B-Raf (V600E) (IC50 = 0.7 nM); B-Raf (IC50 = 5.2 nM); C-Raf (IC50 = 6.3 nM)

Dabrafenib demonstrated strong inhibitory activity in enzyme and cellular mechanistic assays, as well as in cell proliferation assays in B-RafV600E-driven melanoma lines SKMEL28 and A375P F11 (IC50 = 3 and 8 nM, respectively), and colorectal carcinoma line Colo205 (IC50 = 7 nM). On cells with wild-type B-Raf and tumor cells lacking the activating B-RafV600E mutation, dabrafenib has little effect in vitro (HFF IC50 = 3.0 μM). Compared to the majority of kinases screened, it is more selective than 500-fold for B-RafV600E. One of the kinases in the panel, Alk5, showed significant activity (<100-fold selectivity), but GSK2118436 is significantly less effective at inhibiting SMAD2/3 phosphorylation (IC50 = 3.7 μM) than it is at inhibiting ERK phosphorylation (IC50 = 4 nM) in a cellular setting[1]. Dabrafenib reduced the activity of the BRAFV600E kinase in cells, which in turn caused a decrease in MEK and ERK phosphorylation, as well as a G1 cell cycle arrest and cell death.

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Dabrafenib has a 400-fold preference for B-Raf over 91% of the other kinases tested when it comes to Raf kinase selectivity. Dabrafenib reduces ERK phosphorylation and inhibits cell proliferation by initially arresting the cell cycle in the G1 phase in cancer cells that specifically encode the B-RafV600E mutation. [1]

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Recent results from clinical trials with the BRAF inhibitors GSK2118436 (Dabrafenib) and PLX4032 (vemurafenib) have shown encouraging response rates; however, the duration of response has been limited. To identify determinants of acquired resistance to GSK2118436 and strategies to overcome the resistance, we isolated GSK2118436 drug-resistant clones from the A375 BRAFV600E and the YUSIT1 BRAFV600K melanoma cell lines. These clones also showed reduced sensitivity to the allosteric mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitor GSK1120212 (trametinib). Genetic characterization of these clones identified an in-frame deletion in MEK1 (MEK1K59del) or NRAS mutation (NRASQ61K and/or NRASA146T) with and without MEK1P387S in the BRAFV600E background and NRASQ61K in the BRAFV600K background. Stable knockdown of NRAS with short hairpin RNA partially restored GSK2118436 sensitivity in mutant NRAS clones, whereas expression of NRASQ61K or NRASA146T in the A375 parental cells decreased sensitivity to Dabrafenib/GSK2118436. Similarly, expression of MEK1K59del, but not MEK1P387S, decreased sensitivity of A375 cells to GSK2118436. The combination of GSK2118436 and GSK1120212 effectively inhibited cell growth, decreased ERK phosphorylation, decreased cyclin D1 protein, and increased p27kip1 protein in the resistant clones. Moreover, the combination of GSK2118436 or GSK1120212 with the phosphoinositide 3-kinase/mTOR inhibitor GSK2126458 enhanced cell growth inhibition and decreased S6 ribosomal protein phosphorylation in these clones. Our results show that NRAS and/or MEK mutations contribute to BRAF inhibitor resistance in vitro, and the combination of GSK2118436 and GSK1120212 overcomes this resistance. In addition, these resistant clones respond to the combination of GSK2126458 with GSK2118436 or GSK1120212. Clinical trials are ongoing or planned to test these combinations.[2]

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GSK2118436/Dabrafenib (12) displayed compelling inhibitory activity in enzyme and cellular mechanistic assays, and in cell proliferation assays in B-RafV600E-driven melanoma lines, SKMEL28 and A375P F11 (IC50 = 3 and 8 nM, respectively), and colorectal carcinoma line Colo205 (IC50 = 7 nM). Gratifyingly, GSK2118436 was found to have a minimal effect in vitro on cells with wild-type B-Raf (HFF IC50 = 3.0 μM) and in tumor cells not harboring the activating B-RafV600E mutation [3].

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GSK2118436/Dabrafenib was screened against a set of 61 kinases representing broad coverage of the kinome and was found to be a potent biochemical inhibitor of B-RafV600E, wild-type B-Raf, and c-Raf, displaying subnanomolar or nanomolar potencies (see the Supporting Information). Importantly, GSK2118436 was also found to be highly selective, exhibiting >500-fold selectivity for B-RafV600E compared to most kinases screened.11 Significant activity (<100-fold selectivity) was observed for a single kinase in the panel, Alk5. The impact of Alk5 enzyme activity on cellular signaling was further investigated by measuring the downstream phosphorylation level of SMAD2/3 in HepG2 cells.12 GSK2118436 was significantly less effective at inhibiting SMAD2/3 phosphorylation (IC50 = 3.7 μM) compared with inhibiting ERK phosphorylation (IC50 = 4 nM) in a cellular context. These data underscore the remarkable selectivity achieved by this uniquely potent and selective inhibitor of B-RafV600E.[3]

Dabrafenib, when taken orally, prevented ERK activation, downregulated Ki67, and upregulated p27 in a BRAFV600E-containing xenograft model of human melanoma, which inhibited tumor growth. Dabrafenib is orally bioavailable, does not significantly accumulate after multiple doses, and reduces pERK after 7 and 14 days of dosing[2]. This effect is sustained for up to 18 hours post-dosing. Dabrafenib (orally administrated) inhibits the development of B-RafV600E mutant colon cancer (Colo205) and melanoma (A375P) human tumor xenografts that are grown subcutaneously in immunodeficient mice. [1]

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GSK2118436/Dabrafenib was also studied in vivo and found to dramatically reduce tumor growth in mice bearing B-RafV600E human melanoma tumors. In this model, CD1 nu/nu mice bearing A375P F11 (B-RafV600E) tumors were dosed orally with GSK2118436 at doses of 0.1, 1, 10, and 100 mg/kg once daily for 14 days (Figure 3). Dose-proportional reductions in tumor growth were observed. Body weight was also measured, and no significant changes were observed at any of the doses tested. Notably, in the 100 mg/kg group, complete tumor regression was observed in 50% of treated animals.[3]

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Additionally, GSK2118436/Dabrafenib reduced the levels of pERK in A375P F11 (B-RafV600E) tumor tissue in vivo in a dose-dependent manner after a single oral dose. Tumors were collected 2, 6, and 24 h postdose, and pERK levels in each were measured and normalized to the total ERK (tERK) present. The MEK inhibitor PD0325901 was used as a control. Figure 4 shows pERK/tERK levels compared to those of vehicle-treated animals and pharmacokinetic data in a composite of two separate studies. Levels of pERK/tERK were substantially reduced 2 and 6 h postdose, with a notable pharmacodynamic effect (>50% inhibition) at doses of ≥3 mg/kg in this single-dose study, which returned to untreated levels by 24 h at doses of ≤30 mg/kg. The concentration of drug measured in the blood and tumor during the course of the study correlated with the observed pharmacodynamic effects. These data suggest that under the conditions tested, a single oral dose of ≥3 mg/kg Dabrafenib/GSK2118436 can maintain ≥50% B-Raf inhibition for at least 6 h.[3]

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Raf kinases signal via extracellular signal-regulated kinases 1/2 (ERK1/2) to drive cell division. Since activating mutations in BRAF (B-Raf proto-oncogene, serine/threonine kinase) are highly oncogenic, BRAF inhibitors including Dabrafenib have been developed for cancer. Inhibitors of ERK1/2 signalling used for cancer are cardiotoxic in some patients, raising the question of whether dabrafenib is cardiotoxic. In the heart, ERK1/2 signalling promotes not only cardiomyocyte hypertrophy and is cardioprotective but also promotes fibrosis. Our hypothesis is that ERK1/2 signalling is not required in a non-stressed heart but is required for cardiac remodelling. Thus, dabrafenib may affect the heart in the context of, for example, hypertension. In experiments with cardiomyocytes, cardiac fibroblasts and perfused rat hearts, Dabrafenib inhibited ERK1/2 signalling. We assessed the effects of Dabrafenib (3 mg/kg/d) on male C57BL/6J mouse hearts in vivo. Dabrafenib alone had no overt effects on cardiac function/dimensions (assessed by echocardiography) or cardiac architecture. In mice treated with 0.8 mg/kg/d angiotensin II (AngII) to induce hypertension, dabrafenib inhibited ERK1/2 signalling and suppressed cardiac hypertrophy in both acute (up to 7 d) and chronic (28 d) settings, preserving ejection fraction. At the cellular level, dabrafenib inhibited AngII-induced cardiomyocyte hypertrophy, reduced expression of hypertrophic gene markers and almost completely eliminated the increase in cardiac fibrosis both in interstitial and perivascular regions. Dabrafenib is not overtly cardiotoxic. Moreover, it inhibits maladaptive hypertrophy resulting from AngII-induced hypertension. Thus, Raf is a potential therapeutic target for hypertensive heart disease and drugs such as Dabrafenib, developed for cancer, may be used for this purpose [4].

Biochemical and Cellular Assays. [3]
Details of the B-Raf biochemical enzyme and cellular assays have been reported in manuscript Reference 6. The inhibition of 61 protein kinases by Dabrafenib was characterized at GlaxoSmithKline. In general, the assays were configured so that the IC50 values approximate the intrinsic binding constant (Ki or Kd) of Dabrafenib to each enzyme and can therefore be compared for selectivity against these kinases. A375P-F11 assay: A375P cells were plated in 96-well plates by limiting dilution and single cell-derived clones were harvested and tested for sensitivity to B-Raf inhibitors. The F11 clone was selected for future studies and was named A375P-F11. Cellular pSmad Assay to Measure Anti-TGF-β Activity: Activity of compounds was tested in a mechanistic assay in HepG2 cells. Cells were seeded in 12-well plates at a density of 500,000 cells/well and allowed to adhere overnight at 37o C/5% CO2. Media (BME+10% serum) was removed and compound in serum free media was added to the cells for 45 minutes at 37o C/5% CO2. Cells were stimulated with 1 ng/ml TGF-β for 60 minutes. Cells were lysed in buffer (25 mM Tris-HCl ph: 7.5, 2 mM EDTA, 2 mM EGTA,1% Triton X-100, 0.1 % SDS, 50 mM sodium-β-glycerophosphate, 2 mM sodium orthovanadate, 12.5 mM sodium pyrophosphate, protease and phosphatase inhibitor cocktails) for 30 minutes, scraped, collected, clarified by centrifugation and prepared for western blots in LDS/reducing reagent. Samples were resolved on 4-12% Bis-Tris gels, transferred to PVDF, and probed for total and phospho-Smad2 using antibodies from Cell Signaling. Gels were imaged using the odyssey blot scanner and quantified using Licor software. Phospho:total Smad2 ratios were determined and the IC50 was defined as the concentration of compound which decreased the phospho:total ratio by 50%.

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Metabolite Identification. [3]
Metabolite identification study in dog liver microsomes: Dog liver microsomes were purchased from Xenotech. The incubation mixture (800 µL) containing 50 mM potassium phosphate buffer, hepatic microsomal fraction (1.0 mg/mL protein) and 10 µM study compound in a 1.5 mL Eppendorf tube was pre-warmed to 37 °C. The cofactor was preincubated at 37 °C for 5 minutes. For 0 minute time point, aliquots of incubation mixture (200 µL) and cofactor solution (50 µL) were removed and crashed with stop solution [250 µL, (80/20/1, acetonitrile/ethanol/acetic acid, v/v/v)]. The reaction was started by addition of 150 µL NADPH generating system [2.2 mM NADP, 28 mM glucose-6-phosphate and glucose-6-phosphate dehydrogenase (6 units/mL) and 4.0 mM UDPGA in 2 % sodium bicarbonate] and the samples (final volume 750 µL) were incubated at 37 °C. No drug control incubations were conducted in parallel using blank incubation medium with an appropriate amount of methanol in place of the compound for liver microsomes. After a 30 minute incubation the reaction was terminated by the addition of one volume of stop solution. Samples were centrifuged at 34000 rpm for 5 min and 50 µL supernatants were injected to LC/MS for metabolite identification. Metabolite identification study in dog hepatocytes: Dog hepatocytes (Db157) were purchased from CellzDirect. The incubation mixtures consisting of William’s medium E (pH = 7.4), hepatocytes suspension (0.7 million cells/mL) and 10 µM study compound in a total volume of 600 µL were placed into a single well of 12-well culture plate and incubated for 4 h at 37 °C. No drug control incubations were conducted in parallel using blank incubation medium with an appropriate amount of methanol in place of active treatment for liver hepatocytes. After incubation, the cells were scraped from the bottom of each well and mixed with 600 µL of stop solution. The mixture was then stored in -80 °C freezer. Samples were centrifuged at 34000 rpm for 5 min and 50 µL supernatants were injected to LC/MS for metabolite identification.

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Binding Mode: [3]
GSK2118436/Dabrafenib is an ATP-competitive inhibitor of B-Raf11, and is postulated to bind to an inactive-like conformation of the kinase (Figure S1), based on a model of GSK2118436 docked into a recently reported crystal structure of B-RafV600E bound to other small molecule ATP-binding site inhibitors.1 In this model, the αC helix ‘shifts out’ relative to an active-like conformation and a salt bridge between the conserved lysine and glutamic acid is broken. While Phe595 is not in a ‘DFG-out’ conformation, it is rotated to form the floor of a pocket similar to that observed in a reported lapatinib/EGFR co-structure.2 The pyrimidine N1 forms the classic hinge interaction with Cys532 in the ATP-binding pocket of the enzyme. The t-butyl group and thiazole core bind underneath the P-loop leaving only a relatively small portion of the inhibitor as solvent exposed. The arylsulfonamide headgroup of GSK2118436 is predicted to bind into the lipophilic back pocket allowing the sulfonamide to form two hydrogen bonds with the backbone NHs of Asp594 and Phe595. The sulfonamide NH is depicted in a deprotonated form, leaving the nitrogen to participate as a hydrogen bond acceptor. The significant improvement in potency observed with compounds bearing a fluorine substitution at R2 is hypothesized to be a result of modulating the pKa of the sulfonamide NH which could allow both increased ionization of 12 at cellular pH and a more favorable conformation for headgroup binding. Conversely, compounds containing fluorine substitution at R1 (compounds 4 and 9), may induce an unfavorable conformation of the benzenesulfonamide moiety in the binding pocket, potentially accounting for the reduced target affinity observed with those analogs. Analogous models of aniline-tail containing molecules (like 1) show that the aromatic ring in the tail group resides largely within the B-Raf binding pocket while the morpholino ring extends into the solvent exposed region. Comparing 1 and 2, after complete truncation of the tail to the “naked” aminopyrimidine and a loss of 13 heavy atoms, a 10 fold drop in potency was observed. Despite the small drop in potency, 2 actually exhibits a higher ligand efficiency compared to 1 (LE = 0.30 for 2 vs 0.25 for 1), as the all of the remaining heavy atoms in 2 are contained fully within the binding pocket.

For longer-term proliferation assays, cells are plated in 10% FBS-containing RMPI-1640 for 12 days and treated with a single compound or a combination of compounds. The assay involves at least one compound treatment replacement. Using 0.5% methylene blue in 50% ethanol, cells are stained after 12 days. Using a flatbed scanner, pictures are taken.

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A375PF11 cells (referred to henceforth as A375) were exposed to increasing concentrations of Dabrafenib/GSK2118436 and maintained in a final concentration of 1.2 or 1.6 μmol/L. Similarly, YUSIT1 cells were exposed to 0.1 μmol/L of GSK2118436. Single-cell clones were isolated by limiting dilution from these populations. A375 clones were 95% similar to the parental A375 cells as determined by SNP chip analysis. A representative resistant YUSIT1 clones shared 100% of the parental YUSIT1 SNPs as determined by exome sequencing. All cells were grown in RPMI-1640 medium containing 10% FBS without GSK2118436 for at least one passage before an experiment. [2]

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Affymetrix expression analyses: 16R6-4 was selected for comparison with A375 after compound treatment with Dabrafenib/GSK2118436 and GSK1120212 alone and in combination with each other for 24 hours. Data analysis was done as described in the Supplementary Methods. The microarray data were deposited in NCBI's Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE35230. [2]

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Cell growth inhibition and apoptosis assays: Inhibition of cell growth was estimated after 3-day treatment with compound or combination of compounds using CellTiter-Glo as described previously. Caspase-3/7 activity was determined via Caspase-Glo 3/7 assay 24 hours after similar treatment with compounds. Before 3-day growth assay, cells were transfected or transduced with short interfering RNA (siRNA), short hairpin RNA (shRNA), or expression constructs as described in the Supplementary Methods. For longer term proliferation assays, cells were plated and treated with compound or combination of compounds in RMPI-1640 containing 10% FBS for 12 days. Compound treatments were replaced at least once during the assay. After 12 days, cells were stained with 0.5% methylene blue in 50% ethanol. Images were captured using flatbed scanner [2].

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Neonatal rat cardiomyocytes were prepared and cultured from 2 to 4 d Sprague-Dawley rats as described previously. Human cardiac fibroblasts were grown in Fibroblast growth medium-3. Fibroblasts were seeded the day before experimentation (at a density to achieve 90% confluence after 24 h) and synchronized overnight in M199 medium containing 0.1% (v/v) foetal calf serum and 100 U/ml penicillin and streptomycin. Cells were exposed to the concentrations of Dabrafenib and for the times indicated prior to harvesting for immunoblotting [4].

The 26 10-week-old, time-mated, virus-antibody-free SD (Crl:CD[SD]) female rats that were chosen as the test system gave birth to the rat pups. From Day 20 to Day 23 postpartum, mated females are monitored for spontaneous deliveries (the day parturition is complete is designated PND 0). When parturition is complete, on PNDs 3 and 6, litter examinations are carried out. These examinations include external morphologic examinations, gender determination, and individual pup weights. Clinical signs and body weights are used to select parturient dams and their litters for the study, and chosen dams and their litters are then randomly assigned to study groups based on clinical observations and PND 3 litter mean body weights. On PND 3 or 4, litters are reduced to four or five males and females, with only a small amount of fostering required to achieve the desired sex ratio. This helps to preserve natural litter sizes as much as possible. Records of the pups raised by the original and foster dams are kept. Paw tattoos are used to identify each puppy. Nonlittermates are placed in subsets to the greatest extent possible. Dabrafenibis administered to young male and female rats by oral gavage at a dose volume of 5 ml/kg, based on daily body weight, in a suspension of vehicle, 0.5% hydroxypropylmethylcellulose K15M, and 0.1% (v/v) Tween80 in purified water.

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A375P F11 Melanoma Xenograft Studies. [3]
Cells were implanted in nude mice and grown as tumor xenografts. Dosing began when tumors achieved ~150- 200mm3 volume. Dabrafenib/GSK2118436 was administered by oral gavage at a dose volume of 0.2 mL/20 gram body weight in 0.5% hydroxypropylmethylcellulose and 0.2% Tween-80 in distilled water pH 8.0. Dosing was daily for duration stipulated. Results are reported as mean tumor volume for n=7-8 mice/group. Tumors were measured twice weekly with Vernier calipers, and tumor volume was estimated from two-dimensional measurements using a prolate ellipsoid equation (Tumor volume mm3 = (length x width2 ) x 0.5). Complete tumor regression was defined as a >93% decrease in an individual tumor volume for at least 1 week.

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Pharmacokinetic (PK) Analysis. [3]
Blood was drawn and hemolyzed immediately with an equal volume of water. Concentrations of compound in tumor were determined on polytron homogenized tissues in 4 volumes of water per volume of frozen tissue. Aliquots of the homogenized tumor and blood were flash frozen and subsequently evaluated for compound concentration by HPLC/MS/MS analysis.

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Pharmacodyamic measurement of pERK levels in tissues. [3]
Tissues were harvested and homogenized using Medimachine with 1 mL of lysis buffer (25 mM Tris-HCl (pH 7.5), 2 mM EDTA (pH 8.0), 2 mM EGTA (pH 8.0), 1% Triton X-100, 0.1% SDS, 50 mM sodium glycerol phosphate, 2 mM Na3VO4, 4 mM Na-pyrophosphate, 2x phosphatase inhibitor cocktail), and kept on ice. Following homogenization of all samples, homogenates were centrifuged at 14,000 rpm for 15 min at 4°C and flash frozen for later analysis. All samples were analyzed with a duplex ELISA measuring total ERK1/2 and phospho-ERK1/2 according to manufacturer’s instructions. Plates were read on MSD.SI6000.

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In vivo mouse studies [4]
Wild-type male (7 wk) C57Bl/6J mice were allowed to acclimatize for 2 weeks before experimentation. Mice were randomly allocated to each treatment group; body weights are provided in Supplementary Table S1. Drug delivery used Alzet osmotic pumps (models 1007D or 1004), filled according to the manufacturer’s instructions. Mice received minipumps for delivery of 0.8 mg/kg/d angiotensin II (AngII) or vehicle (acidified PBS) without/with DMSO/PEG mix [50% (v/v) dimethyl sulphoxide (DMSO), 20% (v/v) polyethylene glycol 400, 5% (v/v) propylene glycol, 0.5% (v/v) Tween 80] or 3 mg/kg/d Dabrafenib dissolved in DMSO/PEG mix. Separate minipumps were used for AngII and Dabrafenib delivery. Minipumps were incubated overnight in sterile PBS (37°C), then implanted subcutaneously under continuous inhalation anaesthesia using isoflurane (induction at 5%, maintenance at 2–2.5%) mixed with 2 l/min O2, as previously described

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Histology and assessment of myocyte size and fibrosis [4]
Histological staining and analysis were performed as previously described, assessing general morphology by haematoxylin and eosin (H&E) and fibrosis by Masson’s trichrome and picrosirius red (PSR). Sections for the study of the effects of Dabrafenib on AngII-induced cardiac pathology over 28 d were prepared and stained by HistologiX Limited. Analysis was performed by independent assessors blinded to treatment groups.

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Adult rat heart perfusions [4]
Adult male (300–350 g) Sprague-Dawley rats were used for heart perfusions. Hearts were prepared and perfused in the Langendorff mode as described. Hearts were perfused for 15 min with Krebs-Henseleit bicarbonate-buffered saline (25 mM NaHCO3, 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, pH 7.4, containing 10 mM glucose and equilibrated with 95% O2/5% CO2) without or with Dabrafenib (5 µM) or trametinib (1 µM). Dabrafenib and trametinib were from Selleck Chemicals. Perfusions were continued for 10 min without/with addition of human FGF2 (0.5 µg/ml; Cell Guidance Systems Ltd., U.K.). Hearts were ‘freeze-clamped’ between aluminium tongs cooled in liquid nitrogen and pulverized under liquid N2. Heart powders were stored at −80°C.

Absorption, Distribution and Excretion
After oral administration, the median time to achieve peak plasma concentration (Tmax) is 2 hours. Mean absolute bioavailability of oral dabrafenib is 95%. Following a single dose, dabrafenib exposure (Cmax and AUC) increased in a dose-proportional manner across the dose range of 12 mg to 300 mg, but the increase was less than dose-proportional after repeat twice-daily dosing. After repeated twice-daily dosing of 150 mg, the mean accumulation ratio was 0.73, and the inter-subject variability (CV%) of AUC at steady-state was 38%.
Fecal excretion is the major route of elimination accounting for 71% of radioactive dose while urinary excretion accounted for 23% of total radioactivity as metabolites only.
The apparent volume of distribution (Vc/F) is 70.3 L.Distribution to the brain is restricted because dabrafenib is a substrate and undergoes efflux by P-glycoprotein and breast cancer resistance protein.
The clearance of dabrafenib is 17.0 L/h after single dosing and 34.4 L/h after 2 weeks of twice-daily dosing.

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Metabolism / Metabolites
The metabolism of dabrafenib is primarily mediated by CYP2C8 and CYP3A4 to form hydroxy-dabrafenib. Hydroxy-dabrafenib is further oxidized via CYP3A4 to form carboxy-dabrafenib and subsequently excreted in bile and urine. Carboxy-dabrafenib is decarboxylated to form desmethyl-dabrafenib; desmethyl-dabrafenib may be reabsorbed from the gut. Desmethyl-dabrafenib is further metabolized by CYP3A4 to oxidative metabolites.

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Biological Half-Life
The mean terminal half-life of dabrafenib is 8 hours after oral administration. Hydroxy-dabrafenib's terminal half-life (10 hours) parallels that of dabrafenib while the carboxy- and desmethyl-dabrafenib metabolites exhibit longer half-lives (21 to 22 hours).

Hepatotoxicity
Elevations in serum ALT levels were reported in 11% of patients treated with dabrafenib alone, but all elevations were above 5 times ULN. When dabrafenib was given in combination with trametinib, serum ALT elevations occurred in 35% to 42% of patients and were above 5 times ULN in 4%. Similarly, serum alkaline phosphatase elevations occurred in 26% of patients given dabrafenib alone, but in 60% to 67% given dabrafenib and trametinib. These abnormalities were largely asymptomatic and fully reversible. There were no instances of clinically apparent acute liver injury or hepatic failure reported in prelicensure studies of dabrafenib and, since its approval and more wide spread use, there have been no published reports of dabrafenib hepatotoxicity.
Likelihood score: E* (unproven but suspected cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of dabrafenib during breastfeeding. Because dabrafenib is more than 99% bound to plasma proteins, the amount in milk is likely to be low. The manufacturer recommends that breastfeeding be discontinued during dabrafenib therapy and for 2 weeks 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
Dabrafenib is 99.7% bound to human plasma proteins.

[1]. Sylvie Laquerre, et al. 2009, EORTC International Conference. Abst B88.

[2]. Mol Cancer Ther. 2012 Apr;11(4):909-20.

[3]. ACS Med Chem Lett. 2013 Feb 7;4(3):358-62.

[4]. Clin Sci (Lond). 2021 Jul 30;135(14):1631-1647.

Dabrafenib mesylate is a methanesulfonate (mesylate) salt prepared from equimolar amounts of dabrafenib and methanesulfonic acid. Used for treatment of metastatic melanoma. It has a role as an antineoplastic agent and a B-Raf inhibitor. It contains a dabrafenib.
Dabrafenib Mesylate is the mesylate salt form of dabrafenib, an orally bioavailable inhibitor of B-raf (BRAF) protein with potential antineoplastic activity. Dabrafenib selectively binds to and inhibits the activity of B-raf, which may inhibit the proliferation of tumor cells which contain a mutated BRAF gene. B-raf belongs to the raf/mil family of serine/threonine protein kinases and plays a role in regulating the MAP kinase/ERKs signaling pathway, which may be constitutively activated due to BRAF gene mutations.
See also: Dabrafenib (has active moiety).

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Drug Indication
Treatment of melanoma, Treatment of solid malignant tumours (excluding melanoma)

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Pharmacodynamics
Dabrafenib is a kinase inhibitor that is mainly used to target BRAF V600E mutation in multiple types of cancer. Although dabrafenib and [trametinib] both inhibit the RAS/RAF/MEK/ERK pathway, they inhibit different effectors of the pathway, thus increasing response rate and mitigating resistance without cumulative toxicity. The melanoma approval for use with [trametinib] is based on results from COMBI-AD, a Phase III study of 870 patients with Stage III BRAF V600E/K mutation-positive melanoma treated with dabrafenib + trametinib after complete surgical resection. Patients received doses of dabrafenib (150 mg BID) + trametinib (2 mg QD) combination (n = 438) or matching placebos (n = 432). After a median follow-up of 2.8 years, the primary endpoint of relapse-free survival (RFS) was met. In the case of thyroid cancer, Dabrafenib plus Trametinib is the first regimen demonstrated to have potent clinical activity in BRAF V600E–mutated anaplastic thyroid cancer and is well tolerated. These findings represent a meaningful therapeutic advance for this orphan disease.

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Dabrafenib is an organofluorine compound and antineoplastic agent, used as its mesylate salt in treatment of metastatic melanoma. It has a role as an antineoplastic agent, a B-Raf inhibitor and an anticoronaviral agent. It is a sulfonamide, an organofluorine compound, a member of 1,3-thiazoles and an aminopyrimidine.
Dabrafenib mesylate (Tafinlar) is a reversible ATP-competitive kinase inhibitor and targets the MAPK pathway. It was approved on May 29, 2013, for the treatment of melanoma with V600E or V6000K mutation. It was also used for metastatic non-small cell lung cancer with the same mutation. In May 2018, Tafinlar (dabrafenib), in combination with Mekinist ([DB08911]), was approved to treat anaplastic thyroid cancer caused by an abnormal BRAF V600E gene.
Dabrafenib is a Kinase Inhibitor. The mechanism of action of dabrafenib is as a Protein Kinase Inhibitor, and Cytochrome P450 3A4 Inducer, and Cytochrome P450 2B6 Inducer, and Cytochrome P450 2C8 Inducer, and Cytochrome P450 2C9 Inducer, and Cytochrome P450 2C19 Inducer, and Organic Anion Transporting Polypeptide 1B1 Inhibitor, and Organic Anion Transporting Polypeptide 1B3 Inhibitor, and Organic Anion Transporter 1 Inhibitor, and Organic Anion Transporter 3 Inhibitor, and Breast Cancer Resistance Protein Inhibitor.

Dabrafenib is a selective inhibitor of mutated forms of BRAF kinase and is used alone or in combination with trametinib in the treatment of advanced malignant melanoma. Dabrafenib therapy is associated with transient elevations in serum aminotransferase during therapy, but has not been linked to instances of clinically apparent acute liver injury.
Dabrafenib is an orally bioavailable inhibitor of B-raf (BRAF) protein with potential antineoplastic activity. Dabrafenib selectively binds to and inhibits the activity of B-raf, which may inhibit the proliferation of tumor cells which contain a mutated BRAF gene. B-raf belongs to the raf/mil family of serine/threonine protein kinases and plays a role in regulating the MAP kinase/ERKs signaling pathway, which may be constitutively activated due to BRAF gene mutations.
DABRAFENIB is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 2013 and has 7 approved and 19 investigational indications.

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Activation of the Ras‐Raf‐MEK‐ERK pathway has been implicated in a large range of human cancers. Growth factor receptor stimulation by extracellular ligands activates Ras, which then sets in motion a signal transduction cascade through the Raf, MEK and ERK serine/threonine kinases. Mutation of the B‐Raf kinase constitutively activates MAPK signalling, thus bypassing the need for upstream stimuli. This has been genetically associated with several human cancers, especially occurrence of the B‐RafV600E mutant and its high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. The ability to selectively and potently inhibit B‐Raf should provide a potential therapy for patients with mutant B‐Raf tumors, for which addictive dependency on this pathway is observed. We have identified a novel, potent, and selective Raf kinase inhibitor that is capable of inhibiting the kinase activity of wild‐type B‐Raf, B‐RafV600E and c‐Raf with IC50 values of 3.2, 0.8, and 5.0 nM, respectively. Kinase panel screening for over 270 kinases has indicated that this inhibitor is selective for Raf kinase, with ∼400 fold selectivity towards B‐Raf over 91% of the other kinases tested. Specific cellular inhibition of B‐RafV600E kinase by this inhibitor leads to decreased ERK phosphorylation and inhibition of cell proliferation by an initial arrest in the G1 phase of the cell cycle, followed by cell death. This inhibition is selective for cancer cells that specifically encode the mutation for B‐RafV600E. Oral compound administration inhibits the growth of B‐RafV600E mutant melanoma (A375P) and colon cancer (Colo205) human tumor xenografts, growing subcutaneously in immuno‐compromised mice. This cell‐specific B‐RafV600E inhibitor is currently being evaluated in a human Phase I clinical trial. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B88.[1]

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Hyperactive signaling of the MAP kinase pathway resulting from the constitutively active B-RafV600E mutated enzyme has been observed in a number of human tumors, including melanomas. Herein we report the discovery and biological evaluation of Dabrafenib/GSK2118436, a selective inhibitor of Raf kinases with potent in vitro activity in oncogenic B-Raf-driven melanoma and colorectal carcinoma cells and robust in vivo antitumor and pharmacodynamic activity in mouse models of B-RafV600E human melanoma. GSK2118436 was identified as a development candidate, and early clinical results have shown significant activity in patients with B-Raf mutant melanoma. [3]

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In summary, lead molecule 1, with high potency and poor pharmacokinetic properties in higher species, was rapidly evolved into clinical development candidate Dabrafenib/GSK2118436 (12). Identification of the key B-RafV600E potency contributors (sulfonamide head, R2 fluorination, and tert-butyl thiazole core) through detailed and iterative SAR studies allowed for the elimination of nonessential portions of the lead structure and high selectivity to be achieved across the kinome. With high enzyme and cellular potency engineered into the original lead, pharmacokinetic issues were addressed by combining multiple changes in the template, resulting in the improved multispecies PK properties observed for 12. With high rodent exposure realized in GSK2118436, we were able to demonstrate target-related pharmacology in two in vivo models, gaining a high level of confidence in the progression of this molecule into clinical studies. Additionally, vast improvements in higher-species PK allowed for the achievement of exposures necessary to fully explore the safety of the molecule preclinically. Finally, a >20% decrease in molecular weight compared to that of 1 resulted in improved physicochemical properties, expediting the identification of a salt version (mesylate) and form suitable for early clinical studies.
The efforts described herein ultimately led to the discovery of GSK2118436 (Dabrafenib), which is currently in advanced clinical studies in B-Raf mutant tumors. GSK2118436 has shown remarkable efficacy in melanoma patients with activating B-Raf mutations, including patients with brain metastases, and has further shown enhanced clinical activity in combination with the MEK inhibitor, trametinib. Details of these studies will be reported in due course. [3]

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The final considerations for our work are the potential implications of using drugs such as Dabrafenib for cardiac diseases. Dabrafenib itself does not appear to be cardiotoxic and is generally well-tolerated. There are side effects in cancer patients which are generally managed by dose reduction. The most severe effect is probably an increase in cutaneous squamous cell carcinoma (∼12% of patients. Thus, if dabrafenib were to be used as a therapy for hypertensive heart disease, it may be important to consider dosage monitoring and whether patients have a predisposition for other diseases such as cancer (in the context, perhaps, of ‘onco-cardiology’). Nevertheless, reducing cardiac fibrosis with a drug such as dabrafenib may be such a powerful tool in treating cardiac diseases that the benefits could outweigh these costs.[4]

C24H24F3N5O5S3

615.66

615.089

C, 46.82; H, 3.93; F, 9.26; N, 11.38; O, 12.99; S, 15.62

1195768-06-9

Dabrafenib;1195765-45-7

44516822

White to off-white solid powder

7.033

3

14

6

40

910

0

S1C(C2C([H])=C([H])N=C(N([H])[H])N=2)=C(C2C([H])=C([H])C([H])=C(C=2F)N([H])S(C2C(=C([H])C([H])=C([H])C=2F)F)(=O)=O)N=C1C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H].S(C([H])([H])[H])(=O)(=O)O[H]

YKGMKSIHIVVYKY-UHFFFAOYSA-N

InChI=1S/C23H20F3N5O2S2.CH4O3S/c1-23(2,3)21-30-18(19(34-21)16-10-11-28-22(27)29-16)12-6-4-9-15(17(12)26)31-35(32,33)20-13(24)7-5-8-14(20)25;1-5(2,3)4/h4-11,31H,1-3H3,(H2,27,28,29);1H3,(H,2,3,4)

N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1,3-thiazol-4-yl]-2-fluorophenyl]-2,6-difluorobenzenesulfonamide;methanesulfonic acid

GSK2118436A Mesylate; GSK 2118436A Mesylate; Dabrafenib mesylate; 1195768-06-9; Taflinar; UNII-B6DC89I63E; GSK 2118,436B; GSK-2118436-B; Dabrafenib methanesulfonate; GSK2118436B; GSK-2118436A; GSK2118436B ( Dabrafenib Mesylate); GSK-2118436B Mesylate; GSK 2118436B. Trade name: Tafinlar.

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.06 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 25.0 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 2: ≥ 2.5 mg/mL (4.06 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 25.0 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 3: ≥ 2.5 mg/mL (4.06 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2.5 mg/mL (4.06 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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: ≥ 2.5 mg/mL (4.06 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: 8 mg/mL

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