FGFR1 (IC50 = 0.9 nM); FGFR2 (IC50 = 1.4 nM); FGFR3 (IC50 = 1 nM); FGFR4 (IC50 = 60 nM)

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]

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.

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 [γ³³P]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-[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 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 ³³P 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.

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.

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.

Absorption, Distribution and Excretion
Mean (%CV) Cmax is 282.5 ng/mL (54%) and AUC0-24h is 3780 ngxh/mL (59%) for infigratinib. Infigratinib Cmax and AUC increase more than proportionally across the dose range of 5 to 150 mg and steady state is achieved within 15 days. At steady state, median time to achieve peak infigratinib plasma concentration (Tmax) is six hours, with a range between two and seven hours. Mean (%CV) Cmax is 42.1 ng/mL (65%) for BHS697 and 15.7 ng/mL (92%) for CQM157. Mean (%CV) AUC0-24h is 717 ngxh/mL (55%) for BHS697 and 428 ngxh/mL (72%) for CQM157. In healthy subjects, a high-fat and high-calorie meal increased AUCinf of infigratinib by 80%-120% and Cmax by 60%-80%. The median Tmax also shifted from four hours to six hours. A low-fat low-calorie meal increased the mean AUCinf of infigratinib by 70% and Cmax by 90%/
Following administration of a single oral dose of radiolabeled infigratinib in healthy subjects, approximately 77% of the dose was recovered in feces, where 3.4% of the dose was in the unchanged parent form. About 7.2% was recovered in urine with 1.9% of the dose was unchanged.
At steady state, the geometric mean (CV%) apparent volume of distribution of infigratinib was 1600 L (33%). In rats receiving a single oral dose, infigratinib had brain-to-plasma concentration ratios (based on AUC_0-inf) of 0.682.
The geometric mean (CV%) total apparent clearance (CL/F) of infigratinib was 33.1 L/h (59%) at steady state.

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Metabolism / Metabolites
According to _in vitro_ findings, about 94% of infigratinib is metabolized by CYP3A4 and about 6% of the drug is metabolized by flavin-containing monooxygenase 3 (FMO3). About 38% of the dose is circulating parent drug in the plasma and BHS697 and CQM157 are two major metabolites of infigratinib that are each found at >10% of the dose. They are pharmacologically active, with BHS697 representing about 16% to 33% of the overall pharmacological activity of infigratinib and CQM157 contributing to about 9% to 12%. BHS697 undergoes further metabolism mediated by CYP3A4 and CQM157 is metabolized through both Phase I and Phase II biotransformation pathways. The exact metabolic pathways and the structure of BHS697 and CQM157 are not fully characterized.

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Biological Half-Life
The geometric mean (CV%) terminal half-life of infigratinib was 33.5 h (39%) at steady state.

Hepatotoxicity
In the open label clinical trials of infigratinib for advanced or metastatic cholangiocarcinoma, adverse events were common and led to dose interruptions in 64%, dose reductions in 60% of patients, and permanent discontinuations in 15% largely for hyperphosphatemia, infections and sepsis rather than liver injury. In preregistration trials in 108 patients, ALT elevations arose in 51% and to above 5 times ULN in 6%. The elevations were typically self-limited and resolved rapidly with or without dose adjustments. No patients developed clinically apparent liver injury or jaundice. Since its approval, there have been no reports clinically apparent liver injury attributed to infigratinib. However, the total clinical experience with its use has been limited and the frequency of serum aminotransferase elevations during therapy suggest that clinically significant liver injury may occur.
Likelihood score: E* (unproven but possible, rare cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Infigratinib is no longer marketed in the US. No information is available on the clinical use of infigratinib during breastfeeding. Because infigratinib is 96.8% bound to plasma proteins, the amount in milk is likely to be low. However, because of its potential toxicity in the breastfed infant and its half-life of 33.5 hours, the manufacturer recommends that breastfeeding be discontinued during infigratinib therapy and for 1 month after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Infigratinib is about 96.8% bound to plasma proteins, primarily to lipoprotein. 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-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 anti-tumour agent that works to suppress tumour growth in cholangiocarcinoma. It exhibits anti-tumour activity in mouse and rat xenograft models of human tumours with activating FGFR2 or FGFR3 alterations, such as FGFR2-TTC28 or FGFR2-TRA2B fusions. In clinical trials, patients with cholangiocarcinoma who were treated with infigratinib had an overall response rate of 23% - where one patient had a complete response - and a duration of response of 5.5 months, with a range between 0.03 and 28.3 months. Some patients with cancers with FGFR mutations display intrinsic resistance to infigratinib, leading to negligible therapeutic efficacy: investigations are ongoing to target molecular pathways to combat drug resistance.

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.)