FAP (fibroblast activation protein)

Chemical Modification of the FAPI Framework Resulting in Increased FAP Binding In Vitro.[2]
To determine the FAP-binding affinities of the radiotracers (Supplemental Table 1), radioligand binding assays were performed using human FAP-expressing HT-1080 cells. To compensate for varying rates of FAP expression and allow a direct comparison with the lead structure, all experiments were conducted in parallel with FAPI-04. All compounds demonstrated robust binding to human FAP, with binding values equal to or higher than those of FAPI-04 after 1 and 4 h of incubation (Fig. 1). Internalization rates were comparable to those of FAPI-04 for all compounds except for FAPI-38 (63.1% internalized after 24 h; FAPI-04, 97.1%; Supplemental Table 2). Although most derivatives revealed higher binding values after 24 h than for FAPI-04, the compounds FAPI-38, -39, -40, and -41 were eliminated significantly faster from FAP-expressing cells and were, therefore, not considered for a more detailed characterization. Similar to FAPI-04, all compounds demonstrated negligibly low binding to the structurally related membrane protein CD26 (data not shown).

FAPI-4 (intravenous injection; 30 nmol per mouse; once) exhibits excellent tumor food effects in BALB/c nu/nu mice [2].

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Fibroblast activation protein (FAP) is overexpressed in cancer-associated fibroblasts of several tumor entities. The recent development of quinoline-based PET tracers that act as FAP inhibitors (FAPIs) demonstrated promising results preclinically and already in a few clinical cases. Consequently, these tracers are now applied in our hospital to amend the diagnostics of cancer patients facing the limitations of standard examinations. Here, we analyze the tissue biodistribution and preliminary dosimetry of 2 members of this new class of PET radiopharmaceutical. Methods: A preliminary dosimetry estimate for 68Ga-FAPI-2 and 68Ga-FAPI-4 was based on 2 patients examined at 0.2, 1, and 3 h after tracer injection using the QDOSE dosimetry software suit. Further PET/CT scans of tumor patients were acquired 1 h after injection of either 68Ga-FAPI-2 (n = 25) or 68Ga-FAPI-4 (n = 25); for 6 patients an intraindividual related 18F-FDG scan (also acquired 1 h after injection) was available. For the normal tissue of 16 organs, a 2-cm spheric volume of interest was placed in the parenchyma; for tumor lesions, a threshold-segmented volume of interest was used to quantify SUVmean and SUVmax. Results: Similar to literature values for 18F-FDG, 68Ga-DOTATATE, and 68Ga-PSMA-11, an examination with 200 MBq of 68Ga-FAPI-2 or 68Ga-FAPI-4 corresponds to an equivalent dose of approximately 3–4 mSv. After a fast clearance via the kidneys, the normal organs showed a low tracer uptake with only minimal changes between 10 min and 3 h after injection. In 68Ga-FAPI-2, the tumor uptake from 1 to 3 h after injection decreased by 75%, whereas the tumor retention was prolonged with 68Ga-FAPI-4 (25% washout). Regarding tumor-to-background ratios, at 1 h after injection both 68Ga-FAPI tracers performed equally. In comparison to 18F-FDG, the tumor uptake was almost equal (average SUVmax, 7.41 for 18F-FDG and 7.37 for 68Ga-FAPI-2; not statistically significant); the background uptake in brain (11.01 vs. 0.32), liver (2.77 vs. 1.69), and oral/pharyngeal mucosa (4.88 vs. 2.57) was significantly lower with 68Ga-FAPI. Other organs did not relevantly differ between 18F-FDG and 68Ga-FAPI. Conclusion: FAPI PET/CT is a new diagnostic method in imaging cancer patients. In contrast to 18F-FDG, no diet or fasting in preparation for the examination is necessary, and image acquisition can potentially be started a few minutes after tracer application. Tumor-to-background contrast ratios were equal to or even better than those of 18F-FDG.[1]

Cell Culture[2]
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/Disease Models: 8weeks old BALB/c nu/nu (nude) mice were inoculated with HT-1080-FAP cells [2].
Doses: 30 nmol per mouse.
Route of Administration: intravenous (iv) (iv)injection; 30 nmol per mouse; one time.
Experimental Results: Display demonstrated higher overall tumor uptake (9.44% ID/g 4 hrs (hrs (hours)) after injection).

Improvement of Pharmacokinetics and Image Contrast for PET[2]
To assess a potential increase in tumor retention and to evaluate their pharmacokinetic behavior, the most promising candidates were analyzed in vivo. To this end, small-animal PET imaging was performed on HT-1080-FAP xenografted mice. All compounds demonstrated rapid tumor accumulation with overall low background activity and predominantly renal elimination (Supplemental Fig. 4). Tumor uptake was highest for FAPI-55 (SUVmax of 1.8 after 60 min and 1.7 after 120 min), followed by FAPI-36 (1.5 after 60 min and 1.3 after 120 min) and FAPI-21 (1.3 after both 60 and 120 min) (Fig. 2, Supplemental Fig. 5). Because the absolute uptake values allow only a limited comparison of the radiotracers, AUCs were calculated from the time–activity curves, representing the accumulated radioactivity within the interval up to 2 h after injection. As shown in Table 1, 7 of 10 compounds demonstrated higher tumor uptake than that for FAPI-04, headed by FAPI-21, -36, -46, and -55. Yet, FAPI-36 showed a prolonged systemic circulation, resulting in unfavorable tumor-to-blood ratios and a poorer image contrast than that for FAPI-04 (Supplemental Fig. 4). Although the tumor-to-blood and tumor-to-liver ratios for FAPI-35 were comparable to those for FAPI-04, the tumor-to-muscle ratio was slightly improved (Fig. 3). FAPI-21 and -55 demonstrated higher accumulation in liver and muscle tissue than did FAPI-04. From all tested compounds, FAPI-46 displayed the highest tumor-to-blood, tumor-to-muscle, and tumor-to-liver ratios.

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On the basis of the observations in the imaging studies, FAPI-21, -35, -46, and -55 were selected for a more detailed characterization in biodistribution studies using 177Lu-labeled radiotracers. As shown in Figure 4, all compounds demonstrated robust tumor accumulation with overall low uptake into healthy tissue. Moderate radioactivity (1.8–3.5 %ID/g 1 h after injection) was measured only in the kidneys, because of the predominantly renal elimination of the radiotracers, with activity mostly in the renal calyx system. In comparison to FAPI-04, FAPI-21 and -46 demonstrated higher tumor accumulation 1 and 4 h after injection. Although all other compounds displayed their highest intratumoral radioactivity 1 h after injection, tumor uptake was increasing even from 1 to 4 h for FAPI-21. In addition, FAPI-21 revealed the highest tumor retention 24 h after injection (6.03 ± 0.68 %ID/g), followed by FAPI-35 (2.47 ± 0.23 %ID/g) and -46 (2.29 ± 0.16 %ID/g), featuring uptake rates similar to FAPI-04 (2.86 ± 0.31 %ID/g). Accordingly, 64% of the maximum tumor activity was still present 24 h after injection for FAPI-21, followed by FAPI-35 (37%), FAPI-46, and FAPI-55 (almost 20% each). In comparison to FAPI-04, radioactivity levels in the blood were equal or marginally higher at all specified times, except for FAPI-55, which demonstrated the highest blood activities of all compounds up to 6 h after injection. However, blood activity was decreasing steadily, reaching values similar to FAPI-04 after 24 h. All derivatives demonstrated an increased liver uptake as compared with FAPI-04, except for FAPI-46, which displayed comparable activities up to 6 h after injection but narrowed to lower levels in the course of 24 h. The renal activity of the compounds was comparable for FAPI-04, -21, and -35 but significantly reduced for FAPI-46 and -55 at all specified times. A comparison of AUCs, determined from the time–activity curves from 1 to 24 h after injection, revealed the highest overall tumor uptake to be for FAPI-21, followed by FAPI-46 (Table 2). A calculation of tumor-to-organ ratios, based on the overall AUCs, evinced a general improvement in pharmacokinetics for FAPI-21 and -46 and no considerable change for any of the other radiotracers, except for FAPI-35 (Fig. 5, Supplemental Table 3). Notably, FAPI-46 displayed substantially improved ratios of tumor to liver, kidney, and brain uptake.[2]

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Biodistribution[1]
The 2 patients examined 10 min to 3 h after injection demonstrated that both FAPI tracers rapidly reached their stable physiologic biodistribution. In normal tissue, changes between 10 min and 3 h after injection were minimal. Tumor uptake declined by a mean of 75% from 1 h to 3 h after injection using 68Ga-FAPI-2; less washout, only 25% (mean), between 1 h and 3 h after injection (i.e., longer tumor retention) was observed with 68Ga-FAPI-4 (Fig. 2, bottom). However, at 1 h after injection (the time point also chosen for comparison to 18F-FDG), both 68Ga-FAPI tracers performed equally with regard to tumor-to-background ratios.[1]
The quantitative tumor uptake of FAPI PET was similar to that of the current oncologic PET standard of reference, 18F-FDG (average SUVmax, 7.41 for 18F-FDG and 7.37 for 68Ga-FAPI-2; not statistically significant). In pancreatic, esophageal, lung, head and neck, and colorectal cancer, the quantitative tumor uptake was noninferior to that of 18F-FDG. In contrast, dedifferentiated thyroid cancer with flip-flop uptake of 18F-FDG was not accumulating 68Ga-FAPI (Fig. 3). Regarding background activity, the average SUVmax of 68Ga-FAPI-2 was significantly lower in brain (0.32 vs. 11.01), liver (1.69 vs. 2.77), and oral/pharyngeal mucosa (2.57 vs. 4.88), thus improving the contrast ratios for liver metastases of pancreatic and colorectal cancer and delineation of the esophageal cancer (Fig. 3). For all other organs, 68Ga-FAPI-2 presented no significant difference from 18F-FDG (Fig. 4A).

[1]. 68Ga-FAPI PET/CT: Biodistribution and Preliminary Dosimetry Estimate of 2 DOTA-Containing FAP-Targeting Agents in Patients with Various Cancers. J Nucl Med. 2019 Mar;60(3):386-392.

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

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.[2]

C40H54F2N10O10

872.93

872.4

C, 55.04; H, 6.24; F, 4.35; N, 16.05; O, 18.33

2374782-02-0

138454803

White to off-white solid powder

1.46±0.1 g/cm3(Predicted)

1144.1±65.0 °C(Predicted)

-6.8

4

19

16

62

1580

1

FC1(C([H])([H])N(C(C([H])([H])N([H])C(C2C([H])=C([H])N=C3C([H])=C([H])C(=C([H])C=23)OC([H])([H])C([H])([H])C([H])([H])N2C([H])([H])C([H])([H])N(C(C([H])([H])N3C([H])([H])C([H])([H])N(C([H])([H])C(=O)O[H])C([H])([H])C([H])([H])N(C([H])([H])C(=O)O[H])C([H])([H])C([H])([H])N(C([H])([H])C(=O)O[H])C([H])([H])C3([H])[H])=O)C([H])([H])C2([H])[H])=O)=O)[C@]([H])(C#N)C1([H])[H])F

ICWDAESAANBIGG-LJAQVGFWSA-N

InChI=1S/C40H54F2N10O10/c41-40(42)21-29(22-43)52(28-40)34(53)23-45-39(61)31-4-5-44-33-3-2-30(20-32(31)33)62-19-1-6-46-15-17-51(18-16-46)35(54)24-47-7-9-48(25-36(55)56)11-13-50(27-38(59)60)14-12-49(10-8-47)26-37(57)58/h2-5,20,29H,1,6-19,21,23-28H2,(H,45,61)(H,55,56)(H,57,58)(H,59,60)/t29-/m0/s1

(S)-2,2',2''-(10-(2-(4-(3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)propyl)piperazin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

FAPI-04; FAPI 04; FAPI04; FAPI-4; FAPI-4; 2374782-02-0; DOTA-fapi-04; (S)-2,2',2''-(10-(2-(4-(3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)propyl)piperazin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid; D7X5DKW7N9; FAPI-04; UNII-D7X5DKW7N9; SCHEMBL21257058; FAPI 4; FAPI4;

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 (e.g. under nitrogen), 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)

DMSO : ~100 mg/mL (~114.56 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.86 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 25.0 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: ≥ 2.5 mg/mL (2.86 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 25.0 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (2.86 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 4 mg/mL (4.58 mM) in Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
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.)