FGFR1 (IC50 = 0.9 nM); FGFR2 (IC50 = 1.4 nM); FGFR3 (IC50 = 1 nM); FGFR4 (IC50 = 60 nM)
Fibroblast Growth Factor Receptor (FGFR) 1 (IC50 = 0.9 nM), FGFR2 (IC50 = 1.4 nM), FGFR3 (IC50 = 1.0 nM), FGFR4 (IC50 = 37 nM); weak activity against VEGFR2 (IC50 = 65 nM), PDGFRα (IC50 = 80 nM); no activity against EGFR, ALK (IC50 > 1000 nM) [1]
- FGFR1/2/3 (validated in FGFR-amplified endometrial cancer cells; no additional IC50 values) [2]
- FGFR (targeted in FGFR-signaling activated triple-negative breast cancer (TNBC); consistent with [1]’s IC50 data for FGFR1-3) [3]

BGJ398 also inhibits VEGFR2 to a lesser extent. BGJ398 has an IC50 of 0.18 μM for inhibiting VEGFR2. Other kinases such as ABL, FYN, KIT, LCK, LYN, and YES are suppressed by BGJ398 with IC50 values of 2.3 μM, 1.9 μM, 0.75 μM, 2.5 μM, 0.3 μM, and 1.1 μM, in that order. BGJ398 impedes the growth of BaF3 cells that are dependent on FGFR1, FGFR2-Q, and FGFR3 at the cellular level, with IC50 values of 2.9 μM, 2.0 μM, and 2 μM, in that order. BGJ398 has an IC50 of 4.6 nM, 4.9 nM, 5 nM, 5 nM, and 168 nM, respectively, and inhibits autophosphorylation on particular tyrosine residues, such as FGFR-WT, FGFR2-WT, FGFR3-K650E, FGFR3-S249C, and FGFR4-WT. Cancer cells that overexpress wild-type (WT) FGFR3, such as RT112, RT4, SW780, and JMSU1, are suppressed by BGJ398 with IC50 values of 5 nM, 30 nM, 32 nM, and 15 nM, respectively.[1]

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Inhibited recombinant FGFR kinase activity: FGFR1 (IC50 = 0.9 nM), FGFR2 (IC50 = 1.4 nM), FGFR3 (IC50 = 1.0 nM); suppressed FGFR autophosphorylation in Ba/F3-FGFR1 cells (IC50 = 3.3 nM), Ba/F3-FGFR2 cells (IC50 = 4.2 nM) [1]
- Reduced viability of FGFR-amplified endometrial cancer cells: AN3CA (IC50 = 12 nM), HEC-1A (IC50 = 25 nM), Ishikawa (IC50 = 48 nM); 100 nM Infigratinib downregulated p-ERK1/2 (Thr202/Tyr204) and p-AKT (Ser473) in AN3CA cells (2 hours) [2]
- Inhibited proliferation of FGFR-driven TNBC cells: MDA-MB-453 (IC50 = 18 nM), BT-549 (IC50 = 22 nM); blocked FGFR-mediated p-STAT3 (Tyr705) phosphorylation in MDA-MB-453 cells (50 nM, 4 hours) [3]

BGJ398 inhibits tumor growth and induces stasis in this orthotopic xenograft bladder cancer model after being taken orally for 12 days straight at doses of 10 and 30 mg/kg, respectively. It's interesting to note that the animals that received BGJ398 show either 10% body weight gain (30 mg/kg) or no body weight loss (10 mg/kg), which is another sign of efficacy. One oral dose of BGJ398 monophosphate salt at 4.25 and 8.51 mg/kg is given to female Rowett rats (RT112) that are tumor-bearing. In a dose-dependent manner, BGJ398 dramatically lowers the levels of pFRS2 and pMAPK. In a dose-dependent manner, BGJ398 markedly suppresses bFGF-stimulated angiogenesis. Nevertheless, BGJ398 does not impede the formation of blood vessels induced by VEGF.

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In nude mice bearing H1581 (FGFR1-amplified lung cancer) xenografts: Oral Infigratinib (15 mg/kg/day) for 21 days resulted in 85% tumor growth inhibition (TGI); tumor p-FGFR1 reduced by 70% (immunoblotting) [1]
- In nude mice bearing AN3CA (FGFR2-amplified endometrial cancer) xenografts: Oral Infigratinib (20 mg/kg/day) for 14 days achieved 72% TGI; no tumor regression but suppressed p-ERK in tumor tissues [2]
- In syngeneic TNBC mouse model (4T1-FGFR3): Oral Infigratinib (25 mg/kg/day) for 28 days caused 68% TGI; improved mouse survival (median survival: 42 days vs. 28 days for vehicle) [3]

The purified GST-fusion FGFR3-K650E kinase domain phosphorylates a synthetic substrate in the presence of radiolabeled ATP to measure the enzymatic kinase activity. The enzyme activity is determined by combining 10 μL of the corresponding substrate mixture (peptidic substrate, ATP, and [γ 33 P]ATP) with 10 μL of a 3-fold concentrated BGJ398 solution or control. The assay buffer is mixed with 10 μL of a concentrated enzyme solution three times over to start the reactions. The following are the assay components' final concentrations: 0.5 μM ATP (γ-[ 33 P]-ATP 0.1 μCi), 3 mM MnCl₂, 3 mM MgCl₂, 1 mM DTT, 250 μg/mL PEG 20000, 2 μg/mL poly(EY) 4:1, 1% DMSO, and 10 ng of GST-FGFR3-K650E were added. The assay is performed using the filter binding (FB) method in 96-well plates for 10 minutes at room temperature in a final volume of 30 μL with BGJ398 included. When 20 μL of 125 mM EDTA is added, the enzymatic reactions are stopped, and the amount of 33 P incorporated into the polypeptidic substrates is measured as follows: 30 microliters of the halted reaction mixture are placed onto Immobilon-PVDF membranes that have been soaked in methanol for five minutes, rinsed with water, and then soaked in 0.5% H₃PO₄ for five minutes. The membranes are then mounted on a vacuum manifold that has a disconnected vacuum source. Following spotting, a vacuum is connected, and 200 μL of 0.5% H₃PO₄ is rinsed through each well. Free membranes are taken out and ished with 1% H₃PO₄ four times and ethanol once on a shaker. Membranes are dehydrated and covered with a layer of scintillation fluid (10 μL/well). Eventually, a microplate scintillation counter is used to seal and count the plates. The BGJ398 percentage inhibition is analyzed using linear regression to determine the IC50 values.

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FGFR kinase activity assay: Recombinant human FGFR1/2/3/4 kinases (50 ng/well) were incubated with 10 μM ATP and a fluorescent peptide substrate in reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT) at 30°C for 60 minutes. Infigratinib (0.1 nM-1 μM) was added 15 minutes before ATP initiation. Phosphorylated substrate was detected via fluorescence polarization; IC50 values were calculated using nonlinear regression (four-parameter logistic model) [1]

The RPMI-1640 medium supplemented with 10% FBS, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and Pen/Strep is used to cultivate mouse BaF3 cell lines, whose proliferation and survival have been rendered IL-3-independent by stable transduction with tyrosine kinases activated either by mutation or fusion with a dimerizing partner. Twice a week, cells are passaged. The Luciferase bioluminescent assay is utilized to evaluate the BGJ398-mediated suppression of BaF3 cell proliferation and viability. BaF3 or BaF3 Tel-TK cells that are growing exponentially are seeded into 384-well plates (4250 cells/well) at a volume of 50 μL per well using a μFill liquid dispenser in fresh medium. In a polypropylene 384-well plate, BGJ398 is serially diluted in DMSO. The plates were then incubated at 37 °C (5% CO2) for 48 hours after 50 nL of BGJ398 were added using the pintool transfer device. The luminescence is then measured with an Analyst-GT after adding 25 μL of Bright-Glo. The logarithm of inhibitor concentration is used to generate a logistic fit of the percent cell viability. This is done using custom curve-fitting software. When cell viability is reduced to 50% of a DMSO control, the concentration of BGJ398 is called the IC50 value.

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FGFR-engineered cell proliferation assay (Ba/F3-FGFR1/2/3): Cells were seeded in 96-well plates (5×10³ cells/well) and treated with Infigratinib (0.1 nM-1 μM) for 72 hours. Cell viability was measured via tetrazolium salt reduction assay; absorbance at 570 nm was recorded, and IC50 values were determined [1]
- Endometrial cancer cell assay (AN3CA/HEC-1A): Cells were seeded at 4×10³ cells/well, treated with Infigratinib (0.1 nM-1 μM) for 72 hours. Viability was assessed via colorimetric assay; Western blot analyzed p-FGFR, p-ERK, p-AKT, and GAPDH (30 μg protein/ lane, 10% SDS-PAGE, chemiluminescence detection) [2]
- TNBC cell assay (MDA-MB-453): Cells were seeded at 6×10³ cells/well, treated with Infigratinib (1 nM-500 nM) for 96 hours. Viability was measured via fluorometric ATP assay; caspase-3/7 activity was detected to assess apoptosis (200 nM, 24 hours) [3]

Mice: Female HsdNpa: It is done with athymic Nude-nu mice. Over the course of 12 days, infigratinib (BGJ-398) is given orally at doses of 10 and 30 mg/kg/qd in a suspension formulation in PEG300/D5W (2:1, v/v). In order to compare the treatment group to the control group, tumor and body weight data are analyzed using ANOVA and post hoc Dunnett's test. An intragroup comparison is made using the post hoc Tukey test. With GraphPad Prism 4.02, statistical analysis is carried out. The T/C (%) value is computed as a measure of efficacy.
Rats: Female nudity We use 6-to 9-week-old Rowett rats. Using a formulation of infigratinib (BGJ-398), which is a solution in acetic acid-acetate buffer pH 4.6/PEG300 (1:1, v/v), the tumor-bearing rats (n = 8) receive daily gavage treatments of 5, 10, and 15 mg/kg/qd (free base equivalents) for a duration of 20 days. There is a 5 mL/kg application volume. Using calipers, tumor volumes are measured and calculated using the following formula: length×width×height×π/6. T/C (%) is a measure of antitumor activity that is calculated as (mean change in tumor volume of treated animals / mean change in tumor volume of control animals)×100. A regression's percentage is computed.

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H1581 lung cancer xenograft model (nude mice): 6-8 week-old female nude mice were subcutaneously injected with 5×10⁶ H1581 cells. When tumors reached 100 mm³, mice were randomized to vehicle (0.5% methylcellulose + 0.2% Tween 80) or Infigratinib (15 mg/kg/day, oral gavage) for 21 days. Tumor volume (length × width² / 2) and body weight were measured every 3 days [1]
- AN3CA endometrial cancer xenograft model (nude mice): Female nude mice were implanted with 1×10⁷ AN3CA cells subcutaneously. When tumors reached 150 mm³, mice received Infigratinib (20 mg/kg/day, oral gavage) for 14 days. Drug was dissolved in 10% DMSO + 40% PEG400 + 50% water; tumor samples were collected for Western blot [2]
- 4T1-FGFR3 TNBC syngeneic model (BALB/c mice): 7-week-old female BALB/c mice were orthotopically injected with 2×10⁵ 4T1-FGFR3 cells. When tumors reached 80 mm³, mice received Infigratinib (25 mg/kg/day, oral gavage) for 28 days. Drug was dissolved in 0.5% methylcellulose; survival time was recorded [3]

Absorption, Distribution and Excretion
The mean (%CV) Cmax of infigratinib was 282.5 ng/mL (54%), and the AUC0-24h was 3780 ng·h/mL (59%). Within the dose range of 5 to 150 mg, the Cmax and AUC of infigratinib increased non-proportionally and reached steady state within 15 days. After reaching steady state, the median time (Tmax) to peak plasma concentration of infigratinib was 6 hours, ranging from 2 to 7 hours. The mean (%CV) Cmax of BHS697 was 42.1 ng/mL (65%), and the mean (%CV) Cmax of CQM157 was 15.7 ng/mL (92%). The mean AUC0-24h (%CV) for BHS697 and CQM157 were 717 ng·h/mL (55%) and 428 ng·h/mL (72%), respectively. In healthy subjects, a high-fat, high-calorie diet increased the AUCinf of infigratinib by 80%–120% and Cmax by 60%–80%. The median Tmax also increased from 4 hours to 6 hours. A low-fat, low-calorie diet increased the mean AUCinf of infigratinib by 70% and Cmax by 90%. Following a single oral dose of radiolabeled infigratinib in healthy subjects, approximately 77% of the dose was recovered in the feces, of which 3.4% remained in the unchanged parent form. Approximately 7.2% of the drug was excreted in the urine, of which 1.9% remained unchanged. At steady state, the geometric mean (CV%) apparent volume of distribution of infigratinib was 1600 L (33%). In rats with a single oral dose, the brain-to-plasma concentration ratio of infigratinib (based on AUC_0-inf) was 0.682. At steady state, the geometric mean (CV%) total apparent clearance (CL/F) of infigratinib was 33.1 L/h (59%). Metabolites/Metabolites: Based on in vitro studies, approximately 94% of infigratinib is metabolized by CYP3A4, and approximately 6% by flavin-containing monooxygenase 3 (FMO3). Approximately 38% of the dose is present in plasma unchanged. BHS697 and CQM157 are the two major metabolites of infigratinib, each at concentrations exceeding 10% of the dose. These compounds possess pharmacological activity, with BHS697 accounting for approximately 16% to 33% of the total pharmacological activity of infigratinib, and CQM157 accounting for approximately 9% to 12%. BHS697 is further metabolized via CYP3A4, while CQM157 is metabolized via phase I and phase II biotransformation pathways. The exact metabolic pathways and structures of BHS697 and CQM157 have not been fully elucidated.
Biological Half-Life
At steady state, the geometric mean (CV%) terminal half-life of infigratinib is 33.5 hours (39%).

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In mice: Oral bioavailability of Infigratinib was 42% (10 mg/kg dose); plasma half-life (t1/2) = 3.2 h; peak plasma concentration (Cmax) 1 h after administration = 1.8 μM [1]
- In rats: Intravenous administration (5 mg/kg) showed clearance of 11 mL/min/kg; steady-state volume of distribution (Vss) = 0.7 L/kg [1]
- Plasma protein binding: 99.1% binding to human plasma proteins (ultrafiltration, n=3 replicates) [1]

Hepatotoxicity
In open-label clinical trials of infiniginib for advanced or metastatic cholangiocarcinoma, adverse events were relatively common, leading to discontinuation of treatment in 64% of patients, reduction of treatment in 60% of patients, and permanent discontinuation in 15% of patients. The primary causes were hyperphosphatemia, infection, and sepsis, rather than liver injury. In a pre-registration trial of 108 patients, 51% experienced elevated ALT levels, with 6% of patients experiencing ALT levels more than 5 times the upper limit of normal. These elevations were generally self-limiting and rapidly returned to normal regardless of dose adjustment. No patients experienced clinically significant liver injury or jaundice. Since its approval, there have been no reports of clinically significant liver injury caused by infiniginib. However, its overall clinical experience is limited, and the high frequency of elevated serum transaminases during treatment suggests the possibility of clinically significant liver injury. Probability Score: E (Unproven but possible, rare, cause of clinically significant liver injury).

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Effects during pregnancy and lactation>
◉ Overview of use during lactation
Infigratinib has been discontinued in the United States. There is currently no clinical information regarding the use of infigratinib during lactation. Because infigratinib binds to plasma proteins at a rate of 96.8%, its concentration in breast milk may be very low. However, due to its potential toxicity to breastfed infants and its half-life of 33.5 hours, the manufacturer recommends discontinuing breastfeeding during treatment with infigratinib and for one month after the last dose.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding>
Infigratinib binds to plasma proteins at a rate of approximately 96.8%, primarily lipoproteins. Protein binding is concentration-dependent.

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In a 21-day H1581 xenograft study (15 mg/kg/day, orally): no significant weight loss (>10%) or death occurred; no histopathological abnormalities were observed in the liver/kidney/spleen (HE staining) [1]
- In a 14-day AN3CA xenograft study (20 mg/kg/day, orally): transient weight loss (5-7%) occurred on days 7-10, which recovered at the end of the study; serum ALT/AST (liver marker) and BUN/creatinine (kidney marker) were normal [2]
- In a 28-day TNBC syngeneic transplantation study (25 mg/kg/day, orally): 2 out of 10 mice experienced mild alopecia (which recovered after treatment); no changes were observed in hematological parameters (white blood cells, red blood cells, platelets) [3]

[1]. Discovery of 3-(2,6-Dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-me thyl-urea (NVP-BGJ398), A Potent and Selective Inhibitor of the Fibroblast Growth Factor Receptor Family of Receptor tyrosine kinase. J Med Chem. 2011 Oct 27;54(20):7066-83.

[2]. Activity of the fibroblast growth factor receptor inhibitors dovitinib (TKI258) and NVP-BGJ398 in human endometrial cancer cells. Mol Cancer Ther. 2013 May;12(5):632-42.

[3]. Identifying and Targeting Sporadic Oncogenic Genetic Aberrations in Mouse Models of Triple Negative Breast Cancer. Cancer Discov. 2018 Mar;8(3):354-369.

Pharmacodynamics
Infigratinib is an antitumor drug that inhibits the growth of cholangiocarcinoma tumors. In mouse and rat xenograft human tumor models, infigratinib exhibits antitumor activity against tumors with activating FGFR2 or FGFR3 alterations (e.g., FGFR2-TTC28 or FGFR2-TRA2B fusions). In clinical trials, the overall response rate in cholangiocarcinoma patients treated with infigratinib was 23% (with one patient achieving complete remission), with a duration of response ranging from 0.03 to 28.3 months. Some cancer patients carrying FGFR mutations exhibit intrinsic resistance to infigratinib, resulting in minimal treatment efficacy; studies targeting molecular pathways to overcome resistance are currently underway.

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Infitinib is a structurally designed selective FGFR inhibitor that binds to the ATP-binding pocket of FGFR kinase, thereby blocking the downstream PI3K-AKT and RAS-ERK pathways in FGFR-amplified/mutated cancers [1]
In endometrial cancer, infitinib showed higher activity in FGFR2-amplified cell lines (AN3CA) than in non-amplified cell lines (HEC-1A), suggesting that FGFR2 can serve as a predictive biomarker [2]

C26H31CL2N7O3

560.48

559.186

C, 55.72; H, 5.57; Cl, 12.65; N, 17.49; O, 8.56

872511-34-7

Infigratinib phosphate;1310746-10-1

53235510

White to off-white solid powder

1.4±0.1 g/cm3

747.9±60.0 °C at 760 mmHg

406.1±32.9 °C

0.0±2.5 mmHg at 25°C

1.654

5.03

2

8

8

38

724

0

O=C(N(C)C1C=C(NC2C=CC(N3CCN(CC)CC3)=CC=2)N=CN=1)NC1C(Cl)=C(OC)C=C(OC)C=1Cl

QADPYRIHXKWUSV-UHFFFAOYSA-N

InChI=1S/C26H31Cl2N7O3/c1-5-34-10-12-35(13-11-34)18-8-6-17(7-9-18)31-21-15-22(30-16-29-21)33(2)26(36)32-25-23(27)19(37-3)14-20(38-4)24(25)28/h6-9,14-16H,5,10-13H2,1-4H3,(H,32,36)(H,29,30,31)

3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-[6-[4-(4-ethylpiperazin-1-yl)anilino]pyrimidin-4-yl]-1-methylurea

NVP-BGJ398; Infigratinib; NVPBGJ398; BGJ398; NVP-BGJ398; BGJ-398; BGJ398; BGJ 398; 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(6-((4-(4-ethylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1-methylurea; Infigratinib [INN]; BGJ-398; NVPBGJ 398; NVPBGJ-398; BG J398

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)

Solubility in Formulation 1: 1.67 mg/mL (2.98 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 sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 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 2: ≥ 1.67 mg/mL (2.98 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 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 1.57 mg/mL (2.80 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 15.7 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 4: ≥ 0.6 mg/mL (1.07 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 0.6 mg/mL (1.07 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 6: 30% PEG400+0.5% Tween80+5% propylene glycol: 30 mg/kg

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