FAP (fibroblast activation protein)
FAPI-46 targets fibroblast activation protein (FAP). FAP is a serine protease that is abundantly expressed in cancer-associated fibroblasts (CAFs) in over 90% of epithelial tumors. By binding to FAP, FAPI-46 allows for the visualization of FAP-positive tumors using PET imaging. FAP is also a key molecule in tissue fibrosis.

In HT-1080 cells producing human FAP, FAPI-46 (1–24 h) transiently binds to human FAP [1].

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In vitro, FAPI-46 shows high affinity and specificity for FAP. Cellular uptake experiments demonstrate good FAP binding specificity. The compound's affinity for FAP is typically measured using surface plasmon resonance or binding assays on FAP-expressing cells. These in vitro activities support its use as a PET tracer for imaging FAP expression.

FAPI-46 (iv) greatly improves the ratio of tumors to food in the liver, intestines and intestines, hence boosting image restoration in PET imaging [1].

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In vivo, [⁶⁸Ga]Ga-FAPI-46 has demonstrated favourable pharmacokinetics for PET imaging. It has been used in preclinical and clinical studies for imaging various cancers, including pancreatic cancer, glioblastoma, and for evaluating FAP expression in hypertension-induced tissue changes. [⁶⁸Ga]Ga-FAPI-46 PET is a novel tool for cancer imaging.

The in vitro FAP binding assay for FAPI-46 is conducted using FAP-expressing cell lines. Cells are incubated with radiolabeled FAPI-46 (e.g., ⁶⁸Ga-FAPI-46). The bound radioactivity is measured, and the binding affinity (Kd) is calculated. Competitive binding assays are performed using unlabeled FAPI ligands to confirm specificity. Cellular uptake and internalization studies are also performed.

Cell Culture[1]
HT-1080 cells transfected with the human FAP gene, as well as murine FAP- and CD26-transfected human embryonic kidney cells, were cultivated in Dulbecco modified Eagle medium containing 10% fetal calf serum at 37°C/5% carbon dioxide.
For radioligand binding studies, cells were seeded in 6-well plates and cultivated for 48 h to a final confluence of about 80%–90% (1.2–2 million cells per well). The medium was replaced by 1 mL of fresh medium without fetal calf serum. The radiolabeled compound was added to the cell culture and incubated for different intervals ranging from 10 min to 24 h. Competition experiments were performed by simultaneous exposure to unlabeled (10−5 to 10−10 M) and radiolabeled compound for 60 min. Cell efflux was determined after incubation of the cells with the tracer for 60 min. Thereafter, the radioactive medium was removed, and the cells were washed and incubated with nonradioactive medium for 1, 2, 4, and 24 h. In all experiments, the cells were washed twice with 1 mL of phosphate-buffered saline, pH 7.4, and subsequently lysed with 1.4 mL of lysis buffer (0.3 m NaOH, 0.2% sodium dodecyl sulfate). Radioactivity was determined in a γ-counter, normalized to 1 million cells, and calculated as percentage applied dose. Each experiment was performed 3 times, and 3 repetitions per independent experiment were acquired.

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In vitro cellular uptake assays for FAPI-46 are performed using FAP-positive cells. Cells are incubated with radiolabeled FAPI-46 at 37°C. The cells are washed, and the internalized radioactivity is measured. The specificity of uptake is confirmed by blocking with an excess of unlabeled ligand (e.g., DOTA-FAPI-04). The uptake is expressed as a percentage of the added dose per milligram of protein.

Animal Studies[1]
For in vivo experiments, 8-wk-old BALB/c nu/nu mice were subcutaneously inoculated into the right trunk with 5 million HT-1080-FAP cells. When the size of the tumor reached approximately 1 cm3, the radiolabeled compound was injected via the tail vein (80 nmol/GBq for small-animal PET imaging; 200 nmol/GBq for organ distribution). In vivo blocking experiments were performed by adding 30 nmol of unlabeled FAPI to the radiolabeled compound directly before injection. For organ distribution, the animals (n = 3 for each time point) were killed 1, 4, 6, and 24 h after tracer administration. The distributed radioactivity was measured in all dissected organs and in blood using a γ-counter. The values are expressed as percentage injected dose per gram of tissue (%ID/g). PET imaging was performed using a small-animal PET scanner. Within the first 60 min, a dynamic scan was performed in list mode, followed by a static scan from 120 to 140 min after injection. Images were reconstructed iteratively using the 3-dimensional ordered-subset expectation maximization maximum a priori method and were converted to SUV images. For the dynamic data, 28 frames were reconstructed: 4 × 5 s, 4 × 10 s, 4 × 20 s, 4 × 60 s, 4 × 120 s, 6 × 300 s, and 2 × 470 s. Quantification was done using a region-of-interest technique and expressed as SUV. All animal experiments were conducted in compliance with the German animal protection laws (approval 35-91185.81/G-158/15).[1]

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Clinical PET/CT Imaging[1]
Imaging of 8 patients was performed under the conditions of the updated Declaration of Helsinki, section 37 (unproven interventions in clinical practice) and in accordance with the German Pharmaceuticals Law, section 13 (2b), for medical reasons using 68Ga-FAPI-21 and -46, which were applied intravenously (20 nmol, 210–267 MBq for FAPI-21 and 216–242 MBq for FAPI-46). Imaging took place at 10 min, 1 h, and 3 h after tracer administration. The PET/CT scans were obtained with a Biograph mCT Flow PET/CT scanner using the following parameters: slice thickness of 5 mm, increment of 3–4 mm, soft-tissue reconstruction kernel, and CARE Dose. Immediately after CT scanning, a whole-body PET scan was acquired in 3 dimensions (matrix, 200 × 200) in FlowMotion with 0.7 cm/min. The emission data were corrected for randoms, scatter, and decay. Reconstruction was conducted with an ordered-subset expectation maximization algorithm with 2 iterations and 21 subsets and Gauss-filtered to a transaxial resolution of 5 mm in full width at half maximum. Attenuation was corrected using the low-dose nonenhanced CT data. SUVs were quantitatively assessed using a region-of-interest technique. The data were analyzed retrospectively with approval of the local ethics committee (approval S016/2018).

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In vivo imaging studies for FAPI-46 are conducted in mouse models bearing FAP-positive tumor xenografts. The radiolabeled compound (e.g., ⁶⁸Ga-FAPI-46) is administered via intravenous injection. PET/CT imaging is performed at various time points post-injection to visualize the biodistribution and tumor uptake of the tracer. The tumor-to-background ratio is calculated to assess the imaging contrast.

Serum stability [1]
The treated solvent-free radioactive compounds (¹⁷⁷Lu-FAPI-21 and ¹⁷⁷Lu-FAPI-46) were incubated with human serum at 37°C. After incubation for a certain period of time, samples were taken, proteins were removed by acetonitrile precipitation, centrifuged, and the supernatant was taken for radioactive high-performance liquid chromatography analysis. Supplementary Figure 1 shows that even after 24 hours, only the initial (radioactive) peak was detected, and no radioactive degradation products or free radioactivity were observed. These results indicate that neither substance was affected by the enzyme components of human serum.

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Pharmacokinetic data for FAPI-46 indicates that it has favourable pharmacokinetics for PET imaging when labeled with ⁶⁸Ga. The compound has a CAS number of 2374782-04-2. The pharmacokinetic properties depend on the specific radioisotope and the formulation used. For radiolabeled compounds, the biodistribution and clearance are typically evaluated in animal models. Detailed PK parameters are not extensively reported.

Toxicity data for FAPI-46 is not extensively reported. As a research compound, FAPI-46 is intended for research use only and not for human therapeutic applications. The toxicity would depend on the specific radioisotope used for labeling. Standard in vitro cytotoxicity assays and in vivo tolerability studies would be required for a complete toxicity assessment.

[1]. Development of Fibroblast Activation Protein-Targeted Radiotracers with Improved Tumor Retention. J Nucl Med. 2019 Oct;60(10):1421-1429.

[2]. Targeting fibroblast activation protein (FAP): next generation PET radiotracers using squaramide coupled bifunctional DOTA and DATA 5m chelators. EJNMMI Radiopharm Chem. 2020 Jul 29;5(1):19.

Cancer-associated fibroblasts are an important component of the tumor stroma, present in over 90% of epithelial carcinomas. Overexpression of the serine protease fibroblast activating protein (FAP) enables inhibitor-based radiopharmaceuticals (FAPIs) to selectively target a variety of tumors. Among these compounds, FAPI-04 has recently been introduced as a therapeutic radiotracer and has demonstrated high uptake rates in various FAP-positive tumors in cancer patients. To achieve higher dose delivery and thus improve therapeutic efficacy, researchers designed several FAPI variants to further enhance the uptake and retention of these tracers in tumors. Methods: Novel quinoline-based radiotracers were synthesized using organic chemical methods, and radioligand binding assays were performed using HT-1080 cells expressing FAP. Based on their in vitro performance, small animal PET imaging and biodistribution studies were conducted on mice bearing HT-1080-FAP tumors. The most promising compounds were used for clinical PET imaging in 8 cancer patients. Results: Compared with FAPI-04, 11 of the 15 FAPI derivatives showed higher FAP binding capacity in vitro. Of these, seven compounds showed higher tumor uptake in tumor-bearing mice. In addition, most compounds showed an increased tumor/normal organ uptake ratio, resulting in higher contrast images. Notably, the tumor/blood, liver, muscle and intestine uptake ratios of the two radiotracers, FAPI-21 and FAPI-46, were significantly increased. In the first diagnostic application in cancer patients, both radiotracers showed high intratumoral uptake within 10 minutes after administration, but FAPI-21 showed higher uptake in the oral mucosa, salivary glands and thyroid gland. Conclusion: Chemical modification of the FAPI backbone enhances the FAP binding capacity and pharmacokinetic properties of most derivatives, resulting in high contrast images. In addition, higher doses of radioactive material can be delivered while minimizing damage to healthy tissues, which may improve therapeutic effects. [1]
Background: Fibroblast activating protein (FAP) is a proline-selective serine protease that is overexpressed in the tumor matrix and many other disease lesions characterized by tissue remodeling. In 2014, a highly efficient FAP inhibitor (designated UAMC1110) was developed, exhibiting low nanomolar affinity for FAP and high selectivity for related enzymes such as prolyl oligopeptidase (PREP) and dipeptidyl peptidases (DPPs): DPP4, DPP8/9, and DPP2. Recently, other research teams have used this inhibitor to prepare radiopharmaceuticals via bifunctional chelator-linker systems. This paper reports bifunctional chelators DATA5m and DOTA, containing squaric acid (SA) with UAMC1110 as the pharmacophore. We characterized the novel radiopharmaceuticals DOTA.SA.FAPi and DATA5m.SA.FAPi, and their non-radioactive derivatives, in vitro for inhibiting FAP and PREP activity, and conducted radiochemical studies using gallium-68. Furthermore, we performed preliminary in vivo animal experiments using [68Ga]Ga-DOTA.SA.FAPi and determined its in vitro biodistribution. The results showed that [68Ga]Ga-DOTA.SA.FAPi and [68Ga]Ga-DATA5m.SA.FAPi achieved radiochemical yields of over 97% within 10 minutes and maintained good stability for 2 hours. DOTA.SA.FAPi, DATA5m.SA.FAPi, and their natGa and natLu-labeled derivatives exhibited excellent affinity for fibroblast activation protein (FAP), with IC50 values as low as nanomolar levels (0.7–1.4 nM). Furthermore, all five compounds showed low affinity for the related protease PREP (higher IC50 values, 1.7–8.7 μM). In the HT-29 human colorectal cancer xenograft mouse model, the [68Ga]Ga-DOTA.SA.FAPi precursor was used in the first in vivo PET imaging animal experiment, demonstrating high accumulation in tumors (SUVmean = 0.75) with low background signal, indicating promising application prospects. In vitro biodistribution experiments showed that the highest uptake was observed in tumors (5.2% ID/g) 60 minutes after injection, while the overall uptake in healthy tissues was lower. Conclusion: This study synthesized and biochemically investigated novel PET radiotracers targeting fibroblast activation proteins. The key substructures of these novel compounds include a squamous amide linker derived from the squamous acid motif, a DOTA and DATA5m bifunctional chelator, and an FAP targeting moiety. In summary, these novel FAP ligands show promising potential for further research and development as well as for first-time human applications. [2]

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FAPI-46 (CAS 2374782-04-2) is a quinoline-based radiopharmaceutical that targets fibroblast activation protein (FAP). FAP is highly expressed in cancer-associated fibroblasts in over 90% of epithelial tumors. [⁶⁸Ga]Ga-FAPI-46 has demonstrated favourable pharmacokinetics for PET imaging and is a novel tool for cancer imaging. It is for research use only.

885.43

C, 55.58; H, 6.49; F, 4.29; N, 17.39; O, 16.25

2374782-04-2

139400499

Light yellow to yellow solid powder

1.45±0.1 g/cm3(Predicted)

1148.7±65.0 °C(Predicted)

-6.7

4

19

16

63

1620

1

SDBGUEFOSXNKBX-HKBQPEDESA-N

InChI=1S/C41H57F2N11O9/c1-47(30-3-4-34-33(21-30)32(5-6-45-34)40(63)46-24-35(55)54-29-41(42,43)22-31(54)23-44)7-2-8-48-17-19-53(20-18-48)36(56)25-49-9-11-50(26-37(57)58)13-15-52(28-39(61)62)16-14-51(12-10-49)27-38(59)60/h3-6,21,31H,2,7-20,22,24-29H2,1H3,(H,46,63)(H,57,58)(H,59,60)(H,61,62)/t31-/m0/s1

2-[4,7-bis(carboxymethyl)-10-[2-[4-[3-[[4-[[2-[(2S)-2-cyano-4,4-difluoropyrrolidin-1-yl]-2-oxoethyl]carbamoyl]quinolin-6-yl]-methylamino]propyl]piperazin-1-yl]-2-oxoethyl]-1,4,7,10-tetrazacyclododec-1-yl]acetic acid

FAPI-46; 2374782-04-2; 59QC5DY68A; UNII-59QC5DY68A; (10-(2-(4-(3-((4-(((2-((2S)-2-Cyano-4,4-difluoro-1-pyrrolidinyl)-2-oxoethyl)amino)carbonyl)-6-quinolinyl)methylamino)propyl)-1-piperazinyl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)-kappaN1,kappaN4,kappaN7,kappaN10)-; 2-[4,7-bis(carboxymethyl)-10-[2-[4-[3-[[4-[[2-[(2S)-2-cyano-4,4-difluoropyrrolidin-1-yl]-2-oxoethyl]carbamoyl]quinolin-6-yl]-methylamino]propyl]piperazin-1-yl]-2-oxoethyl]-1,4,7,10-tetrazacyclododec-1-yl]acetic acid; [10-[2-[4-[3-[[4-[[[2-[(2S)-2-Cyano-4,4-difluoro-1-pyrrolidinyl]-2-oxoethyl]amino]carbonyl]-6-quinolinyl]methylamino]propyl]-1-piperazinyl]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)-kappaN1,kappaN4,kappaN7,kappaN10]-; SCHEMBL21257093;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~100 mg/mL (~112.87 mM)
DMSO : ~100 mg/mL (~112.87 mM)

Solubility in Formulation 1: ≥ 5.75 mg/mL (6.49 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 57.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 5 mg/mL (5.64 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)