FAP (fibroblast activation protein)
The compound selectively targets fibroblast activation protein (FAP), a type II transmembrane serine protease that is overexpressed on cancer-associated fibroblasts (CAFs) in over 90% of epithelial carcinomas (including breast, pancreatic, colorectal, lung, and ovarian cancers). FAP is minimally expressed in normal adult tissues, making it an excellent target for tumor-specific imaging and therapy.

Of 15 synthesized FAPIs, FAPI-04 was identified as the most promising tracer for clinical application. Compared with the previously published ligand, FAPI-02, FAPI-04 showed excellent stability in human serum, higher affinity for FAP as opposed to CD26, and slower excretion in vitro. https://pubmed.ncbi.nlm.nih.gov/29626119/

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FAPI-2 functions by binding with high affinity (typically low nanomolar KD) to the active site of FAP on CAFs, acting as an antagonist that inhibits FAP's enzymatic activity. The quinoline core structure provides specificity, while chelator conjugation (e.g., DOTA) enables radiolabeling. The TFA salt enhances solubility for radiochemistry applications.

In vivo, a higher SUV was reached in tumor-bearing animals, leading to larger areas under the curve as calculated from biodistribution experiments. Finally, PET/CT scans with 68Ga-FAPI-04 in 2 patients with metastasized breast cancer revealed high tracer uptake in metastases and a reduction in pain symptoms after therapy with a considerably low dose of 90Y-FAPI-04. Conclusion: FAPI-04 represents a promising tracer for both diagnostic imaging and, possibly, targeted therapy of malignant tumors with a high content of activated fibroblasts, such as breast cancer.https://pubmed.ncbi.nlm.nih.gov/29626119/

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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]

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In vivo, when labeled with 68Ga, FAPI-2 PET tracers exhibit rapid tracer kinetics with imaging possible within 1 hour post-injection, low background uptake in liver, oral mucosa, and brain, and no requirement for fasting preparation or blood glucose control. Tumor uptake of 68Ga-FAPI-2 is high and specific to FAP-expressing tissues. 68Ga-FAPI-2 demonstrates 75% washout from tumor from 1-3 hours post-injection, with favorable tumor-to-background ratios superior to 18F-FDG in many cancer types.

FAP enzymatic activity assays: incubate purified recombinant FAP (0.1-1 nM) with the fluorogenic substrate Z-Gly-Pro-AMC (10-100 uM) in 50 mM Tris-HCl buffer (pH 7.5, 100 mM NaCl, 1 mM EDTA) for 10-30 minutes at 37degC. Add FAPI-2 TFA at varying concentrations (1 pM-1 uM) to reaction mixture and measure fluorescence at excitation 355 nm, emission 460 nm to determine IC50. Alternatively, use radioligand binding assays with 68Ga-labeled FAPI-2.

FAPIs based on a quinoline structure were synthesized and characterized with respect to binding, internalization, and efflux in cells expressing human and murine FAP as well as CD26.https://pubmed.ncbi.nlm.nih.gov/29626119/

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For competitive binding and uptake studies, culture FAP-expressing cell lines (e.g., HT-1080 human fibrosarcoma cells or U87MG glioma cells) in DMEM with 10% FBS. Incubate cells with 68Ga-FAPI-2 (0.1-10 uCi/well) in the presence or absence of unlabeled FAPI-2 (1 nM-10 uM) as competitor for 1-2 hours at 4degC or 37degC. Wash cells with cold PBS, lyse with 0.1 M NaOH, and measure cell-associated radioactivity in a gamma counter to determine binding specificity and IC50.

Preclinical pharmacokinetics were determined in tumor-bearing animals with biodistribution experiments and small-animal PET. Finally, a proof-of-concept approach toward imaging and therapy was chosen for 2 patients with metastasized breast cancer. https://pubmed.ncbi.nlm.nih.gov/29626119/

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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[1]

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For PET imaging studies, use FAP-positive tumor-bearing mouse models (subcutaneous xenografts of HT-1080, U87MG, or patient-derived xenografts in nude or SCID mice). Administer 3-10 MBq (approximately 5-25 ug/kg) of 68Ga-FAPI-2 TFA via intravenous injection. Perform dynamic or static PET/CT scans at 10 min, 30 min, 1 h, 2 h, and 3 h post-injection. After final scan, euthanize animals, harvest major organs and tumor, weigh, and measure radioactivity in a gamma counter to calculate %ID/g for biodistribution analysis.

Preclinical pharmacokinetics determined in tumor-bearing animals show rapid blood clearance (t1/2 distribution minutes), renal excretion as primary elimination pathway, and limited hepatobiliary clearance. Tumor uptake peaks at approximately 1 hour post-injection with SUVmean values of 3-10 depending on FAP expression level. The compound exhibits low non-specific tissue retention and minimal off-target accumulation.

68Ga-FAPI-2 TFA used at tracer doses (microgram quantities of ligand, approximately 5-25 ug/kg) has negligible pharmacological toxicity. The primary radiation dose from 68Ga (half-life 68 minutes) is low and suitable for diagnostic imaging. In preclinical studies, no adverse effects were observed at imaging doses. Higher doses for radionuclide therapy (e.g., 177Lu-FAPI-2) may have myelotoxicity and renal toxicity concerns requiring dose optimization.

[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.

Currently, several radiopharmaceuticals targeting fibroblast activation proteins (FAPs) based on the highly effective FAP inhibitor UAMC1110 are under investigation. Preclinical and clinical studies have shown the potential of these imaging agents. However, the monomeric small molecules have short retention times in tumors and high renal clearance. Therefore, our strategy is to develop a homodimer system containing two FAP inhibitors to prolong retention time and enhance tumor accumulation. We synthesized the homodimers DOTA·(SA·FAPi)₂ and DOTAGA·(SA·FAPi)₂, which contain two squaramide-conjugated FAP inhibitors, and evaluated them radiochemically using gallium-68. We tested the in vitro stability, lipophilicity, and affinity of [⁶⁸Ga]Ga-DOTAGA·(SA·FAPi)₂. Furthermore, [68Ga]Ga-DOTAGA.(SA.FAPi)2 was subjected to human PET/CT scans and directly compared with [68Ga]Ga-DOTA.SA.FAPi and [18F]FDG. Gallium-68 labeling showed a higher radiochemical yield. Inhibition experiments demonstrated that the compound exhibits excellent affinity and selectivity for FAP, with IC50 values down to the nanomolar level. In human PET/CT studies, [68Ga]Ga-DOTAGA.(SA.FAPi)2 showed significantly higher tumor uptake and longer tumor retention time compared to [68Ga]Ga-DOTA.SA.FAPi. Therefore, the introduction of the dimer has advanced human PET imaging technology, manifested in increased tumor accumulation and prolonged in vivo retention time. Thus, the application of the dimer structure may be the next step in prolonging the uptake time of FAP inhibitors, thereby developing radiotherapy analogs of FAP inhibitors. [2]

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Fibroblast activating protein (FAP) is a type II membrane-bound glycoprotein that is overexpressed in cancer-associated fibroblasts and activated fibroblasts at wound healing/inflammatory sites. Since the first clinical application of quinoline-based FAP ligands in 2018, FAP inhibitor-based PET imaging and radiotherapy have been used to study a variety of diseases, including malignant and non-malignant ones. Therefore, remarkable progress has been made, especially in deepening the understanding of FAPI-based PET imaging and the potential value of FAPI-based tumor radiotherapy. This article provides a comprehensive review of radiolabeled FAPIs and their clinical translation, aiming to elucidate the current and future potential roles of such molecules in nuclear medicine. In particular, this article highlights the value of FAPI radiopharmaceuticals in the diagnosis and treatment of tumors or benign diseases. However, current research limitations hinder the accurate evaluation of FAPI radiopharmaceuticals. Nevertheless, it remains important to further explore the clinical value of FAPIs in diagnosis and treatment through the design of more sophisticated and larger-sample clinical trials in the future. [3]

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FAPI-2 TFA is also designated FAPI-02. It is one of the most extensively studied FAP-targeting ligands, with numerous clinical trials ongoing for PET imaging and radionuclide therapy (theranostics) of various cancers. 68Ga-FAPI-2 has advantages over 18F-FDG including lower physiological uptake in brain, liver, and oral mucosa, and no need for fasting, making it particularly valuable for detecting peritoneal carcinomatosis and primary brain tumors. The compound is for research use only.

C42H57F3N10O12

950.96

Off-white to light yellow solid powder

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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