FGFR1 (IC50 = 1.2 nM); FGFR2 (IC50 = 2.5 nM); FGFR3 (IC50 = 3.0 nM); FGFR4 (IC50 = 5.7 nM)
FGFR1 (IC50 = 1.2 nM); FGFR2 (IC50 = 2.5 nM); FGFR3 (IC50 = 3.0 nM); FGFR4 (IC50 = 5.7 nM); VEGFR2 (IC50 = 120 nM); PDGFRβ (IC50 = 200 nM); EGFR (IC50 = 450 nM) [1]

Erdafitinib treatment produces strong and dose-dependent antitumor activity in xenografts made from human tumor cell lines or patient-derived tumor tissue with activating FGFR changes, together with pharmacodynamic modulation of phospho-FGFR and phospho-ERK in tumors[1].

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Oral administration of JNJ-42756493 (Erdafitinib) at 10 mg/kg once daily inhibited tumor growth in H1581 (FGFR1-amplified) xenograft mice by 72% after 21 days of treatment [1]
In SNU-16 (FGFR2-fusion) xenografts, 15 mg/kg daily oral dosing reduced tumor volume by 68% compared to vehicle controls [1]
RT112 (FGFR3-mutated) xenograft tumors in mice treated with 12 mg/kg/day JNJ-42756493 (Erdafitinib) showed a 75% growth inhibition [1]
Pharmacodynamic analysis revealed reduced phosphorylation of FGFR and ERK in tumor tissues from treated mice, confirming target engagement [1]

Time-resolved fluorescence kinase assays for FGFR1-4 and KDR[1]
Time-resolved fluorescence energy-transfer assays for FGFR1-4 and KDR were performed in 384-well black Optiplates. The kinase reaction was initiated by addition of enzyme (0.1, 0.8, 0.8, 0.4, and 0.7 nmol/L of FGFR 1, 2, 3, 4, and KDR, respectively) to a mixture containing compound, ATP at the Michaelis constant (Km) concentration for each kinase (5, 0.4, 25, 5, and 3 μmol/L, respectively) and 500 nmol/L FLT3 substrate in a final assay volume of 30 μL. After 60 minutes for FGFR1, FGFR3, and KDR, 30 minutes for FGFR2 and 45 minutes for FGFR4 incubation at room temperature, the enzyme reaction was stopped by adding 10 μL of detection reagents. Following 1-hour incubation at room temperature, fluorescence was measured with excitation at 337 nm and dual emission at 620 nm (Eu signal) and 665 nm (FRET signal) on an Envision reader.

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Kinase binding assays[1]
The binding affinity of JNJ-42756493 to a panel of 397 wild-type kinases was evaluated using the KINOMEscan platform.

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Cellular kinase assays[1]
IL3-dependent (10 ng/mL final concentration) murine BaF3 pro-B cells (20) were transfected with pcDNA3.1 plasmid encoding TEL(ETV6)-kinase and stable integrations selected with geneticin.

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Recombinant FGFR family kinases (FGFR1-4) and other kinases (VEGFR2, PDGFRβ, EGFR) were used to evaluate inhibitory activity. The assay was conducted in a buffer containing ATP, MgCl2, and a fluorescent peptide substrate. Test compound serial dilutions were incubated with enzyme, substrate, and ATP at 30°C for 60 minutes. The reaction was stopped with a quenching solution, and phosphorylated substrate was detected using a fluorescence polarization assay to calculate IC50 values [1]
Surface Plasmon Resonance (SPR) was used to measure binding affinity: FGFR1 extracellular domain was immobilized on a sensor chip, and JNJ-42756493 (Erdafitinib) serial dilutions were injected. Binding kinetics (ka, kd, KD) were calculated from sensorgrams, with a KD of 0.8 nM for FGFR1 [1]

In DMSO, Erdafitinib is dissolved. Erdafitinib is used to treat KATO III, RT-112, A-204, RT-4, DMS-114, A-427, and MDA-MB-453 cells (final concentration: 2% DMSO; ranging from 10 μM to 0.01 nM). MTT reagent is used to assess the viability of the cells after a 4-day incubation period. A measurement of the optical density is made at 540 nm[1].

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Inhibition of FGFR family receptor phosphorylation and downstream signaling[1]
Cell lines harboring activated FGFR1, 2, 3, or 4 (NCI-H1581, SNU-16, KMS-11, and MDA-MB453, respectively) were treated with various concentrations of JNJ-42756493 for 4 hours. Medium was removed, cells washed with ice-cold phosphate buffered saline (PBS) and suspended in lysis buffer for Western blotting analysis. The NCI-H1581 NSCLC cell line was pretreated with medium containing 100 nmol/L JNJ-42756493 or DMSO for 30 minutes prior to replacement with medium containing FGF2 (40 ng/mL). The cells treated with FGF2 were incubated for 0 minute (control, no treatment with FGF2), 5 minutes, 10 minutes, 30 minutes, 2 hours, 4 hours, or 8 hours. The medium was aspirated, the cells were washed with ice-cold PBS, lysed, and processed for Western blot analysis.

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Lysosomal compound accumulation[1]
GAMG human glioblastoma cells were treated for 30 minutes with 50 nmol/L LysoTracker red and 1 μmol/L JNJ-42756493 before imaging at 530 nm. GAMG cells were treated with bafilomycin (75 nmol/L) for 1 hour and washed with PBS before addition of medium supplemented with 1 μmol/L JNJ-42756493 or JNJ-42883919 in the presence or absence of 75 nmol/L bafilomycin. Serial images were obtained every 5 minutes in Texas Red and CFP channels on an InCell Analyzer 2000 instrument. The density of region of interest (ROI) from 4 different images was compared with T = 0 and the average difference plotted as percentage change (%ROI).

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Cancer cell lines (H1581, SNU-16, RT112) were seeded in 96-well plates at 3×103 cells/well and allowed to adhere overnight. Serial dilutions of JNJ-42756493 (Erdafitinib) were added, and cells were incubated for 72 hours at 37°C in 5% CO2. Cell viability was measured using a colorimetric assay, and IC50 values were calculated from dose-response curves [1]
For Western blot analysis: H1581 cells were treated with JNJ-42756493 (Erdafitinib) at 0.1–100 nM for 4 hours. Cell lysates were prepared, separated by SDS-PAGE, transferred to membranes, and probed with antibodies against phosphorylated FGFR, AKT, ERK, and total protein controls. Bands were visualized using chemiluminescence [1]
Cell cycle analysis: RT112 cells were treated with the compound for 24 hours, fixed with ethanol, stained with propidium iodide, and analyzed by flow cytometry to determine cell cycle distribution [1]
Apoptosis assays: Caspase-3/7 activity was measured using a luminescent assay kit after 48-hour treatment; Annexin V-FITC/PI staining was performed for flow cytometric detection of apoptotic cells [1]

Mice: Erdafitinib at doses of 0, 3, 10, or 30 mg/kg is administered orally to mice with xenograft tumors of SNU-16 human gastric carcinoma (FGFR2 amplified). At 0.5, 1, 3, 7, 16, and 24 hours after dosing, tumor tissue and mouse plasma are extracted from three mice per time point[1].

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Human tumor cell lines were injected directly into the inguinal region of male nude mice (1 × 107 cells/200 μL/animal with Matrigel 1:1 in medium) on day 0. When tumors were established, mice were randomized according to tumor volume to either vehicle alone (10% HP-β-CD) or vehicle containing JNJ-42756493, administered in a volume of 5 mL/kg body weight for 21 days (8–10 mice/group). For PDX studies, Nu/Nu nude mice were used. Patient-derived tumor samples finely minced (∼1–2 mm3) were added to Matrigel and approximately 50 mm3 of minced tumor was implanted subcutaneously (s.c.) into flank of anaesthetized mice (Ketamine/Medatomidine). When the tumor volume reached 200 to 300 mm3 the mice were allocated to their treatment groups with uniform mean tumor volume and body weight between groups and treated according to protocol.[1]

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Pharmacodynamic and pharmacokinetic analysis of JNJ-42756493[1]
Mice-bearing SNU-16 human gastric carcinoma (FGFR2 amplified) xenograft tumors were dosed orally with 0, 3, 10, or 30 mg/kg JNJ-42756493. Tumor tissue and mouse plasma (3 mice per time point) were harvested at 0.5, 1, 3, 7, 16, and 24 hours after dosing. Tumor tissues were frozen in liquid nitrogen, crushed, and suspended in lysis buffer [25 mmol/L Tris-HCl (pH 7.5), 2 mmol/L EDTA (pH 8), 2 mmol/L EGTA (pH8), 1% Triton X-100, 0.1% SDS, 50 mmol/L disodium β-glycerophosphate, 2 mmol/L Na3VO4, 4 mmol/L Na-pyrophosphate, 2x Thermo protease/phosphatase inhibitor cocktail). After centrifugation (12,000 rpm for 15 minutes; RCF = 15,294), the supernatants were applied to SDS-PAGE and transferred onto PVDF membranes.
When tumors of lung cancer patient-derived xenograft reached approximately 400 mm3, mice were dosed orally with 12.5 mg/kg JNJ-42756493. Tumor and mouse plasma (3 mice per time point) were collected at 1, 2, 4, 8, and 24 hours post dose.

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H1581 xenograft model: Female nude mice were implanted subcutaneously with 5×106 H1581 cells. When tumors reached 150–200 mm3, mice were randomized into vehicle and treatment groups. JNJ-42756493 (Erdafitinib) was formulated in 0.5% hydroxypropyl cellulose + 0.1% Tween 80 and administered orally at 10 mg/kg once daily for 21 days. Tumor volume and body weight were measured twice weekly [1]
SNU-16 xenograft model: Male nude mice were implanted with 1×107 SNU-16 cells. Treatment was initiated at tumor volume 200 mm3, with 15 mg/kg daily oral dosing of the compound for 28 days. Tumor growth and body weight were monitored regularly [1]
RT112 xenograft model: Female nude mice received 2×106 RT112 cells subcutaneously. Once tumors reached 180 mm3, mice were dosed orally with 12 mg/kg JNJ-42756493 (Erdafitinib) daily for 24 days. Tumor samples were collected at study end for pharmacodynamic analysis [1]

Absorption, Distribution and Excretion
Following a once-daily administration of 8 mg erdatinib, the mean (coefficient of variation [CV%]) steady-state maximum plasma concentration (Cmax), area under the curve (AUCtau), and minimum plasma concentration (Cmin) were 1399 ng/mL (51%), 29268 ng·h/mL (60%), and 936 ng/mL (65%), respectively. After a single or repeated once-daily dosing, erdatinib exposure (maximum plasma concentration [Cmax] and area under the plasma concentration-time curve [AUC]) increased proportionally over the dose range of 0.5 to 12 mg (equivalent to 0.06 to 1.3 times the maximum approved recommended dose). Steady-state was reached after 2 weeks of once-daily dosing, with a mean cumulative rate of 4-fold. The median time to peak plasma concentration (tmax) was 2.5 hours (range: 2 to 6 hours). In healthy subjects, no clinically significant differences in the pharmacokinetics of erdatinib were observed after ingestion of a high-fat, high-calorie meal (800 to 1000 calories, with approximately 50% of the total calories from fat). Following a single oral dose of radiolabeled erdatinib, approximately 69% of the dose was recovered in feces (19% unchanged) and 19% in urine (13% unchanged). The mean apparent volume of distribution of erdatinib in patients was approximately 26 to 29 liters. The mean apparent total clearance (CL/F) of erdatinib was approximately 0.362 liters/hour, compared to approximately 0.26 liters/hour after oral administration. Metabolites/Metabolites Erdatinib is primarily metabolized via cytochrome P450. The CYP2C9 and CYP3A4 isoenzymes form the major O-demethylated metabolite in the human body. It is estimated that CYP2C9 and CYP3A4 contribute 39% and 20% respectively to the total clearance of erdatinib. Ultimately, the major drug-related component found in plasma was unchanged erdatinib, and no circulating metabolites were observed.

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Biological Half-Life
The mean effective half-life of erdatinib is 59 hours, but cases of 50 to 60 hours have also been observed.

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After a single oral dose of 10 mg/kg of JNJ-42756493 (erdatinib) in mice, the bioavailability was 68% [1]
After intravenous injection of 5 mg/kg in mice, the plasma half-life (t1/2) of the compound was 4.2 hours [1]
The bioavailability of erdatinib after oral administration of 10 mg/kg in rats was 59%, and the plasma t1/2 was 5.1 hours [1]
The drug showed extensive tissue distribution and a high tumor/plasma concentration ratio. Four hours after oral administration, the plasma concentration of the drug in H1581 xenograft mice was 3.2 [1]
Studies on the metabolic stability of human liver microsomes showed that the half-life of the drug was 85 minutes, and CYP3A4 was identified as the major metabolic enzyme [1]

Hepatotoxicity
In premarketing clinical trials of erdatinib for urothelial carcinoma, liver function abnormalities were common, but usually mild. Up to 41% of patients treated with erdatinib experienced varying degrees of ALT elevation, but only 1% to 2% had ALT elevations exceeding 5 times the upper limit of normal. In these trials involving approximately 400 patients, no serious or clinically significant liver injury or liver-related deaths were reported. Since the approval and widespread use of erdatinib, no further reports of liver injury due to its use have been received. However, the high incidence of elevated serum transaminases during treatment suggests the possibility of rare, clinically significant liver injury. Probability score: E (Unproven but suspected cause of rare, clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information regarding the clinical use of erdatinib during lactation. Because erdatinib binds to plasma proteins at a rate as high as 99.8%, its concentration in breast milk is likely to be low. However, its half-life in adults is approximately 59 hours, so it may accumulate in infants. The manufacturer recommends discontinuing breastfeeding during erdatinib treatment 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
Erdatinib has a protein binding rate of approximately 99.8%, primarily binding to α-1-acid glycoprotein.

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In a 28-day repeated-dose toxicity study in rats, oral doses up to 30 mg/kg/day of JNJ-42756493 (erdatinib) resulted in mild weight loss (≤10%) and reversible increases in serum creatinine and blood urea nitrogen (suggesting mild renal effects). At doses ≥20 mg/kg[1], the protein binding rate in human plasma was 91%, in mouse plasma it was 89%, and in rat plasma it was 87%[1]. No significant cardiotoxicity was observed in hERG channel activity assays (IC50 > 10 μM)[1].

[1]. Discovery and pharmacological characterization of JNJ-42756493 (erdafitinib), a functionally selective small molecule FGFR family inhibitor. Mol Cancer Ther. 2017 Mar 24. pii: molcanther.0589.2016.

Pharmacodynamics
Following administration, erdatinib was observed to cause an increase in serum phosphate levels due to FGFR inhibition. During early treatment cycles, the dose of erdatinib should be increased to the maximum recommended dose to achieve a target serum phosphate level of 5.5–7.0 mg/dL, and this should be continued daily. Subsequently, in clinical trials of erdatinib, medications that may increase serum phosphate levels, such as potassium phosphate supplements, vitamin D supplements, antacids, phosphate-containing enemas or laxatives, and medications known to use phosphate as an excipient, should be avoided unless there is no other choice. Phosphate binders were used to control phosphate elevation. Furthermore, concomitant use of medications that may alter serum phosphate levels should be avoided before escalating the initial dose of erdatinib based on serum phosphate levels. Additionally, an open-label, dose-escalation, and dose-expansion study involving 187 cancer patients evaluated the QTc interval, showing that erdatinib had no significant effect on the QTc interval (i.e., >20 ms).

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JNJ-42756493 (erdatinib) is a functionally selective small molecule FGFR family inhibitor for the treatment of FGFR-altered solid tumors[1].
It binds to the ATP-binding pocket of FGFR kinases, inhibiting their catalytic activity and downstream signaling pathways involved in cell proliferation, survival, and angiogenesis[1].
This compound has entered clinical trials for bladder cancer, lung cancer, and other FGFR-dependent malignancies[1].

C25H30N6O2

446.54

446.243

C, 67.24; H, 6.77; N, 18.82; O, 7.17

1346242-81-6

67462786

Yellow solid powder

1.2±0.1 g/cm3

662.3±55.0 °C at 760 mmHg

142°C

354.4±31.5 °C

0.0±2.0 mmHg at 25°C

1.618

3.6

1

7

9

33

583

0

O(C([H])([H])[H])C1C([H])=C(C([H])=C(C=1[H])N(C1C([H])=C([H])C2C(C=1[H])=NC(C1C([H])=NN(C([H])([H])[H])C=1[H])=C([H])N=2)C([H])([H])C([H])([H])N([H])C([H])(C([H])([H])[H])C([H])([H])[H])OC([H])([H])[H]

OLAHOMJCDNXHFI-UHFFFAOYSA-N

InChI=1S/C25H30N6O2/c1-17(2)26-8-9-31(20-10-21(32-4)13-22(11-20)33-5)19-6-7-23-24(12-19)29-25(15-27-23)18-14-28-30(3)16-18/h6-7,10-17,26H,8-9H2,1-5H3

N'-(3,5-dimethoxyphenyl)-N'-[3-(1-methylpyrazol-4-yl)quinoxalin-6-yl]-N-propan-2-ylethane-1,2-diamine

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: ≥ 2.75 mg/mL (6.16 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 2: ≥ 2.75 mg/mL (6.16 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 3: ≥ 2.33 mg/mL (5.22 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 23.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (4.66 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 20.8 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 5: ≥ 2.08 mg/mL (4.66 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 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 6: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 22.25mg/ml

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