FAP (fibroblast activation protein)

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

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].

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.

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).

Serum Stability[1]
Processed and solvent-free radioactive compounds (177Lu-FAPI-21 and 177Lu-FAPI-46) were incubated in human sera at 37°C. After the respective incubation time, samples were taken, freed from proteins by precipitation with acetonitrile, and centrifuged, and the supernatant was analyzed via radio–high-performance liquid chromatography. Supplemental Figure 1 shows that even at 24 h, only the initial (radioactive) peaks were detected and neither radioactive degradation products nor free radioactivity were observed. These findings demonstrate that both substances were unhampered by enzymatic components of human sera.

[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 constitute a vital subpopulation of the tumor stroma and are present in more than 90% of epithelial carcinomas. The overexpression of the serine protease fibroblast activation protein (FAP) allows a selective targeting of a variety of tumors by inhibitor-based radiopharmaceuticals (FAPIs). Of these compounds, FAPI-04 has been recently introduced as a theranostic radiotracer and demonstrated high uptake into different FAP-positive tumors in cancer patients. To enable the delivery of higher doses, thereby improving the outcome of a therapeutic application, several FAPI variants were designed to further increase tumor uptake and retention of these tracers. Methods: Novel quinoline-based radiotracers were synthesized by organic chemistry and evaluated in radioligand binding assays using FAP-expressing HT-1080 cells. Depending on their in vitro performance, small-animal PET imaging and biodistribution studies were performed on HT-1080-FAP tumor-bearing mice. The most promising compounds were used for clinical PET imaging in 8 cancer patients. Results: Compared with FAPI-04, 11 of 15 FAPI derivatives showed improved FAP binding in vitro. Of these, 7 compounds demonstrated increased tumor uptake in tumor-bearing mice. Moreover, tumor-to-normal-organ ratios were improved for most of the compounds, resulting in images with higher contrast. Notably two of the radiotracers, FAPI-21 and -46, displayed substantially improved ratios of tumor to blood, liver, muscle, and intestinal uptake. A first diagnostic application in cancer patients revealed high intratumoral uptake of both radiotracers already 10 min after administration but a higher uptake in oral mucosa, salivary glands, and thyroid for FAPI-21. Conclusion: Chemical modification of the FAPI framework enabled enhanced FAP binding and improved pharmacokinetics in most of the derivatives, resulting in high-contrast images. Moreover, higher doses of radioactivity can be delivered while minimizing damage to healthy tissue, which may improve therapeutic outcome.[1]
Background: Fibroblast activation protein (FAP) is a proline selective serine protease that is overexpressed in tumor stroma and in lesions of many other diseases that are characterized by tissue remodeling. In 2014, a most potent FAP-inhibitor (referred to as UAMC1110) with low nanomolar FAP-affinity and high selectivity toward related enzymes such as prolyl oligopeptidase (PREP) and the dipeptidyl-peptidases (DPPs): DPP4, DPP8/9 and DPP2 were developed. This inhibitor has been adopted recently by other groups to create radiopharmaceuticals by coupling bifunctional chelator-linker systems. Here, we report squaric acid (SA) containing bifunctional DATA5m and DOTA chelators based on UAMC1110 as pharmacophor. The novel radiopharmaceuticals DOTA.SA.FAPi and DATA5m.SA.FAPi with their non-radioactive derivatives were characterized for in vitro inhibitory efficiency to FAP and PREP, respectively and radiochemical investigated with gallium-68. Further, first proof-of-concept in vivo animal study followed by ex vivo biodistribution were determined with [68Ga]Ga-DOTA.SA.FAPi. Results: [68Ga]Ga-DOTA.SA.FAPi and [68Ga]Ga-DATA5m.SA.FAPi showed high complexation > 97% radiochemical yields after already 10 min and high stability over a period of 2 h. Affinity to FAP of DOTA.SA.FAPi and DATA5m.SA.FAPi and its natGa and natLu-labeled derivatives were excellent resulting in low nanomolar IC50 values of 0.7-1.4 nM. Additionally, all five compounds showed low affinity for the related protease PREP (high IC50 with 1.7-8.7 μM). First proof-of-principle in vivo PET-imaging animal studies of the [68Ga]Ga-DOTA.SA.FAPi precursor in a HT-29 human colorectal cancer xenograft mouse model indicated promising results with high accumulation in tumor (SUVmean of 0.75) and low background signal. Ex vivo biodistribution showed highest uptake in tumor (5.2%ID/g) at 60 min post injection with overall low uptake in healthy tissues. Conclusion: In this work, novel PET radiotracers targeting fibroblast activation protein were synthesized and biochemically investigated. Critical substructures of the novel compounds are a squaramide linker unit derived from the basic motif of squaric acid, DOTA and DATA5m bifunctional chelators and a FAP-targeting moiety. In conclusion, these new FAP-ligands appear promising, both for further research and development as well as for first human application.[2]

C41H57F2N11O9

885.97

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

CN(CCCN1CCN(CC1)C(=O)CN2CCN(CCN(CCN(CC2)CC(=O)O)CC(=O)O)CC(=O)O)C3=CC4=C(C=CN=C4C=C3)C(=O)NCC(=O)N5CC(C[C@H]5C#N)(F)F

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.)