice with PBS, and the PBS solution after washing was collected and mixed with the incubation solution in the corresponding wells above for the determination of the radioactivity bound to the cell surface. Finally, the cells were lysed with 1M NaOH solution, and the solution containing cell fragments was collected in a plastic tube for determination of radioactivity taken into the cells. Finally, the radioactivity on the cell surface and inside the cells was measured with a γ counter.

2) Cellular internalization experiments of ⁶⁴ Cu-FAP-NOX and ⁶⁴ Cu-FAPI-04

A549-FAP cells (8×10 ⁴ cells/well) were seeded in 24-well plates 24 hours in advance. Before the experiment, the culture medium was removed and the cells were washed twice with PBS. Then, serum-free DMEM basal medium containing ⁶⁴ Cu-FAP-NOX/ ⁶⁴ Cu-FAPI-04 (74 kBq/well, 1 mL/well) was added to the well plate and incubated with the cells at 37°C for 0.5, 1, 2, 4, 12, and 24 hours. After reaching the designated time point, the culture medium containing the radioactive drug was immediately removed, the cells were washed twice with PBS, and 1 mL of pre-cooled glycine-HCl (0.05 M, pH = 2.8) solution was added to each well and incubated with the cells for 10 minutes. The incubated solution in each well was collected, the cells were washed twice with PBS, and the PBS solution after washing was collected and mixed with the incubation solution in the corresponding wells for the determination of the radioactivity bound to the cell surface. Finally, the cells were lysed with 1 M NaOH solution, and the solution containing cell fragments was collected in a plastic tube for the determination of the radioactivity taken into the cells. Finally, a γ counter was used to measure the radioactivity on the cell surface and inside the cells.

3) Cellular internalization experiment of ^177Lu -FAP-DOX

HT1080-FAP cells (5×10 ⁴ cells/well) were seeded in 24-well plates 24 hours in advance. Before the experiment, the culture medium was removed and the cells were washed twice with PBS. Subsequently, serum-free DMEM basal medium containing ¹⁷⁷ Lu-FAP-DOX (74 kBq/well, 1 mL/well) was added to the well plate and incubated with the cells at 37°C for 0.5, 1, 2, 4, 8, 24 and 72 hours. After reaching the designated time point, the culture medium containing the radioactive drug was immediately removed, the cells were washed twice with PBS, and 1 mL of pre-cooled glycine-HCl (0.05 M, pH = 2.8) solution was added to each well and incubated with the cells for 10 minutes. The incubated solution in each well was collected, the cells were washed twice with PBS, and the PBS solution after washing was collected and mixed with the incubation solution in the corresponding wells for the determination of the radioactivity bound to the cell surface. Finally, the cells were lysed with 1 M NaOH solution, and the solution containing cell fragments was collected in a plastic tube for the determination of the radioactivity taken into the cells. Finally, a γ counter was used to measure the radioactivity on the cell surface and inside the cells.

(5) Cell efflux experiment

1) Cell efflux experiments of Al ¹⁸ F-FAP-NUR, Al ¹⁸ F-FAP-NOX, and ⁶⁸ Ga-FAP-2286

HT1080-FAP cells (5×10 ⁴ cells/well) were seeded in 24-well plates 24 hours in advance. Before the experiment, the culture medium was removed and the cells were washed twice with PBS. Then, serum-free DMEM basal medium (74 kBq/well, 1 mL/well) containing Al ¹⁸ F-FAP-NUR/ ⁶⁸ Ga-FAP-2286/Al ¹⁸ F-FAP-NOX was added to the well plate and incubated at 37°C for 60 minutes. After reaching the designated time point, the culture medium containing the radioactive drug was immediately removed, and the cells were washed twice with PBS. Then, 1 mL of DMEM basal medium without radioactive drugs and serum was added to each well and continued to be incubated with the cells at 37°C for 5, 15, 30, 60 and 120 minutes. After reaching the designated time point, the culture medium was immediately removed and the cells were washed twice with PBS. Finally, the cells were lysed with 1M NaOH solution, and the solution containing cell fragments was collected in a plastic tube, and the radioactivity of the cells was measured using a γ counter.

2) Cell efflux experiments of ⁶⁴ Cu-FAP-NOX and ⁶⁴ Cu-FAPI-04

A549-FAP cells (8×10 ⁴ cells/well) were seeded in 24-well plates 24 hours in advance. Before the experiment, the culture medium was removed and the cells were washed twice with PBS. Then, serum-free DMEM basal medium (74 kBq/well, 1 mL/well) containing ⁶⁴ Cu-FAP-NOX/ ⁶⁴ Cu-FAPI-04 was added to the well plate and incubated at 37°C for 1 hour. After reaching the designated time point, the culture medium containing the radioactive drug was immediately removed, and the cells were washed twice with PBS. Then, 1 mL of DMEM basal medium without radioactive drugs and serum was added to each well and continued to be incubated with the cells at 37°C for 0.5, 1, 2, 4, 12, and 24 hours. After reaching the designated time point, the culture medium was immediately removed and the cells were washed twice with PBS. Finally, the cells were lysed with 1M NaOH solution, and the solution containing cell fragments was collected in a plastic tube, and the radioactivity of the cells was measured with a γ counter.

3) Cell efflux experiment of ^177Lu -FAP-DOX

HT1080-FAP cells (5×10 ⁴ cells/well) were seeded in 24-well plates 24 hours in advance. Before the experiment, the culture medium was removed and the cells were washed twice with PBS. Then, serum-free DMEM basal medium containing ¹⁷⁷ Lu-FAP-DOX (74 kBq/well, 1 mL/well) was added to the well plate and incubated at 37°C for 1 hour. After reaching the designated time point, the medium containing the radioactive drug was immediately removed and the cells were washed twice with PBS. Then, 1 mL of medium without radioactive drugs and serum was added to each well and continued to be incubated with cells at 37°C for 0.5, 1, 2, 4, 8, 24, 48 and 72 hours. After reaching the designated time point, the DMEM basal medium containing the radioactive drug was immediately removed and the cells were washed twice with PBS. Finally, the cells were lysed with 1M NaOH solution, the solution containing cell fragments was collected in a plastic tube, and the radioactivity of the cells was measured with a γ counter.

result:

(1) Cell binding affinity experiment

1) NOTA-FAP-NUR:

As shown in Figure 2a, the _IC50 values of DOTA-FAP-2286 and NOTA-FAP-NUR were 31.7 nM and 202.9 nM, respectively. It can be seen that compared with DOTA-FAP-2286, NOTA-FAP-NUR has a significantly higher cell binding affinity.

2)NOTA-FAP-NOX:

As shown in Figure 3a, the _IC50 values of NOTA-FAP-NOX and DOTA-FAP-2286 were 0.078 nM and 0.876 nM, respectively.

(2) Cell uptake and inhibition experiments

1) Al ¹⁸ F-FAP-NUR:

As shown in Figure 4a, at 60 minutes, the uptake of Al ¹⁸ F-FAP-NUR and ⁶⁸ Ga-FAP-2286 by HT1080-FAP cells was 56.92±1.98%ID/mio cells and 46.29±6.84%ID/mio cells, respectively; at 120 minutes, the uptake of Al ¹⁸ F-FAP-NUR and ⁶⁸ Ga-FAP-2286 by HT1080-FAP cells was 65.77%ID/mio cells and 42.47%ID/mio cells, respectively. It can be seen that compared with ⁶⁸ Ga-FAP-2286, the uptake of Al ¹⁸ F-FAP-NUR by HT1080-FAP cells was significantly increased.

As shown in Figure 4b, in the cell inhibition experiment, at 120 minutes, the uptake of Al ¹⁸ F-FAP-NUR and ⁶⁸ Ga-FAP-2286 by HT1080-FAP cells was 0.75% ID/mio cell and 4.61% ID/mio cell, respectively, which was similar to the uptake of HT1080 cells. It can be seen that the uptake of Al ¹⁸ F-FAP-NUR by HT1080-FAP cells can be effectively inhibited by DOTA-FAP-2286, which is almost similar to the drug uptake level observed in FAP-negative cells.

2) Al ¹⁸ F-FAP-NOX:

As shown in Figure 5a, at 15 minutes, the uptake of Al ¹⁸ F-FAP-NOX by HT1080-FAP cells was 7.31% ID/mio cells, and then the uptake was in a plateau phase. At 120 minutes, the uptake was still 7.90% ID/mio cells. In the inhibition experiment, the uptake of Al ¹⁸ F-FAP-NOX by HT1080-FAP cells could be significantly inhibited by DOTA-FAP-2286, indicating that Al ¹⁸ F-FAP-NOX highly targeted FAP.

3) ⁶⁴ Cu-FAP-NOX:

As shown in Figure 6a, cells had obvious uptake in the first hour of incubation with ⁶⁴ Cu-FAP-NOX and ⁶⁴ Cu-FAPI-04. At subsequent time points, the cell uptake rate of ⁶⁴ Cu-FAP-NOX was faster than that of ⁶⁴ Cu-FAPI-04. At 24 hours, the cell uptake of ⁶⁴ Cu-FAP-NOX (37.66% ID/mio cells) was more significant than that of ⁶⁴ Cu-FAPI-04 (13.72% ID/mio cells). As shown in Figure 6b, ⁶⁴ Cu-FAP-NOX had a higher absorption rate in FAP-positive A549-FAP cells. At the 4-hour incubation time point, it could be effectively inhibited by the inhibitor DOTA-FAP-2286, indicating that ⁶⁴ Cu-FAP-NOX highly targeted FAP. In contrast, ⁶⁴ Cu-FAP-NOX had no obvious uptake in FAP-negative A549 cells.

4) ^177Lu -FAP-DOX:

As shown in Figure 7a, the uptake of ¹⁷⁷ Lu-FAP-DOX by HT1080-FAP cells continued to increase within 24 hours, reaching a maximum at the 24th hour (6.52% ID/mio cell). At the 4th hour, the uptake of ¹⁷⁷ Lu-FAP-DOX by HT1080-FAP cells was significantly inhibited by DOTA-FAP-2286, and the uptake was 0.58% ID/mio cell, indicating that ¹⁷⁷ Lu-FAP-DOX highly targeted FAP.

(3) Cell internalization experiment

1) Al ¹⁸ F-FAP-NUR:

As shown in Figure 4c, after 60 minutes of co-incubation of cells and radioactive drugs, the internalization rates of Al ¹⁸ F-FAP-NUR and ⁶⁸ Ga-FAP-2286 were 61% ID/mio cell and 32% ID/mio cell, respectively. It can be seen that the internalization rate of Al ¹⁸ F-FAP-NUR is higher than that of ⁶⁸ Ga-FAP-2286.

2) Al ¹⁸ F-FAP-NOX:

As shown in FIG. 5 b , in the cell internalization experiment, at 120 minutes, the internalization rate of Al ¹⁸ F-FAP-NOX by HT1080-FAP cells remained at a high level (>75%).

3) ⁶⁴ Cu-FAP-NOX:

As shown in Figure 6c, the internalization rates of both tracers were comparable, with 70% retention observed in A549-FAP at the 12 h time point.

4) ^177Lu -FAP-DOX:

As shown in FIG. 7 b , in the cell internalization experiment, the amount of ¹⁷⁷ Lu-FAP-DOX internalized by cells continued to increase within 24 hours. At the 24th hour, the amount of ¹⁷⁷ Lu-FAP-DOX internalized by HT1080-FAP cells reached the highest level (4.57% ID/mio cell).

(4) Cell efflux experiment

1) Al ¹⁸ F-FAP-NUR:

As shown in Figure 4d, at 120 minutes, the ratios of Al ¹⁸ F-FAP-NUR and ⁶⁸ Ga-FAP-2286 excreted from the cells were <52% and <75%, respectively, indicating that the rate of Al ¹⁸ F-FAP-NUR excretion from the cells was slower than that of ⁶⁸ Ga-FAP-2286 excretion.

2) Al ¹⁸ F-FAP-NOX:

As shown in Figure 5c, in the cell efflux experiment, the efflux rate of Al ¹⁸ F-FAP-NOX was <80% in HT1080-FAP cells at 120 min.

3) ⁶⁴ Cu-FAP-NOX:

As shown in Figure 6d, the cell efflux experiment at the 12 h time point showed that both tracers were gradually released within the first 6 h and then reached a plateau. The cell efflux half-lives of ⁶⁴ Cu-FAP-NOX and ⁶⁴ Cu-FAPI-04 in A549-FAP were 16.08 h and 6.45 h, respectively, indicating that the efflux rate of ⁶⁴ Cu-labeled cyclic peptide was significantly slower.

4) ^177Lu -FAP-DOX:

As shown in FIG. 7 c , in the cell efflux experiment, the efflux rate of ¹⁷⁷ Lu-FAP-DOX was relatively slow. At the 24th hour, the efflux rate of ¹⁷⁷ Lu-FAP-DOX was <84%.

Example 7. Biodistribution

Steps:

1) Tumor-bearing model preparation

Normal Kunming (KM) mice (6 weeks old, weighing 25-34 g) were purchased from Zhuhai Baishitong Biotechnology Co., Ltd., and BALB/c nude mice (4-5 weeks old, weighing 16-22 g) were purchased from Guangdong Yaokang Biotechnology Co., Ltd. For the tumor model, 293T-FAP or 293T cells (5 × 10 ⁶ /mouse) were subcutaneously injected into the right axilla of BALB/c nude mice.

2) Biodistribution of Al ¹⁸ F-FAP-NUR and Al ¹⁸ F-FAP-NOX:

To characterize the normal biodistribution of Al ¹⁸ F-FAP-NUR in normal KM mice, mice were divided into 5 groups (n=4/group) and injected intravenously with 100 μL of Al ¹⁸ F-FAP-NUR (3.70 MBq/mouse). At different time points after injection, mice were sacrificed, blood samples and organs of interest were collected, weighed and counted using a gamma counter.

To further verify the biodistribution of Al ¹⁸ F-FAP-NUR and ⁶⁸ Ga-FAP-2286 in tumor mice, mice bearing 293T-FAP tumors were divided into 3 groups (n=4/group) and intravenously injected with 100 μL of ⁶⁸ Ga-FAP-2286 or Al ¹⁸ F-FAP-NUR (2.22-3.70 MBq/mouse). One hour after injection, they were subjected to the same biodistribution operation as normal KM mice.

To further verify the biodistribution of Al ¹⁸ F-FAP-NOX in tumor mice, mice bearing 293T-FAP tumors were divided into 2 groups (n=4/group), normal group and inhibition group, and intravenously injected with 100 μL of "Al ¹⁸ F-FAP-NOX" and "Al ¹⁸ F-FAP-NOX+DOTA-FAP-2286" (radiopharmaceutical: 2.22-3.70 MBq/mouse; DOTA-FAP-2286: 3 μM/mouse), respectively. One hour after injection, they were subjected to the same biodistribution operation as normal KM mice.

3) Biodistribution of ⁶⁴ Cu-FAP-NOX and ⁶⁴ Cu-FAPI-04:

Mice were divided into 6 groups (n=4/group), and each mouse was injected with 1.85MBq (50μCi) of ^64Cu -FAP-NOX/ ^64Cu -FAPI-04 through the tail vein. Mice were euthanized by cervical dislocation under anesthesia at 1, 4 and 24 hours after injection. Specific tissues were dissected, weighed and radioactivity was counted by a gamma counter. The results are expressed as mean ± standard deviation in %ID/gram, three mice per time point. The total injected activity of each mouse was quantified based on a known aliquot of the injected solution to obtain accurate quantification of the injected radiotracer activity.

4) Biodistribution of ^177Lu -FAP-DOX:

Mice were divided into 5 groups (n=4/group), and each mouse was injected with 1.85MBq (50μCi) of ^177Lu -FAP-DOX through the tail vein. Mice were euthanized by cervical dislocation under anesthesia at 1, 4, 8, 24 and 72 hours after injection. Specific tissues were dissected, weighed and radioactivity was counted by a gamma counter. The results are expressed as mean ± standard deviation in %ID/gram, three mice per time point. The total injected activity of each mouse was quantified based on a known aliquot of the injected solution to obtain accurate quantification of the injected radiotracer activity.

result:

(1) Biodistribution of Al ¹⁸ F-FAP-NUR:

1) Biodistribution of Al ¹⁸ F-FAP-NUR in normal KM mice

As shown in Figure 8a, in vivo biodistribution experiments were conducted in normal KM mice. The uptake of Al ¹⁸ F-FAP-NUR by normal KM mice gradually decreased over time in most organs, and it was concluded that the drug was mainly excreted through the kidneys, and the uptake in the kidneys had been reduced by half at 30 minutes (the uptake in the kidneys was 18.33±1.75%ID/g at 5 minutes and 5.58±0.89%ID/g at 30 minutes).

As shown in FIG8 b , according to the biodistribution results, it can be obtained that the drug blood concentration half-life of Al ¹⁸ F-FAP-NUR is about 25.48 minutes.

2) Biodistribution of Al ¹⁸ F-FAP-NUR in 293T-FAP tumor-bearing mice

As shown in Figure 8c, according to the biodistribution data of 293T-FAP tumor-bearing mice at 1 hour, the tumor uptake of Al ¹⁸ F-FAP-NUR and ⁶⁸ Ga-FAP-2286 were 26.99% ID/g and 11.55% ID/g, respectively. It can be seen that the tumor uptake of Al ¹⁸ F-FAP-NUR is twice that of ⁶⁸ Ga-FAP-2286.

As shown in Figure 8d, the tumor-muscle ratios of Al ¹⁸ F-FAP-NUR and ⁶⁸ Ga-FAP-2286 were 61.31±18.53%ID/g and 22.66±8.08%ID/g, and the tumor-kidney ratios were 5.09±2.60%ID/g and 1.63±0.58%ID/g, respectively. It can be seen that except for the tumor-bone ratio, the other tumor-background ratios of Al ¹⁸ F-FAP-NUR were higher than those ^{of 68} Ga-FAP-2286, especially the tumor-muscle ratio and tumor-kidney ratio.

(2) Biodistribution of Al ¹⁸ F-FAP-NOX in 293T-FAP tumor-bearing mice

Figure 9 summarizes the biodistribution results of Al ¹⁸ F-FAP-NOX in 293T-FAP tumor-bearing mice. At 1 hour, Al ¹⁸ F-FAP-NOX was highly uptaken in the tumor, reaching 14.59% ID/g, and the drug uptake in the tumor could be significantly inhibited by DOTA-FAP-2286, with an uptake of 0.67% ID/g. In normal organs, the uptake values of Al ¹⁸ F-FAP-NOX were all at a low level.

(3) Biodistribution of ^64Cu -FAP-NOX in 293T-FAP tumor-bearing mice

Figures 10a-10d summarize the biodistribution results of ⁶⁴ Cu-FAP-NOX and ⁶⁴ Cu-FAPI-04 in 293T-FAP tumor-bearing mice. The tumor uptake of ⁶⁴ Cu-FAP-NOX and ⁶⁴ Cu-FAPI-04 reached peak values at 4h (12.60±2.24%ID/g) and 1h (10.69±1.70%ID/g), respectively, and the tumor uptake at 24h was 5.47±0.25%ID/g and 2.32±1.37%ID/g, respectively. Compared with ⁶⁴ Cu-FAPI-04 at 4h, the accumulation of ⁶⁴ Cu-FAP-NOX in the liver and intestine was slightly higher, but the tumor uptake of ⁶⁴ Cu-FAP-NOX was 34% stronger than that of ⁶⁴ Cu-FAPI-04, and the bone uptake was significantly reduced. At 24 hours, the tumor-to-muscle ratio of ⁶⁴ Cu-FAP-NOX was 4.4 times that of ⁶⁴ Cu-FAPI-04, indicating that ⁶⁴ Cu-FAP-NOX has great potential for delayed imaging with enhanced contrast (Figures 11a-11f). In addition, both ⁶⁴ Cu-FAP-NOX and ⁶⁴ Cu-FAPI-04 showed rapid renal excretion within 1 hour. However, at 24 hours, the renal uptake of ⁶⁴ Cu-FAPI-04 was approximately twice that of ⁶⁴ Cu-FAP-NOX (2.27±1.22% ID/g and 1.23±0.16% ID/g, respectively).

(4) In vivo biodistribution experiment of ^177Lu -FAP-DOX in 293T-FAP tumor-bearing mice

Figure 12 summarizes the biodistribution results of ¹⁷⁷ Lu-FAP-DOX in 293T-FAP tumor-bearing mice. At 1 hour, ¹⁷⁷ Lu-FAP-DOX was in high uptake in the tumor, reaching 6.66% ID/g, and was in a plateau phase within 24 hours, and the uptake remained stable. In normal organs, the uptake values of ¹⁷⁷ Lu-FAP-DOX were all at a low level. From the trend of the uptake value of the kidney, it can be concluded that the drug is mainly excreted through the kidney.

Example 8. Mouse PET/CT imaging

Steps:

(1) Small animal PET/CT imaging of Al ¹⁸ F-FAP-NUR, Al ¹⁸ F-FAP-NOX, ⁶⁸ Ga-FAP-DOX and ⁶⁸ Ga-FAP-2286:

Small animal PET/CT imaging was performed when the tumor diameter reached 5-8 mm. PET/CT imaging was performed using a small animal PET scanner (MadicLab PSA071) from Shandong Mai De Ying Hua Technology Co., Ltd. 293T-FAP tumor-bearing mice and 293T tumor-bearing mice were intravenously injected with radiopharmaceuticals (0.1 ml, 7.4 MBq) and image acquisition was performed by PET scanning for 120 minutes, and then framed at a rate of 5 minutes per frame. FAP-negative tumor-bearing mice were subjected to a 10-minute static scan 60 minutes after receiving an equivalent drug injection. The CT scanning parameters were a tube voltage of 80 kV, a tube current of 0.7 mA, and a voxel spacing of 200 μm. PET images were used to determine radiotracer uptake, and CT images were used for attenuation correction of PET images and localization of radiotracer uptake sites. Images and volumes of interest were processed using PMOD software (version 4.3, PMOD Technology Co., Ltd., Switzerland).

(2) ⁶⁴ Cu-FAP-NOX and ⁶⁴ Cu-FAPI-04 Small Animal PET/CT Imaging:

⁶⁴ Cu-FAP-NOX was injected into the tail vein of 293T-FAP-bearing and 293T-bearing mice (7.4 MBq/mouse, n=3). The mice were placed in a prone position after inhalation of 2 Vol.% isoflurane anesthesia, and dynamic imaging for 2 hours was performed using a small animal PET scanner (MadicLab PSA071) of Shandong Mai De Ying Hua Technology Co., Ltd. At 4, 6 and 24 hours after injection, the mice were placed in a prone position after inhalation of 2 Vol.% isoflurane anesthesia, and a 10-minute static PET scan and a 4.5-minute CT scan were performed using the above-mentioned PET/CT scanner. The counts in the reconstructed images were converted into the percentage of the injected dose in the tissue (% ID/gram). The CT scanning parameters for tomography included 80 kV tube voltage, 0.6 mA tube current, and 200 μm voxel spacing. Image data were processed using PMOD software (version 4.3; PMOD Technologies GmbH, Zurich, Switzerland), and time-activity curves (TACs) of volumes of interest (VOIs) were selected. VOIs were defined on micro-PET coronal images to encompass the entire tumor (or specific organs or tissues), and the activity accumulation of the tumor and other major organs was measured. All values were expressed as a percentage of injected dose per gram (%ID/gram).

result:

(1) Small Animal PET/CT Imaging of Al ¹⁸ F-FAP-NUR and ⁶⁸ Ga-FAP-2286:

The dynamic PET/CT fusion images are shown in Figures 13a and 13c. The biodistribution curves of 293T-FAP tumor-bearing mice based on dynamic PET/CT scanning are shown in Figures 13b and 13d, where at 60 minutes, the tumor uptake of Al ¹⁸ F-FAP-NUR and ⁶⁸ Ga-FAP-2286 were 17.85±5.12%ID/g and 7.62±1.83%ID/g, respectively. It can be seen that compared with ⁶⁸ Ga-FAP-2286, the tumor uptake of Al ¹⁸ F-FAP-NUR is higher, especially in the tumor uptake of 293T-FAP tumor-bearing mice. Al ¹⁸ F-FAP-NUR showed rapid clearance in the kidneys of 293T-FAP tumor-bearing mice, which was reduced by 50% at 45 minutes. At 60 minutes, the 293T-FAP tumor-to-background ratio (TBR) of Al ¹⁸ F-FAP-NUR was higher than that of ⁶⁸ Ga-FAP-2286, which was more obvious in the tumor-to-muscle ratio of 293T-FAP (Figure 13e, the 293T-FAP tumor-to-background ratio (TBR) of Al ¹⁸ F-FAP-NUR and ⁶⁸ Ga-FAP-2286 were 55.13 and 13.60, respectively, P<0.05). In other non-targeted organs, it can be found that the uptake of Al ¹⁸ F-FAP-NUR is extremely low, especially in muscle (Figure 13d, at 60 minutes, the muscle uptake is 0.35±0.13%ID/g).

(2) Al ¹⁸ F-FAP-NOX small animal PET/CT imaging:

As shown in Figure 14, the tumor uptake of Al ¹⁸ F-FAP-NOX in 293T-FAP tumor-bearing mice increased over time and reached a plateau at 60 minutes, which was at a high uptake level. In the MIP image of PET/CT imaging, it can be observed that the drug is mainly excreted through the kidneys. After 30 minutes, it can be observed that part of the drug begins to be excreted through the liver. In 293T tumor mice, the drug uptake value in the tumor is very low.

(3) Small Animal PET/CT Imaging of ⁶⁴ Cu-FAP-NOX, ⁶⁴ Cu-FAPI-04 and ⁶⁸ Ga-FAP-2286:

Figure 15 shows representative coronal microPET images at different time points within 24 hours after about 7.4 MBq (0.2 mCi) of ⁶⁴ Cu-FAP-NOX, ⁶⁴ Cu-FAPI-04 or ⁶⁸ Ga-FAP-2286 radioligand was injected into the tail vein of 293T-FAP tumor-bearing mice (n=3/group). The tumor uptake of ⁶⁴ Cu-FAP-NOX was 15.51±1.39, 18.07±1.24, 20.51±1.04, 19.34±1.95, 19.74±0.61, 18.00±0.83, 12.31±0.19 and 10.06±0.27%ID/g at 0.25, 0.5, 1, 2, 4, 6, 12 and 24 hours, respectively. The results showed that ^64Cu -FAP-NOX accumulated in 293T-FAP tumors. For both ^64Cu -labeled tracers, at 0.25 hours, PET images showed identifiable background organs and tissues due to the presence of the radiotracer in the blood circulation, however, the background organs and tissues were rapidly weakened in the images at later time points.

In addition, the tumor uptake of ⁶⁴ Cu-FAPI-04 and ⁶⁸ Ga-FAP-2286 was lower than that ^{of 64} Cu-FAP-NOX throughout the experiment. Notably, PET images showed that ⁶⁴ Cu-FAP-NOX had higher uptake and sustained tumor retention at the tumor site from the initial time point to 24 hours after intravenous injection. The kidneys showed high uptake and rapid metabolism in the early stages, demonstrating that the tracer was excreted primarily through the renal pathway. The tumor-to-kidney ratio of ⁶⁴ Cu-FAP-NOX was significantly greater than 1 at 1 hour after intravenous injection, and this ratio continued to increase gradually over time. And because the tracer can be quickly cleared in normal non-targeted organs, the tumor-to-background ratios (TBRs) also increased steadily over time, reaching peak values of 39.64±7.93, 5.00±1.94, and 63.72±19.74 in the vertebral body, liver, and muscle at 6 hours, respectively. The hepatic accumulation of ^64Cu -FAP-NOX and ^64Cu -FAPI-04 in PET/CT images corresponded to the biodistribution data.

(4) ⁶⁸ Ga-FAP-DOX small animal PET/CT imaging:

As shown in Figure 16, the tumor uptake of ⁶⁸ Ga-FAP-DOX in 293T-FAP tumor-bearing mice increased with time and reached a plateau at 30 minutes, at a high uptake level; at 240 minutes, the drug uptake in the tumor was still high, indicating that the drug retention time in the tumor was relatively long, up to 240 minutes (ie, 4 hours). In the MIP map of PET/CT imaging, it can be observed that the drug is mainly excreted through the kidneys, and very little is excreted by the liver.

The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims (17)

1. A conjugate targeting fibroblast activation protein (FAP), characterized in that the conjugate comprises: A binding peptide that specifically binds to FAP; a chelating group to which the radionuclide is bound; and, A linker is attached to the polypeptide. 2. The conjugate according to claim 1, characterized in that the chelating group is selected from one or more of DOTA, DOTAGA, NOTA, NODAGA, NODA-MPAA, HBED, TETA, CB-TE2A, DTPA, DFO, Macropa, HOPO, TRAP, THP, DATA, NOTP, sarcophagine, FSC, NETA, H4octapa, Pycup, N _x S _4-x (N4, N2S2, N3S), Hynic, ^99m Tc(CO) ₃ - groups, preferably, the chelating group is selected from DOTA and/or NOTA. 3. The conjugate according to claim 1, characterized in that the binding peptide is a cyclic peptide, Preferably, the binding peptide has 3 to 40 amino acid residues, more preferably 3 to 20 amino acid residues, Preferably, the binding peptide is a single-ring peptide. 4. The conjugate according to claim 1, characterized in that the binding peptide is cyclized by a peptide bond, an alkyl bond, an alkenyl bond, an ester bond, a thioester bond, an ether bond, a thioether bond, a phosphate ether bond, an azo bond, a C-N-C bond, a C=N-C bond, a C=N-O bond, an amide bond, a lactam bridge, a carbamoyl bond, a urea bond, a thiourea bond, an amine bond and/or a thioamide bond, Preferably, the binding peptide is cyclized via a thioether bond, Preferably, the amino acid residues at the N-terminus and the C-terminus of the binding peptide are cyclized via thioether bonds. 5. The conjugate according to claim 1, characterized in that the binding peptide has a (hetero)aromatic or (hetero)alicyclic portion, preferably a tri-substituted (hetero)aromatic or (hetero)alicyclic portion, and more preferably a 1,3,5-trimethylbenzene portion. 6. The conjugate according to claim 1, characterized in that the binding peptide has a structure of formula I, CX ₁ -X ₂ -X ₃ -X ₄ -X ₅ -C (I), wherein X ₁ -X ₅ are selected from any amino acid residues, Preferably, the amino acid residues of _X1 - _X5 are each independently selected from Phe, Pro, Gln, Thr, Cys, Glu, Ala, Gly, Val, Leu, Ile, Trp, Tyr, Asp, Asn, Lys, Met, Ser, His or derivatives thereof, Preferably, _X1 is selected from Phe, Tyr or Trp, more preferably Phe; Preferably, _X2 and _X3 are each independently selected from Ser, Thr, Gln, Asn, Met or Cys, more preferably _X2 is Gln, and/or _X3 is Thr; _X4 and _X5 are each independently selected from Gly, Ala, Pro, Val, Leu or Ile, more preferably, _X4 is Pro, and/or _X5 is Pro. 7. The conjugate according to claim 1, characterized in that the N-terminal and/or C-terminal residues of the binding peptide have one or more modifications, preferably a modification of (C ₁ -C ₆ alkyl)-NH-(C=O) _n -, wherein n is an integer selected from 1 to 5; Preferably, the amino acid residue at the N-terminus of the binding peptide has and / or modification. 8. The conjugate according to claim 1, characterized in that the binding peptide has one or more of the following structures: 9. The conjugate according to claim 1, characterized in that the binding peptide is linked to the chelating group via a thioether bond. 10. The conjugate according to claim 1, characterized in that the radionuclide is one or more of 18F, 68Ga, 64Cu and 177Lu. 11. The conjugate according to claim 1, characterized in that the conjugate has one or more of the following structures: 12 . The conjugate according to claim 1 , characterized in that the conjugate is one or more selected from ¹⁸ F-(NOTA)-FAP-NUR, ¹⁸ F-(NOTA)-FAP-NOX, ⁶⁴ Cu-(NOTA)-FAP-NOX, ¹⁷⁷ Lu-(DOTA)-FAP-DOX, and ⁶⁸ Ga-(DOTA)-FAP-DOX. 13. A composition comprising the conjugate of any one of claims 1 to 12, and a pharmaceutically acceptable carrier. 14. The composition according to claim 13, characterized in that the composition further comprises an additional therapeutic agent and/or an imaging agent. 15. A targeted imaging kit comprising the conjugate according to any one of claims 1 to 12. 16. A method for imaging and/or treating a diseased area using the conjugate according to any one of claims 1 to 12. 17. The method according to claim 16, wherein the lesion comprises a disease or condition associated with fibroblast activation protein, and/or The imaging is positron emission tomography and/or X-ray computed tomography imaging.