FGFR1 (IC50 = 0.9 nM); FGFR2 (IC50 = 1.4 nM); FGFR3 (IC50 = 1 nM); FGFR4 (IC50 = 60 nM)
Infigratinib phosphate suppresses FGFR1, FGFR2, and FGFR3 at IC50 values of ~1 nM, FGFR3^K650E at IC50 values of 4.9 nM, and FGFR4 at IC50 values of 60 nM. With the exception of VEGFR2, KIT, and LYN, which are inhibited at submicromolar concentrations (IC50=0.18, 0.75, and 0.3 μM, respectively), the IC50 values for all other kinases fall within the μM range (FYN, LCK, YES, and ABL, IC50=1.9, 2.5, 1.1, and 2.3 μM, respectively). With IC50 values in the low nanomolar range, infigratinib inhibits the proliferation of the FGFR1-, FGFR2-, and FGFR3-dependent BaF3 cells in a manner similar to that seen in the enzymatic assay for the inhibition of receptor kinase activity. Except for VEGFR2 (IC50 1449 and 938 nM), which has at least a 400-fold selectivity versus FGFR1, FGFR2, and FGFR3, all IC50 values for the remaining cells are greater than 1.5 μM[1]. The growth of FGFR2-mutant endometrial cancer cells is effectively inhibited by infigratinib (at concentrations between 1 nM and 10 μM)[2].

Infigratinib phosphate suppresses FGFR1, FGFR2, and FGFR3 at IC50 values of ~1 nM, FGFR3^K650E at IC50 values of 4.9 nM, and FGFR4 at IC50 values of 60 nM. With the exception of VEGFR2, KIT, and LYN, which are inhibited at submicromolar concentrations (IC50=0.18, 0.75, and 0.3 μM, respectively), the IC50 values for all other kinases fall within the μM range (FYN, LCK, YES, and ABL, IC50=1.9, 2.5, 1.1, and 2.3 μM, respectively). With IC50 values in the low nanomolar range, infigratinib inhibits the proliferation of the FGFR1-, FGFR2-, and FGFR3-dependent BaF3 cells in a manner similar to that seen in the enzymatic assay for the inhibition of receptor kinase activity. Except for VEGFR2 (IC50 1449 and 938 nM), which has at least a 400-fold selectivity versus FGFR1, FGFR2, and FGFR3, all IC50 values for the remaining cells are greater than 1.5 μM[1]. The growth of FGFR2-mutant endometrial cancer cells is effectively inhibited by infigratinib (at concentrations between 1 nM and 10 μM)[2].

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The selective FGFR inhibitor NVP-BGJ398 inhibited proliferation of all 20 tested endometrial cancer cell lines in a concentration-dependent manner, with IC50 values ranging from 1.0 µmol/L in the FGFR2-mutated AN3CA cell line to 10.0 µmol/L in the KRAS-mutated HEC1A cell line. Cell lines with activating FGFR2 mutations (S252W, N550K) had significantly lower mean IC50 values compared to FGFR2 wild-type cell lines (mean IC50 2.96 vs. 5.55, P = 0.021). The BRAF-mutated MFE319 cell line (which also harbors an FGFR2 S252W mutation) was the least sensitive among FGFR2-mutated lines. [2]
At a clinically achievable concentration of 1 µmol/L, NVP-BGJ398 inhibited anchorage-independent growth (≥50% growth inhibition) in 3 out of 4 FGFR2-mutated cell lines, but in only 3 out of 11 (27%) cell lines with wild-type FGFR2, indicating a tighter correlation with FGFR2 mutation status. [2]
NVP-BGJ398 inhibited FGFR2 pathway signaling in FGFR2-mutant cells. It blocked phosphorylation of FRS2α and ERK in a time-dependent manner in cells carrying the FGFR2 N550K mutation (AN3CA, MFE296) and blocked both basal and FGF7-stimulated phosphorylation in cells with the S252W mutation (MFE280). However, restoration of ERK signaling was observed within 24 to 72 hours. The inhibitor caused modest or variable effects on AKT phosphorylation. In FGFR2 wild-type cells (SNGM, HEC1A), NVP-BGJ398 treatment did not alter ERK or AKT signaling. [2]
Treatment with NVP-BGJ398 induced a significant increase in the fraction of cells in G0–G1 phase arrest and a significant increase in apoptosis in FGFR2-mutated cell lines (AN3CA, MFE296, MFE280), but had minimal to no effect on cell cycle or apoptosis in FGFR2 wild-type cell lines (SNGM, HEC1A). [2]

Infigratinib is given to athymic nude mice that have had RT112/luc1 tumors implanted subcutaneously. The administration options include an intravenous bolus of 5 mg/kg in NMP/PEG200 (1:9, v/v) or an oral gavage of a suspension in PEG300/D5W (2:1, v/v) at a dose of 20 mg/kg. According to the pertinent pharmacokinetic (PK) parameters, infigratinib has a 32% oral bioavailability in this investigation. Following intravenous administration, infigratinib exhibits a high volume of distribution (26 L/kg) due to its quick distribution from the vascular compartment into the peripheral tissues. With a clearance of 3.3 L/h/kg (61% of liver blood flow), the plasma level is high. Based on AUC, the ratio of tumor to plasma following oral dosing is found to be 10[1]. The growth of endometrial cancer xenograft models with FGFR2 mutations is markedly inhibited by infigratinib (30 mg/kg)[2].

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In mouse xenograft models derived from FGFR2-mutated endometrial cancer cells (AN3CA, MFE296), daily oral administration of NVP-BGJ398 at 30 mg/kg significantly delayed tumor growth. [2]
In a long-term study using the FGFR2 wild-type SNGM xenograft model, NVP-BGJ398 showed no inhibitory effects. However, in the FGFR2 wild-type HEC1A xenograft model, NVP-BGJ398 treatment did delay tumor growth, despite showing no in vitro efficacy in these cells. [2]

The purified GST-fusion FGFR3-K650E kinase domain phosphorylates a synthetic substrate in the presence of radiolabeled ATP to measure the enzymatic kinase activity. Enzyme activities are determined by combining 10 μL of the corresponding substrate mixture (peptidic substrate, ATP, and [γ³³P]ATP) with 10 μL of a 3-fold concentrated Infigratinib 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-[³³P]-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 and 30 μL of final volume, which includes the components mentioned above. The following measurement is used to determine the amount of ³³P incorporated into the polypeptidic substrates after 20 μL of 125 mM EDTA is added to stop the enzymatic reactions: Using a disconnected vacuum source, mount the Immobilon-PVDF membranes on a vacuum manifold and transfer 30 μL of the stopped reaction mixture onto them after they have been soaked in methanol for 5 minutes, rinsed with water, and soaked in 0.5% H₃PO₄ for 5 minutes. Following spotting, each well is rinsed with 200 μL of 0.5% H₃PO₄ and vacuumed. After extracting the free membranes, they are shaker-washed four times with 1% H₃PO₄ and once with ethanol. After desiccating the membranes, 10 μL of a scintillation fluid are added per well. In the end, the plates are sealed and counted using a microplate scintillation counter. By using linear regression analysis to determine the percentage inhibition of NVP-BGJ398[1], IC50 values are determined.

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. Twice a week, cells are passed through. A Luciferase bioluminescent assay is used to evaluate compound-mediated inhibition of BaF3 cell proliferation and viability. Using a μFill liquid dispenser, exponentially growing BaF3 or BaF3 Tel-TK cells are seeded at 50 μL/well into 384-well plates (4250 cells/well) in fresh medium. After being serially diluted in DMSO, infigratinib is arranged in a 384-well polypropylene plate. The plates were then incubated at 37°C (5% CO₂) for 48 hours after 50 nL of the compound was transferred into them using the pintool transfer device. Next, add 25 μL of Bright-Glo, and use an Analyst-GT to measure the luminescence. A logistic fit of the percent cell viability as a function of the logarithm of inhibitor concentration is generated using specialized curve-fitting software. The concentration of a compound required to lower cell viability to 50% of a DMSO control is known as the IC50 value[1].

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Proliferation Assay: Cells were plated in 24-well plates at a density of 2×10^5 to 5×10^5 and treated with increasing concentrations of NVP-BGJ398 (0.001 to 10 µmol/L). After 7 days, cells were harvested by trypsinization and counted. Growth inhibition was calculated, and IC50 values were interpolated from dose-response curves. [2]
Soft Agar Clonogenic Assay: A bottom layer of 0.5% agar was placed in 24-well plates. Cells (5×10^3) were mixed into a top layer of 0.3% agar prepared with or without 1 µmol/L NVP-BGJ398 and plated in quadruplicate. Plates were incubated for up to 5 weeks. Colonies were stained and counted visually. Percent inhibition was calculated versus untreated controls. [2]
Western Blot Analysis: Cells were treated with NVP-BGJ398, washed, and lysed. Protein concentrations were determined. Proteins were resolved by SDS-PAGE, transferred to membranes, and probed with antibodies against phospho-FRS2α, phospho-AKT, phospho-ERK, and α-Tubulin. For MFE280 cells, stimulation with FGF7 (30 ng/mL for 30 minutes) was performed prior to inhibitor treatment. Detection was performed using chemifluorescence. [2]
Cell Cycle Analysis: Cells in log phase were treated with NVP-BGJ398 for 72 hours. Cells were stained and analyzed by flow cytometry to determine the fraction in different cell cycle phases. [2]
Apoptosis Assay (Annexin V/PI): Cells were treated with NVP-BGJ398 for 72 hours. Apoptosis was detected using Annexin V-FITC and propidium iodide staining followed by flow cytometry. [2]

Mice: HsdNpa female: Athymic Nude-nu mice are employed. Infigratinib is taken orally for 12 straight days at doses of 10 and 30 mg/kg/qd. It is prepared as a suspension in PEG300/D5W (2:1, v/v). ANOVA is used to analyze the tumor and body weight data, and Dunnett's test is used to compare the treatment group to the control group post hoc. For intragroup comparison, the post hoc Tukey test is employed. To perform statistical analysis, use GraphPad Prism 4.02. One calculates the T/C (%) value as a measure of efficacy.
Rats: Woman in the nude 6 to 9-week-old Rowett rats are utilized. The tumor-bearing rats (n=8) receive infigratinib intraperitoneally (gavage) once a day for 20 days at doses of 5, 10, and 15 mg/kg/qd (free base equivalents). The drug is prepared as a solution in acetic acid-acetate buffer pH 4.6/PEG300 (1:1, v/v). It uses five milliliters per kilogram. The formula for determining tumor volumes is length×width×height×π/6, which can be measured using calipers. Antitumor activity is represented as T/C (%), which is calculated as (mean change in tumor volume of treated animals / mean change in tumor volume of control animals)×100. One calculates regressions (%).

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Female athymic mice (4-6 weeks old) were injected subcutaneously in the flank with endometrial cancer cells (2×10^7 cells per mouse). After tumors reached a mean volume of approximately 105 mm³ (7 days), mice were stratified into treatment groups based on tumor volume and weight. Mice (10 per group) were treated daily via oral gavage with: (i) vehicle control, or (ii) NVP-BGJ398 at 30 mg/kg (formulated as 6 mg in 0.5 mL PEG300 and 0.5 mL acetic acid/acetate buffer, pH 4.68). Tumor volumes were measured serially. [2]

Absorption, Distribution and Excretion
Absorption: The mean (%CV) Cmax of infigratinib was 282.5 ng/mL (54%), and the AUC0-24h was 3780 ng·h/mL (59%). Infigratinib showed a non-proportional increase in Cmax and AUC over a dose range of 5 to 150 mg, reaching 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%.
Elimination Pathway
In healthy subjects, after a single oral dose of radiolabeled infigratinib, approximately 77% of the dose was recovered in feces, of which 3.4% was in the unaltered parent form. Approximately 7.2% of the dose was recovered in urine, of which 1.9% was in the unaltered form.
Volume of Distribution
At steady state, the geometric mean (CV%) apparent volume of distribution of infigratinib is 1600 L (33%). In rats with a single oral dose, the brain-to-plasma concentration ratio (based on AUC0-inf) of infigratinib is 0.682.
Clearance
At steady state, the geometric mean (CV%) total apparent clearance (CL/F) of infigratinib is 33.1 L/h (59%).

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Metabolites
According to in vitro studies, approximately 94% of infigratinib is metabolized by CYP3A4, and approximately 6% of the drug is metabolized by flavin-containing monooxygenase 3 (FMO3). Approximately 38% of the dose exists in plasma as the parent drug. BHS697 and CQM157 are the two major metabolites of infigratinib, each at concentrations exceeding 10% of the dose. They are pharmacologically active, 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.

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Biological Half-Life
At steady state, the geometric mean (CV%) terminal half-life of infigratinib is 33.5 hours (39%).

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 exceeding five times the upper limit of normal. These elevations were generally self-limiting and rapidly returned to normal regardless of dose adjustment. No clinically significant liver injury or jaundice was reported. Since the approval of infiniginib, no cases of clinically significant liver injury have been reported. 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 low. However, due to the potential toxicity of this drug 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.

[1]. Discovery of 3-(2,6-dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-methyl-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.

Infigratinib phosphate is the phosphate form of infigratinib, a pan-inhibitor with high oral bioavailability that inhibits human fibroblast growth factor receptor (FGFR) and possesses potential anti-angiogenic and anti-tumor activities. After administration, infigratinib selectively binds to and inhibits FGFR activity, thereby suppressing angiogenesis and cell proliferation, and inducing death in tumor cells with activating FGFR amplification, mutation, or fusion. FGFRs are a class of receptor tyrosine kinases involved in tumor cell differentiation and proliferation, tumor angiogenesis, and tumor cell survival. Activating FGFR amplification, mutation, or fusion occurs in various cancer cell types.
See also: Infigratinib (with active moiety).

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Through the rational design of aromatic ring substitution patterns, we optimized a series of novel N-aryl-N'-pyrimidin-4-ylurea to obtain potent and selective inhibitors of fibroblast growth factor receptor tyrosine kinases 1, 2, and 3. Based on its in vitro activity, we selected compound 1h (NVP-BGJ398) for in vivo evaluation and it showed significant antitumor activity in the RT112 bladder cancer xenograft model overexpressing wild-type FGFR3. These results support the potential therapeutic use of compound 1h as a novel anticancer drug. [1]

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Recently discovered mutations in activating fibroblast growth factor receptor 2 (FGFR2) in endometrial cancer have provided an opportunity for novel targeted therapies. This study explored the potential of two FGFR inhibitors—the multi-kinase inhibitor dovitinib (TKI258) and the more selective FGFR inhibitor NVP-BGJ398—in the treatment of endometrial cancer. We used 20 molecularly identified human endometrial cancer cell lines to examine the effects of these two inhibitors on tumor cell growth, the FGFR2 signaling pathway, cell cycle, and apoptosis. Soft agar colony formation assays were used to study anchorage-independent cell growth. In vivo studies were conducted using an endometrial cancer xenograft model. The results showed that, compared with wild-type FGFR2 cell lines, cell lines carrying FGFR2 activating mutations (S252W, N550K) were more sensitive to dovitinib and NVP-BGJ398 (P values were 0.073 and 0.021, respectively). Both drugs inhibited the FGFR2 signaling pathway, induced cell cycle arrest, and significantly increased apoptosis in FGFR2 mutant cell lines. In vitro experiments showed that both dovitinib and NVP-BGJ398 effectively inhibited the growth of FGFR2 mutant endometrial cancer cells, but dovitinib's activity was less restrictive on FGFR2 mutant cell lines compared to NVP-BGJ398. In vivo experiments showed that both dovitinib and NVP-BGJ398 significantly inhibited the growth of FGFR2 mutant endometrial cancer xenograft models. In addition, in long-term in vivo studies, dovitinib has also shown significant antitumor activity in a xenograft model of FGFR2 wild-type endometrial cancer, including complete tumor regression. Dovitinib and NVP-BGJ398 warrant further clinical evaluation in patients with FGFR2-mutant endometrial cancer. Dovitinib may also have antitumor activity against endometrial cancers other than those with FGFR2 mutations and may provide patients with more flexibility in their choices. [2]

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NVP-BGJ398 is a highly selective pan-FGFR kinase inhibitor optimized through rational drug design. Its selectivity suggests that its toxicity profile may be better compared to non-selective multi-kinase inhibitors. [2]
Some endometrial cancers have activating FGFR2 mutations (e.g., S252W mutations in the ligand-binding domain and N550K mutations in the kinase domain), which confer sensitivity to FGFR inhibitors. These mutations may be more frequent in recurrent/metastatic disease compared to the primary tumor. [2]
The preclinical data from this study support the rationale for conducting a clinical trial of NVP-BGJ398 in patients with recurrent endometrial cancer carrying the FGFR2 mutation. [2]

C26H34CL2N7O7P

658.47

657.163

C, 47.43; H, 5.20; Cl, 10.77; N, 14.89; O, 17.01; P, 4.70

1310746-10-1

Infigratinib;872511-34-7

56669626

White to off-white solid powder

4.574

5

12

8

43

773

0

ClC1C(=C([H])C(=C(C=1N([H])C(N(C([H])([H])[H])C1C([H])=C(N=C([H])N=1)N([H])C1C([H])=C([H])C(=C([H])C=1[H])N1C([H])([H])C([H])([H])N(C([H])([H])C([H])([H])[H])C([H])([H])C1([H])[H])=O)Cl)OC([H])([H])[H])OC([H])([H])[H].P(=O)(O[H])(O[H])O[H]

GUQNHCGYHLSITB-UHFFFAOYSA-N

InChI=1S/C26H31Cl2N7O3.H3O4P/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;1-5(2,3)4/h6-9,14-16H,5,10-13H2,1-4H3,(H,32,36)(H,29,30,31);(H3,1,2,3,4)

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

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)