FGFR1 (IC50 = 3.9 nM); FGFR2 (IC50 = 1.3 nM); FGFR3 (IC50 = 1.6 nM); FGFR4 (IC50 = 8.3 nM); wild-type FGFR2 (IC50 = 0.3 nM); FGFR2 V5651 (IC50 = 1-3 nM); FGFR2 N550H (IC50 = 3.6 nM); FGFR2 E566G (IC50 = 2.4 nM)
Futibatinib (TAS-120) is an irreversible pan-FGFR inhibitor that covalently binds to a highly conserved cysteine residue (C492 in FGFR2-IIIb isoform) within the ATP-binding pocket of FGFR1–4. It exhibits low nanomolar in vitro potency against wild-type FGFR1–4 and retains activity against multiple secondary FGFR2 kinase domain mutations (e.g., N550K, E566A, K660M, L618V, H683L), except for the V565F gatekeeper mutation which confers high-level resistance (103‑fold increase in IC50). [1]

Futibatinib (TAS-120) is a permanent fibroblast growth factor receptor (FGFR) inhibitor that inhibits all four FGFR subtypes, with enzyme half-lives (IC50) for FGFR1, FGFR2, FGFR3, and FGFR4 being 1.8 nM, 1.4 nM, 1.6 nM, and 3.7 nM, respectively.

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Futibatinib shows potent anti-proliferative activity in FGFR-driven intrahepatic cholangiocarcinoma (ICC) cell lines. In ICC13‑7 cells (harboring FGFR2‑OPTN fusion) and CCLP‑1 cells (overexpressing wild‑type FGFR1), the IC50 values were 0.6–1.5 nM, while FGFR‑independent cell lines exhibited IC50 values of 300–8000 nM. [1]
Treatment with 50 nM Futibatinib effectively suppressed phosphorylation of FRS2 (Y196), SHP2 (Y542), MEK1/2 (S217/221), and ERK1/2 (T202/Y204) in ICC13‑7 and CCLP‑1 cells, indicating durable inhibition of the MEK/ERK pathway without reactivation for up to 3 days. No significant inhibition of PI3K/AKT signaling (pAKT T308/S473) was observed. [1]
In engineered CCLP‑1 cells expressing FGFR2‑PHGDH fusions with various kinase domain mutations, Futibatinib maintained activity (2‑ to 7‑fold IC50 increase) against mutations that confer resistance to ATP‑competitive inhibitors (BGJ398, Debio1347), except for V565F (103‑fold IC50 increase). [1]
A pooled clone system containing 1% of each mutant FGFR2‑PHGDH clone and 90% wild‑type cells was treated with 10 nM Futibatinib for 14 days. ddPCR analysis showed outgrowth of V565F, and to a lesser extent E566A and N550K, while other mutants were suppressed. [1]

TAS-120 (3, 30, 100 mg/kg/day, p.o.) exerts an anti-tumor effect in mice. By lowering the blood phosphorus level's sustained elevation and weight suppression, as well as by intermittently administering the drug every other day and twice a week, TAS-120 exhibits anti-tumor effects. Its daily administration is also effective.

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To corroborate these results in vivo, we screened a collection of patient-derived xenograft (PDX) models of ICC for FGFR alterations, and identified a model harboring a FGFR2-KIAA1217 fusion (designated MG69) (Supplemental Figure S1G). Treatment of MG69 PDX tumors with Futibatinib (TAS-120)  (starting when the volume reached ~500 mm3) led to tumor regression and complete proliferative arrest, with prominent effects evident within three days and persisting over a 14-day course (Figure 2E, F). Moreover, FGFR inhibition suppressed MEK/ERK and SHP2 activity, but not PI3K signaling, in MG69 PDX tumors (Figure 2G). Thus, FGFR activated ICC models are highly dependent on FGFR activity to sustain growth and maintain MEK/ERK signaling in vitro and in vivo [1].

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In a patient‑derived xenograft (PDX) model of ICC harboring an FGFR2‑KIAA1217 fusion (MG69), oral administration of Futibatinib (25 mg/kg daily) induced tumor regression and complete proliferative arrest within 3 days, effects that persisted over a 14‑day treatment course. [1]
Immunoblot analysis of PDX tumors after 14 days of Futibatinib treatment showed suppression of pFRS2, pSHP2, pMEK, and pERK, but not PI3K/AKT signaling, consistent with in vitro findings. [1]

1-[(3S)-3-[4-Amino-3-[2-(3,5-dimethoxyphenyl)ethynyl]-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-pyrrolidinyl]-2-propen-1-one (TAS-120) is an irreversible inhibitor of the fibroblast growth factor receptor (FGFR) family, and is currently under phase I/II clinical trials in patients with confirmed advanced metastatic solid tumours harbouring FGFR aberrations. This inhibitor specifically targets the P-loop of the FGFR tyrosine kinase domain, forming a covalent adduct with a cysteine side chain of the protein. Our mass spectrometry experiments characterise an exceptionally fast chemical reaction in forming the covalent complex. The structural basis of this reactivity is revealed by a sequence of three X-ray crystal structures: a free ligand structure, a reversible FGFR1 structure, and the first reported irreversible FGFR1 adduct structure. We hypothesise that the most significant reactivity feature of TAS-120 is its inherent ability to undertake conformational sampling of the FGFR P-loop. In designing novel covalent FGFR inhibitors, such a phenomenon presents an attractive strategy requiring appropriate positioning of an acrylamide group similarly to that of TAS-120 [2].

Overexpressing FGFR in a human gastric cancer cell line The Dulbecco's Modified Eagle (OCUM-2MD3) cells, which contain 10% fetal bovine serum (FBS) in medium (DMEM), are routinely passaged at a cell density of no more than 80%. In order to test the cytostatic activity, 3,000 cells per well are seeded in each well of 96-well flat-bottom plates. The cells are then suspended in the DMEM medium above and cultured for one day at 37°C in an incubator with 5% carbon dioxide gas. It is stage-diluted to 100 times the final concentration of the test compound in DMSO the following day. The test compound is diluted with DMSO solution and added to a final concentration of DMSO in each well of a culture plate containing cells at a rate of 0.5%, 5% carbon dioxide gas incubator, and then cultured for 72 hours at 37°C. Following the suggested protocols provided by Dojindo Laboratories, the number of cells is measured using a cell counting kit-8 72 hours after the test compound is added to the culture. The reagent kit is added to each plate, and the color reaction is carried out for a predetermined amount of time at 37°C in an incubator with 5% carbonic acid gas. A microplate reader is used to measure the absorbance at 450 nm after the reaction is finished. GI50 (nM) is the concentration of the test compound at which 50% inhibition occurs, which is calculated by applying the growth inhibition rate formula.

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For cell viability assays, cells were dissociated and seeded into 384‑well plates (200 viable cells/well). After 24 hours, Futibatinib was added over a 15‑point concentration range using a digital drug dispenser. Cells were cultured for 5 days, and viability was assessed by adding Cell Titer‑Glo reagent, incubating for 20 minutes, and measuring luminescence. IC50 values were determined using a 4‑parameter dose‑response model. [1]
For immunoblot analysis, cells were treated with drugs in 6‑well plates for 5–8 hours. Lysates were prepared in RIPA buffer containing protease and phosphatase inhibitors. Proteins were separated by SDS‑PAGE, transferred, and probed with specific antibodies against phospho‑FRS2 Y196, phospho‑SHP2 Y542, phospho‑MEK1/2 S217/221, phospho‑ERK1/2 T202/Y204, phospho‑AKT T308/S473, and total proteins. [1]
For the clonal pool growth modeling, cell pools containing 1% of each mutant FGFR2‑PHGDH clone and 90% wild‑type cells were seeded in 6‑well plates. Drugs (or DMSO) were added 24 hours later and refreshed every 3–4 days. After 1, 7, or 14 days, genomic DNA was extracted and analyzed by ddPCR to determine fractional abundance of mutant alleles. [1]

The old 6-week-old male nude rats with an intermittent administration schedule are transplanted to the right chest of the anti-tumor effect human gastric cancer strain (OCUM-2MD3). Measuring the tumor's volume after implantation and its major and minor axes in millimeters: The day 0 of the days that are conducted in groups of (n=5) is determined by allocating the mouse average TV to each group after the tumor volume TV has been calculated. Futibatinib (TAS-120)  is prepared so that it contains 3 mg/kg/day and 30 mg/kg/day. 3 mg/kg/day is taken orally every day, while 30 mg/kg/day is taken orally every other day. 100 mg/kg/day is taken orally twice a week starting on day 1, with a 14-day evaluation period and a 15-day final valuation date.

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PDX treatment studies [1]
To develop an FGFR2 fusion human PDX, we obtained tissue from a fresh resection specimen from a patient with an FGFR2-KIAA1217 fusion ICC tumor, per our IRB-approved protocol. The tissue was rinsed in HBSS and cut into 0.3–0.5 mm3 pieces with sterile razor blades. These tumor pieces were implanted subcutaneously into 6–8-week old female NSG mice. Tumor size was measured with a digital caliper. Upon reaching ~500 mm3, mice were randomized to either vehicle control or 25 mg/kg Futibatinib (TAS-120) (in hydroxypropyl methyl cellulose solution) by oral gavage daily for three and fourteen days prior to harvest.

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NSG mice bearing subcutaneous PDX tumors (FGFR2‑KIAA1217 fusion) were randomized when tumor volume reached approximately 500 mm³. Futibatinib was administered orally at 25 mg/kg daily, formulated in hydroxypropyl methyl cellulose solution, for 3 or 14 days before tumor harvest. Control mice received vehicle alone. Tumor size was measured with digital calipers. [1]
For histology and immunohistochemistry, harvested tumors were fixed in 4% buffered formaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin & eosin or an anti‑Ki‑67 antibody (1:400 dilution) to assess proliferation. [1]

Absorption, Distribution and Excretion
The Tmax ranged from 1.2 to 22.8 hours, with a median of 2 hours. In healthy subjects, a high-fat, high-calorie meal (900 to 1000 calories, with approximately 50% of the total calories from fat) reduced the AUC of fabatinib by 11% and Cmax by 42%. Following a single oral dose of 20 mg of radiolabeled fabatinib, approximately 91% of the total recovered radioactive material was found in feces, and 9% in urine. The amount of unmetabolized fabatinib in urine or feces was negligible. The geometric mean (CV%) apparent volume of distribution (Vc/F) was 66 L (18%). The geometric mean (CV%) apparent clearance (CL/F) was 20 L/h (23%).

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Metabolism/Metabolites
In vitro studies have shown that futazine is primarily metabolized by CYP3A, followed by CYP2C9 and CYP2D6. In healthy subjects, unmetabolized futazine was the major drug-related component in plasma (accounting for 59% of radioactivity).

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Biological Half-Life
The mean (CV%) elimination half-life (t_1/2) of futazine was 2.9 hours (27%).

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This study indicates that the irreversible binding mode of futazine permanently inhibits the enzyme activity of FGFR2, potentially prolonging the duration of efficacy without maintaining high drug concentrations. [1]

Hepatotoxicity
Adverse events were relatively common in open-label clinical trials of fubatinib, leading to discontinuation of treatment in 66% of patients, reduction in treatment in 58% of patients, and discontinuation in 5% of patients. However, only a small percentage of these were due to elevated serum transaminases. In a pre-registration trial of 103 patients with cholangiocarcinoma, 50% experienced elevated ALT levels, with 7% of patients experiencing ALT levels more than 5 times the upper limit of normal. These elevations were usually self-limiting and returned to normal rapidly regardless of dose adjustment. No patients experienced clinically significant liver injury or jaundice. The literature on the efficacy and safety of fubatinib rarely mentions elevated serum ALT or hepatotoxicity. Since fubatinib's approval, no cases of clinically significant liver injury have been reported. However, the overall clinical experience with this drug is limited, and the high incidence of elevated serum transaminases during treatment suggests the possibility of clinically significant liver injury. Probability Score: E (Unproven, but likely a rare cause of clinically significant liver injury).

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Effects during pregnancy and lactation>
◉ Overview of use during lactation
There is currently no information regarding the clinical use of flotebatinib during lactation. Because flotebatinib binds to plasma proteins at a rate of up to 95%, its concentration in breast milk may be very low. The manufacturer recommends discontinuing breastfeeding during flotebatinib treatment and for one week after the last dose.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding>
Flotebatinib binds to human plasma proteins at a rate of 95% in vitro, with binding concentrations ranging from 0.2 to 5 µmol/L, primarily albumin and α1-acid glycoprotein.

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Due to the targeted blockade of the FGF23-FGFR1 signaling pathway in the renal tubules, hyperphosphatemia was observed to be a class effect of FGFR inhibitors. In the clinical cohort, one patient taking fubatinib had to discontinue the drug due to hyperphosphatemia, and two patients taking BGJ398 also had to discontinue the drug for the same reason. [1]
Other adverse events leading to dose adjustments in patients taking fubatinib included grade 3 motor neuropathy, grade 2 ALT/AST elevation, and grade 4 creatine kinase elevation. [1]

[1]. TAS-120 Overcomes Resistance to ATP-Competitive FGFR Inhibitors in Patients with FGFR2 Fusion-Positive Intrahepatic Cholangiocarcinoma. Cancer Discov. 2019 Aug;9(8):1064-1079.

[2]. TAS-120 Cancer Target Binding: Defining Reactivity and Revealing the First Fibroblast Growth Factor Receptor 1 (FGFR1) Irreversible Structure. ChemMedChem. 2019 Feb 19;14(4):494-500.

[3]. Molecular targeted therapies: Ready for "prime time" in biliary tract cancer [published online ahead of print, 2020 Mar 12]. J Hepatol. 2020;S0168-8278(20)30165-3.

Pharmacodynamics
Futabatinib is an anticancer drug that has been shown to have antitumor activity in mouse and rat xenograft models of human tumors carrying activating FGFR gene alterations. Futabatinib is not expected to affect cell lines that do not carry FGFR genomic abnormalities. It inhibits tumor growth in a dose-dependent manner. Futabatinib is a third-generation irreversible FGFR inhibitor designed to overcome acquired resistance to ATP-competitive FGFR inhibitors (such as BGJ398/infigratinib and Debio1347) in FGFR2 fusion-positive intrahepatic cholangiocarcinoma. [1] In a phase I basket trial, futabatinib showed an overall response rate of 25.0% and a disease control rate of 78.6% in 28 patients with ICC carrying the FGFR2 fusion gene, including some patients who had previously received ATP-competitive FGFR inhibitors. [1]
Resistance to fumarinib may stem from FGFR2 V565F gating mutations, but no mutations at the covalent binding site (C492) were found in the patients studied. [1]
This study supports strategic sequential therapy with FGFR inhibitors, guided by serial ctDNA and tumor biopsy, to prolong the efficacy of treatment in patients with FGFR2-altered ICC. [1]

C22H22N6O3

418.4485

418.18

C, 63.15; H, 5.30; N, 20.08; O, 11.47

1448169-71-8

71621331

Off-white to light beige solid powder

1.0±0.1 g/cm3

244.0±0.0 °C at 760 mmHg

87.5±21.3 °C

0.0±0.4 mmHg at 25°C

1.490

2.39

1

7

6

31

723

1

O=C(C=C)N1CC[C@@H](C1)N1C2C(=C(N)N=CN=2)C(C#CC2C=C(C=C(C=2)OC)OC)=N1

KEIPNCCJPRMIAX-HNNXBMFYSA-N

InChI=1S/C22H22N6O3/c1-4-19(29)27-8-7-15(12-27)28-22-20(21(23)24-13-25-22)18(26-28)6-5-14-9-16(30-2)11-17(10-14)31-3/h4,9-11,13,15H,1,7-8,12H2,2-3H3,(H2,23,24,25)/t15-/m0/s1

1-[(3S)-3-[4-amino-3-[2-(3,5-dimethoxyphenyl)ethynyl]pyrazolo[3,4-d]pyrimidin-1-yl]pyrrolidin-1-yl]prop-2-en-1-one

Futibatinib; TAS-120; TAS 120; 1448169-71-8; Lytgobi; UNII-4B93MGE4AL; 4B93MGE4AL; Futibatinib [USAN]; TAS120

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: ≥ 29 mg/mL (~69.3 mM)

Solubility in Formulation 1: 2.08 mg/mL (4.97 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: 2.08 mg/mL (4.97 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: ≥ 2.08 mg/mL (4.97 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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