Plixorafenib (PLX8394) is an orally active inhibitor of the serine/threonine protein kinase B-Raf (BRAF) protein.Plixorafenib can selectively bind to and inhibit the activity of wild-type and mutant forms of BRAF, inhibiting the proliferation of tumor cells expressing mutant forms of BRAF.

BRAF:14 nM, Raf:23 nM, B-Raf (V600E):3.8 nM

METHODS: Parental 1205LuTR reporter cells and PRT#3 and #4 were treated with Plixorafenib (PLX8394) (1 μM, 24 hours), then cells were lysed and analyzed by western blot for the indicated cyclins; 1205Lu, PRT #3, and PRT #4 were treated with (PLX8394) (1 μM, 24 hours), EdU was added for the last 16 hours, and EdU incorporation was analyzed by flow cytometry.
RESULTS Plixorafenib effectively reduced the expression of cyclin D3 and cyclin D1, phosphorylated retinoblastoma protein, and cyclin A2 in parental 1205Lu cells; no reduction was observed in PRT #3 and PRT #4 cells; Plixorafenib inhibited EdU incorporation during the S phase in PRT #3 and PRT #4 cells. [1]

METHODS: Plixorafenib (PLX8394) (75, 150, 300 mg/kg, oral) was treated in HCC364 cell model mice, and serum concentrations (ng/mL) were measured at 0, 1, 2, 4, and 8 hours after treatment; HCC364 cells stably expressing firefly luciferase were surgically implanted into immunodeficient mice, and mice were treated with plixorafenib (150 mg/kg, oral) and tumor growth and response to RAF inhibitor treatment were monitored using a bioluminescence imaging (BLI) system.
RESULTS Plixorafenib (150 mg/kg) produced plasma concentrations >150μM without significant toxicity to the animals; Plixorafenib significantly inhibited tumor growth, and a more rapid and substantial initial antitumor response was observed in the treatment, an effect associated with enhanced and sustained inhibition of ERK phosphorylation and tumor cell proliferation. [2]

Dissolvent: DMSO. For MTT assays, 2×103?cells are seeded in triplicate in 96 wells in their regular culture medium (containing PLX4720 for PRT lines). Next day, cells are washed twice with PBS and then the medium is replenished containing the indicated RAF inhibitor. Medium is changed 48 hours later and after a further 48 hours, 10 μL of 5 mg/mL MTT reagent is added to wells and incubated for three hours. Formazan crystals are then solubilized overnight with a 1:10 dilution of 0.1 M glycine (pH 10.5) in DMSO. Wells are then analyzed at 450 nM in a Multiskan Spectrum spectrophotometer. Results depicted are normalized to DMSO conditions and are a composite of three independent experiments. Error bars shown are representative of the standard error of mean (SEM).

PLX8394 is dissolved in PEG 400 [20% (v/v)], TPGS [5% (v/v)], and water [75% (v/v)].H1755 tumor xenografts are generated by injection of 5×106 cells in a 50/50 mixture for matrigel and PBS into 6- to 8-wk-old female NOD/SCID mice. Mice are randomized to treatment groups once tumors reach an average size of 150 mm3. H1755 cells are s.c. implanted and allowed to grow to appr 200 mm3 (4 wk after implantation). Mice are then treated with vehicle, vemurafenib, or PLX8394 for 15 d. The vehicle for daily oral gavage is PEG 400 [20% (vol/vol)], tocopheryl polyethylene glycol succinate (TPGS) [5% (vol/vol)], water [75% (vol/vol)]. PLX8394 is dissolved in PEG 400 [20% (vol/vol)], TPGS [5% (vol/vol)], and water [75% (vol/vol)] and vortexed continuously throughout the dosing period. PLX8394 (p.o.) is given at a dose of 150 mg/kg/d.