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).

Improving Pharmacokinetics and Image Contrast in PET [2]
To assess the potential for increased tumor retention and its pharmacokinetic behavior, we performed in vivo analyses of the most promising candidates. For this purpose, we performed small animal PET imaging on HT-1080-FAP xenograft mice. All compounds showed rapid tumor accumulation with low overall background activity and were primarily excreted via the kidneys (Supplementary Fig. 4). FAPI-55 showed the highest tumor uptake (SUVmax 1.8 at 60 min and 1.7 at 120 min), followed by FAPI-36 (1.5 at 60 min and 1.3 at 120 min) and FAPI-21 (1.3 at both 60 min and 120 min) (Fig. 2, Supplementary Fig. 5). Since the absolute uptake values are limited for comparison with radiotracers, AUC values were calculated based on the time-activity curve, representing the accumulated radioactivity within 2 hours after injection. As shown in Table 1, 7 out of the 10 compounds showed higher tumor uptake than FAPI-04, with FAPI-21, -36, -46, and -55 exhibiting the highest uptake. However, FAPI-36 had a longer systemic circulation time, resulting in an unfavorable tumor/blood ratio and lower image contrast than FAPI-04 (Supplementary Figure 4). Although FAPI-35's tumor/blood and tumor/liver ratios were comparable to FAPI-04, its tumor/muscle ratio was slightly improved (Figure 3). FAPI-21 and -55 accumulated more in liver and muscle tissues than FAPI-04. Among all tested compounds, FAPI-46 had the highest tumor/blood, tumor/muscle, and tumor/liver ratios. Based on observations from imaging studies, we selected FAPI-21, -35, -46, and -55 for more detailed biodistribution studies using 177Lu-labeled radiotracers. As shown in Figure 4, all compounds exhibited significant tumor accumulation, while overall uptake in healthy tissues was lower. Because the radiotracers are primarily cleared by the kidneys, and their activity is concentrated mainly in the renal calyces, only moderate radioactivity was detected in the kidneys (1.8–3.5 %ID/g at 1 hour post-injection). Compared to FAPI-04, FAPI-21 and -46 showed higher tumor accumulation at 1 and 4 hours post-injection. Although all other compounds showed the highest intratumoral radioactivity at 1 hour post-injection, tumor uptake of FAPI-21 continued to increase from 1 to 4 hours post-injection. Furthermore, FAPI-21 had the highest tumor retention rate at 24 hours post-injection (6.03 ± 0.68 %ID/g), followed by FAPI-35 (2.47 ± 0.23 %ID/g) and FAPI-46 (2.29 ± 0.16 %ID/g), whose uptake rates were similar to those of FAPI-04 (2.86 ± 0.31 %ID/g). Correspondingly, FAPI-21 retained 64% of its maximum tumor activity 24 hours post-injection, followed by FAPI-35 (37%), FAPI-46, and FAPI-55 (all close to 20%). Compared to FAPI-04, all compounds except FAPI-55 showed equal or slightly higher blood radioactivity levels at all specified time points. FAPI-55 exhibited the highest blood radioactivity within 6 hours post-injection, but subsequently declined gradually, reaching a level similar to FAPI-04 after 24 hours. Except for FAPI-46, all derivatives showed higher hepatic uptake than FAPI-04. FAPI-46 showed comparable activity to FAPI-04 within 6 hours post-injection, but its activity gradually decreased over 24 hours. Renal activity was comparable for FAPI-04, -21, and -35, while renal activity was significantly reduced for FAPI-46 and -55 at all specified time points. Comparison of AUC calculated from time-activity curves from 1 to 24 hours post-injection showed that FAPI-21 had the highest overall tumor uptake, followed by FAPI-46 (Table 2). The tumor/organ ratios calculated based on the overall AUC indicated that the pharmacokinetics of FAPI-21 and FAPI-46 were generally improved, with no significant changes in other radiotracers except for FAPI-35 (Figure 5, Supplementary Table 3). Notably, FAPI-46 showed significantly increased tumor/liver, kidney, and brain uptake ratios. [2]

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Biodistribution[1]
Two patients were examined 10 minutes to 3 hours post-injection, and the results showed that both FAPI tracers rapidly reached stable physiological biodistribution. In normal tissues, changes were minimal from 10 minutes to 3 hours post-injection. When using 68Ga-FAPI-2, tumor uptake decreased by an average of 75% over 1 to 3 hours post-injection; clearance of 68Ga-FAPI-4 was lower, averaging only 25% (i.e., longer tumor retention time) compared to 1 to 3 hours post-injection (Figure 2, bottom). However, at 1 hour post-injection (which was also used for comparison with 18F-FDG), the two 68Ga-FAPI tracers were comparable in terms of tumor/background ratio. [1] The quantitative tumor uptake of FAPI PET was similar to that of 18F-FDG, the current reference standard for tumor PET (mean SUVmax was 7.41 for 18F-FDG and 7.37 for 68Ga-FAPI-2; the difference was not statistically significant). In pancreatic cancer, esophageal cancer, lung cancer, head and neck cancer, and colorectal cancer, quantitative tumor uptake was non-inferior to 18F-FDG. Conversely, dedifferentiated thyroid carcinomas exhibiting a reversal in 18F-FDG uptake did not accumulate 68Ga-FAPI (Figure 3). Regarding background activity, 68Ga-FAPI-2 showed significantly reduced mean SUVmax in the brain (0.32 vs. 11.01), liver (1.69 vs. 2.77), and oral/pharyngeal mucosa (2.57 vs. 4.88), thereby improving the contrast of pancreatic and colorectal cancer liver metastases and the imaging clarity of esophageal cancer (Figure 3). For all other organs, there was no significant difference between 68Ga-FAPI-2 and 18F-FDG (Figure 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 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 increased tumor/normal organ uptake ratios, resulting in higher contrast images. Notably, the tumor/blood, liver, muscle, and intestinal uptake ratios of the two radiotracers, FAPI-21 and FAPI-46, were significantly increased. In first-time diagnostic applications in cancer patients, both radiotracers showed high intratumoral uptake within 10 minutes of 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. Furthermore, higher doses of radioactive material can be delivered with minimal damage to healthy tissues, which may improve therapeutic efficacy. [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.)