FAP/Fibroblast activation protein

Suppression of Liver Accumulation and Hepatobiliary Excretion by Additional Hydrophilic Amino Acids[2]
To improve the in vivo characteristics, the chelating moiety was modified with the amino acids Asn, Glu, and Gla (γ-carboxyglutamic acid). Additionally, a precursor with triethylene glycol as a spacer between the piperazinyl and quinolinyl moieties was synthesized to increase the hydrophilicity of the underlying FAPI-29 without further modification at the chelating moiety. Compared with the initially synthesized 99mTc-FAPI-19, the derivatives 99mTc-FAPI-33 and -34 revealed higher uptake ratios of up to 45.8% ± 1.3% and 41.86% ± 1.07%, respectively, on HT-1080-FAP cells (Fig. 2A). Furthermore, internalization rates above 95% (Fig. 2A; Supplemental Table 2) and high affinity for FAP, with IC50 values of 10.9 nM for FAPI-33 and 6.9 for FAPI-34, were observed (Fig. 1), as evaluated by competition experiments (Fig. 2B). In contrast, less binding was measured for 99mTc-FAPI-27 (≤12.94% ± 0.77%), 99mTc-FAPI-28 (≤37.52% ± 1.62%), 99mTc-FAPI-29 (≤39.34% ± 1.01%), and 99mTc-FAPI-43 (≤28.83% ± 0.88%) after exposure to HT-1080-FAP cells for 4 h (Fig. 2A). Furthermore, competition experiments revealed a slightly reduced affinity of these derivatives for FAP, with IC50 values of 12.0 nM for FAPI-29 and 12.7 nM for FAPI-28 (Fig. 1).

In Vivo Targeting Properties and Pharmacokinetics of 99mTc-Labeled FAPI Derivatives[2]
To compare the in vivo targeting properties and pharmacokinetics of FAPI-28, -29, -33, -34, and -43 with those of FAPI-19 planar scintigraphy, biodistribution experiments on HT-1080-FAP–xenotransplanted mice were performed. The scintigraphic images demonstrated an improvement in the pharmacokinetics of the FAPI derivatives. Compared with FAPI-19 (Fig. 3A; Supplemental Fig. 3A), an accumulation of radioactivity in the tumor lesion and a reduction in the proportion of the hepatobiliary excretion was noticed at 60 min and lasted until at least 120 min after injection of the compounds (Fig. 3A). 99mTc-FAPI-34 showed the lowest uptake in the liver, biliary gland, and intestine and significant uptake in the tumor lesions of mice (Fig. 3A; Supplemental Fig. 3B), which was prevented by simultaneous injection of the unlabeled analog and confirmed the target specificity of the compound (Fig. 3B). [2]
In accordance with these results, biodistribution experiments with 99mTc-FAPI-34 revealed a tumor uptake of 5.4 ± 2.05 and 4.3 ± 1.95 %ID/g and a liver uptake of 0.91 ± 0.25 and 0.73 ± 0.18 %ID/g at 1 and 4 h, respectively, after injection of the tracer (Fig. 4A). Except for the kidneys, less than 1 %ID/g of the FAPI-34 activity was detected in the blood and organs of xenografts, accounting for tumor-to-tissue ratios above 1 (Fig. 4B). In contrast, we measured a lower tumor uptake of 99mTc-FAPI-29 (2.79 ± 1.19 and 1.43 ± 1.13 %ID/g) and of 99mTc-FAPI-43 (2.41 ± 0.34 and 2.57 ± 0.32 %ID/g) at 1 and 4 h, respectively, after tracer injection (Supplemental Fig. 4). The liver uptake of these derivatives, however, increased from 0.63 ± 0.06 to 1.73 ± 1.33 %ID/g (FAPI-29) or slightly decreased from 1.74 ± 0.28 to 1.56 ± 0.03 %ID/g (FAPI-43) after 1 and 4 h, respectively. In summary, 99mTc-FAPI-34 provided the best pharmacokinetics in xenografts and was, therefore, clinically applied for scintigraphy and SPECT.

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FAPI-34 Accumulation in Human Tumors[2]
Two patients with metastasized ovarian and pancreatic cancer underwent PET with 68Ga-FAPI-46, therapy with 90Y-FAPI-46 in the setting of a last-line treatment, and scintigraphy or SPECT with 99mTc-FAPI-34. The patient with metastasized ovarian cancer underwent 68Ga-FAPI-46 PET/CT on July 7, 2018, followed by therapy with 6 GBq of 90Y-FAPI-46 on July 25, 2018. Therapy follow-up was done using 99mTc-FAPI-34 on September 19, 2018, and showed stable disease. The patient with pancreatic cancer had a previous FAPI therapy in June 2018. The 99mTc-FAPI-34 scintigraphy was done for follow-up. One day after scintigraphy, another therapy was done with 6 GBq of 90Y-FAPI-46. Therapy was done with 90Y because 188Re was not available at that time. Six weeks later, follow-up imaging was done with FAPI-46 PET/CT. In both cases, the tumor lesions could be visualized (Figs. 5 and 6; Supplemental Figs. 5 and 6). Although evidence of a biliary secretion into the intestine was found in animal experiments, this was not the case in these patients.

Cell Culture[2]
The binding properties of 99mTc-labeled FAPI derivatives were evaluated using HT-1080 cells stably transfected with the human FAP gene (HT-1080-FAP), as well as the mouse FAP gene (HEK-muFAP) and human CD26 (HEKCD26)–transfected human embryonic kidney cells. The cells were cultivated in Dulbecco modified Eagle’s medium containing 10% fetal calf serum at 37°C and 5% carbon dioxide.[2]
Radioligand binding studies were performed as described previously. In brief, recombinant cells were seeded in 6-well plates and cultivated for 48 h to a final confluence of approximately 80%–90% (1.2–2 × 106 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 to 240 min. Competition experiments were performed by simultaneous exposure to unlabeled (10−5–10−10 M) and radiolabeled compound for 60 min. In all experiments, the cells were washed twice with 1 mL of phosphate-buffered saline at pH 7.4 and subsequently lysed with 1.4 mL of lysis buffer (0.3 M NaOH, 0.2% sodium dodecyl sulfate).[2]
For internalization experiments, the cells were incubated with the radiolabeled compound for 60 and 240 min at 37°C. Cellular uptake was terminated by removing medium from the cells and washing twice with 1 mL of phosphate-buffered saline. Subsequently, the cells were incubated with 1 mL of glycine-HCl (1 M, pH 2.2) for 10 min at room temperature to harvest the surface-bound peptides (glycine fraction). Thereafter, the cells were washed with 2 mL of ice-cold phosphate-buffered saline and lysed as described (4,5,11) to determine the internalized (lysed) fraction. Radioactivity was determined in a Wizard γ-counter (PerkinElmer), normalized to 1 × 106 cells and calculated as the percentage of the applied dose. Each experiment was performed 3 times, and 3 repetitions per independent experiment were acquired.[2]

Animal Studies[2]
For in vivo experiments, 5 × 106 HT-1080-FAP cells were subcutaneously inoculated into the right trunk of 8-wk-old BALB/c nu/nu mice. When the size of the tumor reached approximately 1 cm3, the radiolabeled compound was injected via the tail vein (2–5 MBq in 100 μL of 0.9% saline for small-animal imaging and 1 MBq in 100 μL of 0.9% saline for organ distribution). For organ distribution, the animals (n = 6 or 3 for each time point) were sacrificed at 1 and 4 h or at different time points (30 min–24 h) after tracer administration. The distributed radioactivity was measured in all dissected organs and in blood using a γ-counter (Cobra Autogamma; Packard). The values are expressed as percentage injected dose per gram of tissue (%ID/g). Scintigraphic images were obtained using a γ-camera (γ-Imager) with a recording time of 10 min per image. For the in vivo blockade experiments, 30 nmol of unlabeled FAPI were added to the radiolabeled compound directly before injection. [2]
All animal experiments were conducted in compliance with the German animal protection laws (permission 35-91185.81/G-158/15).

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Scintigraphy and SPECT/CT Imaging[2]
The patients gave written informed consent to undergo FAPI PET/CT, FAPI therapy, and FAPI scintigraphy following the regulations of the German Pharmaceuticals Act §13(2b). All patients were referred for the experimental diagnostics by their oncologists, who were facing an unmet diagnostic challenge that could not be solved sufficiently with standard diagnostic means. The data were analyzed retrospectively with approval of the local ethics committee (approval S016/2018). [2]
The 99mTc-FAP-34 was applied via intravenous catheter as a bolus injection of 660 MBq via a sterile filter system. Whole-body planar scintigraphy was performed at 10 min, 1 h, 4 h, and 20 h, and 2-bed-position SPECT/CT was performed at 4 h after tracer administration. [2]
Scintigraphic images were obtained using a low-energy high-resolution collimating system with an acquisition time of 1 min/15 cm of body height in a 1,025 × 256 matrix. The SPECT acquisition was performed on an Infinia scanner system using a 128 × 128 matrix, a zoom of 1, step-by-step scanning at 30 s per step, and 120 images with a 3° angle cut in a 128 × 128 matrix. For FAPI-34 imaging, a 4-slice low-dose CT scan (as a part of SPECT/CT) was performed for attenuation correction and general localization of FAPI-positive lesions. [2]
PET/CT imaging was performed on a Biograph mCT Flow scanner. After non–contrast-enhanced low-dose CT (130 keV, 30 mAs, CareDose; reconstructed with a soft-tissue kernel to a slice thickness of 5 mm), PET was acquired in 3-dimensional mode (matrix, 200 × 200) using FlowMotion. The emission data were corrected for randoms, scatter, and decay. Reconstruction was performed with ordered-subset expectation maximization using 2 iterations and 21 subsets, along with Gauss filtering to a transaxial resolution of 5 mm in full width at half maximum. Attenuation correction was performed using the nonenhanced low-dose CT data. The FAPI-46 was synthesized and labeled as described previously. The injected activity for the 68Ga-FAPI-46 (11) examinations was 260 MBq, and the PET scans began 1 h after injection. A 500-mL volume of saline with 20 mg of furosemide was infused from 15 min before to 30 min after tracer application. The patients were asked to self-report any side effects 30 min after finishing the examination.

[1]. Fap inhibitor. WO2019154886A1.

Tumor growth and spread depend not only on cancer cells but also on the non-malignant components of malignant lesions, collectively known as the stroma. In tumors with a pro-fibrotic response (e.g., breast, colon, and pancreatic cancer), the stroma may account for more than 90% of the tumor volume. In particular, a subset of fibroblasts called cancer-associated fibroblasts (CAFs) is known to be involved in tumor growth, migration, and progression. Therefore, these cells are ideal targets for diagnosis and anti-tumor therapy. A prominent feature of CAFs is the expression of serine protease or fibroblast activation protein α (FAP-α), a type II membrane-bound glycoprotein belonging to the dipeptidyl peptidase 4 (DPP4) family. FAP-α possesses both dipeptidyl peptidase and endopeptidase activities. The endopeptidase activity distinguishes FAP-α from other members of the DPP4 family. Known substrates for endopeptidase activity include denatured type I collagen, α1-antitrypsin, and various neuropeptides. FAP-α plays a role in normal developmental processes such as embryogenesis and tissue remodeling. It is expressed at very low or almost no levels in normal adult tissues. However, FAP-α is highly expressed in wound healing, arthritis, atherosclerotic plaques, fibrosis, and over 90% of epithelial carcinomas. FAP-α is present in cancer-associated fibroblasts (CAFs) of many epithelial tumors, and its overexpression is associated with poor prognosis in cancer patients, leading to the hypothesis that FAP-α activity is involved in cancer development and progression, as well as the migration and spread of cancer cells. Therefore, targeting this enzyme for imaging and internal radiotherapy could be considered an effective strategy for detecting and treating malignant tumors. The inventors have developed a small molecule based on a specific FAP-α inhibitor and demonstrated its specific uptake, rapid internalization, and successful imaging of tumors in both animal models and cancer patients. Compared to the commonly used radiotracer 18F-fluorodeoxyglucose (18F-FDG), this novel FAP-α ligand shows significant advantages in patients with locally advanced lung adenocarcinoma. Therefore, the present invention is particularly capable of: (i) detecting smaller primary tumors, thereby enabling early diagnosis; (ii) detecting smaller metastatic lesions, thereby enabling more accurate assessment of tumor staging; (iii) providing precise intraoperative guidance to facilitate complete resection of tumor tissue; (iv) better distinguishing between inflammatory tissue and tumor tissue; (v) more accurately staging tumor patients; (vi) better following up on tumor lesions after antitumor therapy; and (vii) using the molecule as a therapeutic agent for diagnosis and treatment. In addition, these molecules can also be used to diagnose and treat non-malignant diseases such as chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, and keloids. [1]

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Most epithelial tumors recruit fibroblasts and other non-malignant cells and activate them into cancer-associated fibroblasts. This usually leads to overexpression of membrane serine protease fibroblast activator protein (FAP). Studies have shown that DOTA-containing FAP inhibitors (FAPI) can generate high-contrast images in PET/CT scans. Since SPECT is a lower-cost and more widely applicable alternative to PET, 99mTc-labeled FAPI holds promise as an ideal tracer for a wider range of patient imaging applications. Furthermore, the chemical homolog 188Re is readily available from the generator, enabling FAP-targeted internal radiotherapy. Methods: To prepare the 99mTc-tricarbonyl complex, we selected a chelating agent whose carboxylic acid group is readily converted into multiple derivatives in the final product, thus enabling a platform strategy based on the original tracer. We investigated the resulting 99mTc complex in vitro using binding and competition experiments on FAP-transfected HT-1080 cells (HT-1080-FAP) or HEK cells expressing mouse FAP (HEK-muFAP) and CD26 (HEKCD26), and characterized it using planar scintillation and organ distribution studies in tumor-bearing mice. In addition, we conducted the first-in-human application in two patients with ovarian and pancreatic cancer, respectively. Results: 99mTc-FAPI-19 exhibited high affinity and specific binding to recombinant FAP-expressing cells. Unfortunately, no liver accumulation, bile excretion, or tumor uptake was observed in planar scintigraphy of HT-1080-FAP xenograft mice. To improve its pharmacokinetic properties, the researchers linked hydrophilic amino acids to the chelating moiety of the compound. The resulting 99mTc-labeled FAPI tracer showed excellent binding properties (binding rate ≤45%; internalization rate >95%), high affinity (half-maximal inhibitory concentration 6.4–12.7 nM), and significant tumor uptake (≤5.4% per gram of tissue at the injected dose) in biodistribution studies. The lead candidate 99mTc-FAPI-34 was used for diagnostic scintigraphy and SPECT examinations in patients with metastatic ovarian and pancreatic cancer, and for follow-up after 90Y-FAPI-46 treatment. 99mTc-FAPI-34 accumulates in tumor lesions, as confirmed in PET/CT imaging using 68Ga-FAPI-46. Conclusion: 99mTc-FAPI-34 is a potent diagnostic scintillation tracer, particularly useful in situations where PET imaging is unavailable. Furthermore, the chelating agent used in this compound could be used to label the therapeutic radionuclide 188Re, which is expected to be realized in the near future. [2]

C50H57F2N13O18

1166.06

1165.391

2374782-07-5

156060696

White to light yellow solid powder

-6.5

9

26

30

83

2330

3

N1(CC(=O)N[C@H](C(O)=O)CC(C(=O)O)C(=O)O)C(CN(CC(=O)N2CCN(CCCOC3=CC=C4C(=C3)C(C(=O)NCC(=O)N3CC(F)(C[C@H]3C#N)F)=CC=N4)CC2)CC2N(CC(=O)N[C@H](C(O)=O)CC(C(=O)O)C(=O)O)C=CN=2)=NC=C1

FPOZMWSPTRNDPQ-UVXHQIPUSA-N

InChI=1S/C50H57F2N13O18/c51-50(52)19-28(20-53)65(27-50)41(68)21-57-43(70)30-4-5-54-34-3-2-29(16-31(30)34)83-15-1-8-60-11-13-62(14-12-60)42(69)26-61(22-37-55-6-9-63(37)24-39(66)58-35(48(79)80)17-32(44(71)72)45(73)74)23-38-56-7-10-64(38)25-40(67)59-36(49(81)82)18-33(46(75)76)47(77)78/h2-7,9-10,16,28,32-33,35-36H,1,8,11-15,17-19,21-27H2,(H,57,70)(H,58,66)(H,59,67)(H,71,72)(H,73,74)(H,75,76)(H,77,78)(H,79,80)(H,81,82)/t28-,35-,36-/m0/s1

(3S)-3-[[2-[2-[[[2-[4-[3-[4-[[2-[(2S)-2-cyano-4,4-difluoropyrrolidin-1-yl]-2-oxoethyl]carbamoyl]quinolin-6-yl]oxypropyl]piperazin-1-yl]-2-oxoethyl]-[[1-[2-oxo-2-[[(1S)-1,3,3-tricarboxypropyl]amino]ethyl]imidazol-2-yl]methyl]amino]methyl]imidazol-1-yl]acetyl]amino]propane-1,1,3-tricarboxylic acid

FAPI-34; 2374782-07-5; SCHEMBL22966423;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: 50 mg/mL (42.88 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (4.29 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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Solubility in Formulation 2: ≥ 5 mg/mL (4.29 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 50.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)