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. \n

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\nA375P F11 Melanoma Xenograft Studies. [3]
\nCells 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. \n

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\nPharmacokinetic (PK) Analysis. [3]
\nBlood 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. \n

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\nPharmacodyamic measurement of pERK levels in tissues. [3]
\nTissues 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. \n

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\n\nIn vivo mouse studies [4]
\nWild-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\n

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\n\nHistology and assessment of myocyte size and fibrosis [4]
\nHistological 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.\n

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\n\nAdult rat heart perfusions [4]
\nAdult 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.\n\n

Absorption, Distribution and Excretion
Following oral administration, the median time to reach peak plasma concentration (Tmax) of dabrafenib is 2 hours. The mean absolute bioavailability of oral dabrafenib is 95%. Following a single dose, dabrafenib exposure (Cmax and AUC) increases proportionally to the dose range of 12 mg to 300 mg, but the increase is less proportional to the dose after repeated dosing twice daily. After repeated dosing of 150 mg twice daily, the mean cumulative rate was 0.73, and the inter-subject coefficient of variation (CV%) for steady-state AUC was 38%. Fecal excretion is the primary elimination route, accounting for 71% of the radioactive dose, while urinary excretion accounts for only 23% of total radioactivity (in metabolite form). The apparent volume of distribution (Vc/F) is 70.3 L. Because dabrafenib is a substrate and is effluxed via P-glycoprotein and breast cancer resistance protein, its brain distribution is limited.
The clearance of dabrafenib after a single dose is 17.0 L/h, and after two weeks of twice-daily administration, it is 34.4 L/h.

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Metabolism/Metabolites
Dabrafenib metabolism is primarily mediated by CYP2C8. Dabrafenib is metabolized by CYP3A4 to hydroxydabrafenib. Hydroxydabrafenib is further oxidized by CYP3A4 to carboxydabrafenib, which is then excreted in bile and urine. Carboxydabrafenib is decarboxylated to desmethyldabrafenib; desmethyldabrafenib may be reabsorbed by the intestine. Desmethyldabrafenib is further metabolized by CYP3A4 to oxidative metabolites.

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Biological Half-Life
The mean terminal half-life of orally administered dabrafenib is 8 hours. The terminal half-life of hydroxydabrafenib (10 hours) is similar to that of dabrafenib, while the half-lives of carboxydabrafenib and desmethyldabrafenib metabolites are longer (21 to 22 hours).

Hepatotoxicity
In patients receiving dabrafenib monotherapy, 11% experienced elevated serum ALT levels, but all elevations exceeded 5 times the upper limit of normal. When dabrafenib was combined with trametinib, 35% to 42% of patients experienced elevated serum ALT, with 4% of these patients having ALT levels exceeding 5 times the upper limit of normal. Similarly, 26% of patients receiving dabrafenib monotherapy experienced elevated serum alkaline phosphatase, while 60% to 67% of patients receiving dabrafenib combined with trametinib experienced elevated serum alkaline phosphatase. Most of these abnormalities were asymptomatic and completely reversible. No clinically significant cases of acute liver injury or liver failure were reported in pre-marketing studies of dabrafenib; and no published reports of dabrafenib hepatotoxicity have been found since its approval and widespread use. Probability score: E (Unproven but suspected cause of clinically significant liver injury).

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Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information regarding the clinical use of dabrafenib during lactation. Because dabrafenib binds to plasma proteins at a rate exceeding 99%, its concentration in breast milk may be very low. The manufacturer recommends discontinuing breastfeeding during dabrafenib treatment and for two weeks 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
Dabrafenib binds to human plasma proteins at a rate of 99.7%.

[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 mesylate salt prepared from equimolar amounts of dabrafenib and mesylate. It is used to treat metastatic melanoma. It is an antitumor drug and a B-Raf inhibitor. Its main component is dabrafenib. Dabrafenib mesylate is the mesylate form of dabrafenib, a highly bioavailable orally bioavailable B-Raf (BRAF) protein inhibitor with potential antitumor activity. Dabrafenib selectively binds to and inhibits the activity of B-Raf, thereby inhibiting the proliferation of tumor cells containing mutated BRAF genes. BRAF belongs to the RAF/MIL family of serine/threonine protein kinases and plays a role in regulating the MAP kinase/ERK signaling pathway, which may be persistently activated due to BRAF gene mutations. See also: Dabrafenib (with active moiety). Drug Indications: Treatment of melanoma, and solid malignancies (excluding melanoma). Pharmacodynamics Dabrafenib is a kinase inhibitor primarily used to target BRAF V600E mutations in various cancers. While both dabrafenib and trametinib inhibit the RAS/RAF/MEK/ERK pathway, they inhibit different effector molecules within this pathway, thus improving response rates and reducing resistance without cumulative toxicity. Trametinib's approval for melanoma is based on the results of the COMBI-AD study. COMBI-AD was a phase III clinical trial that enrolled 870 patients with BRAF V600E/K mutation-positive stage III melanoma who received dabrafenib in combination with trametinib after complete surgical resection. Patients received either dabrafenib (150 mg twice daily) in combination with trametinib (2 mg once daily) (n = 438) or a matched placebo (n = 432). The primary endpoint of recurrence-free survival (RFS) was met after a median follow-up of 2.8 years. In the treatment of thyroid cancer, dabrafenib in combination with trametinib is the first clinically proven and well-tolerated treatment regimen for BRAF V600E-mutant anaplastic thyroid cancer. These findings represent a significant advancement in the treatment of this rare disease. Dabrafenib is an organofluorine compound and anti-tumor drug; its mesylate form is used to treat metastatic melanoma. It has dual action as an anti-tumor, BRAF inhibitor, and anti-coronavirus agent. It is a sulfonamide, organofluorine compound, 1,3-thiazole compound, and aminopyrimidine compound. Dabrafenib mesylate (Tafinlar) is a reversible ATP-competitive kinase inhibitor targeting the MAPK pathway. It was approved on May 29, 2013, for the treatment of melanoma harboring V600E or V6000K mutations. It has also been used to treat metastatic non-small cell lung cancer harboring the same mutation. In May 2018, Tafinlar (dabrafenib) was approved in combination with Mekinist ([DB08911]) for the treatment of anaplastic thyroid cancer caused by BRAF V600E gene abnormalities. Dabrafenib is a kinase inhibitor. Its mechanism of action is as a protein kinase inhibitor, a cytochrome P450 3A4 inducer, a cytochrome P450 2B6 inducer, a cytochrome P450 2C8 inducer, a cytochrome P450 2C9 inducer, a cytochrome P450 2C19 inducer, an organic anion transporter 1B1 inhibitor, an organic anion transporter 1B3 inhibitor, an organic anion transporter 1 inhibitor, an organic anion transporter 3 inhibitor, and a breast cancer resistance protein inhibitor. Dabrafenib is a selective inhibitor of BRAF kinase mutants and can be used alone or in combination with trametinib for the treatment of advanced malignant melanoma. A transient increase in serum transaminases may occur during dabrafenib treatment, but it has not been found to be associated with clinically significant cases of acute liver injury. Dabrafenib is an orally bioavailable B-raf (BRAF) protein inhibitor with potential antitumor activity. Dabrafenib selectively binds to and inhibits the activity of B-raf, thereby potentially inhibiting the proliferation of tumor cells containing mutated BRAF genes. B-Raf belongs to the Raf/Mil family of serine/threonine protein kinases and plays a role in regulating the MAP kinase/ERK signaling pathway, which may be persistently activated by BRAF gene mutations. Dabrafenib is a small molecule drug, with its clinical trial phase up to Phase IV (covering all indications). It was first approved in 2013 and currently has 7 approved indications and 19 investigational indications. Activation of the Ras-Raf-MEK-ERK pathway is associated with various human cancers. Growth factor receptors (GFRs) activate Ras upon stimulation by extracellular ligands, initiating a signaling cascade involving Raf, MEK, and ERK serine/threonine kinases. Mutations in B-Raf kinase can persistently activate the MAPK signaling pathway, bypassing upstream stimuli. This gene is associated with various human cancers, particularly the prevalence of the B-RafV600E mutant in melanoma, colorectal cancer, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. Selective and potent inhibition of B-Raf holds promise for providing potential therapies for patients with mutant B-Raf tumors, as these tumors are dependent on this pathway. We have identified a novel, potent, and selective Raf kinase inhibitor that inhibits the kinase activity of wild-type B-Raf, B-RafV600E, and c-Raf, with IC50 values of 3.2 nM, 0.8 nM, and 5.0 nM, respectively. Screening of over 270 kinases revealed that this inhibitor exhibits selectivity for Raf kinases, with a selectivity for B-Raf approximately 400-fold higher than 91% of other tested kinases. This inhibitor specifically inhibits intracellular B-RafV600E kinase, leading to decreased ERK phosphorylation levels and inhibiting cell proliferation by first arresting the cell cycle in the G1 phase, ultimately resulting in cell death. This inhibition is selective for cancer cells specifically encoding the B-RafV600E mutation. Oral administration of this compound inhibited the growth of B-RafV600E mutant melanoma (A375P) and colon cancer (Colo205) xenografts implanted subcutaneously in immunodeficient mice. Currently, this cell-specific B-RafV600E inhibitor is undergoing Phase I human clinical trials. Citation information: Mol Cancer Ther 2009;8(12 Suppl):B88.[1]

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Overactivation of the MAP kinase pathway caused by constitutively active B-RafV600E mutant enzymes has been observed in various human tumors, including melanoma. This article reports the discovery and biological evaluation of dabrafenib/GSK2118436. Dabrafenib is a selective Raf kinase inhibitor that exhibits potent in vitro activity against oncogenic B-Raf-driven melanoma and colorectal cancer cells and significant in vivo antitumor and pharmacodynamic activity in a mouse model of B-RafV600E human melanoma. GSK2118436 was identified as a candidate drug, and early clinical results showed significant efficacy in patients with B-Raf mutant melanoma. [3]

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In summary, lead compound 1, which exhibited high activity but poor pharmacokinetic properties in higher animals, was rapidly improved into a clinical development candidate drug, dabrafenib/GSK2118436 (12). Through detailed and iterative structure-activity relationship (SAR) studies, we identified key contributors to the activity of B-RafV600E (sulfonamide head, R2 fluorination, and tert-butylthiazole core), thereby removing non-essential parts of the lead compound structure and achieving high selectivity for the entire kinase group. Due to the high enzymatic and cellular activity of the initial lead compound, we addressed pharmacokinetic issues through multiple modifications to the template, ultimately achieving the improved multi-species pharmacokinetic properties observed in compound 12. GSK2118436 achieved high exposure levels in rodents, and we were able to demonstrate its target-related pharmacological properties in two in vivo models, giving us confidence that this molecule will enter the clinical development stage. Furthermore, the significant improvement in pharmacokinetics of higher species enabled drug exposure to be achieved, thus allowing for a full exploration of the molecule's safety in the preclinical stage. Finally, the molecular weight was reduced by more than 20% compared to compound 1, thereby improving its physicochemical properties and accelerating the identification of the salt form (mesylate) and its form suitable for early clinical studies. The efforts described in this article ultimately led to the discovery of GSK2118436 (dabrafenib), which is currently undergoing late-stage clinical trials in B-Raf-mutant tumors. GSK2118436 has shown significant efficacy in patients with melanoma carrying activating B-Raf mutations, including those with brain metastases, and its clinical activity is further enhanced when used in combination with the MEK inhibitor trametinib. Details of these studies will be published in due course. [3] The final consideration in our work was the potential impact of using drugs such as dabrafenib to treat heart disease. Dabrafenib itself does not appear to be cardiotoxic and is generally well tolerated. Cancer patients taking dabrafenib may experience some side effects, which can usually be managed by reducing the dose. The most serious side effect may be an increased incidence of squamous cell carcinoma of the skin (approximately 12%). Therefore, if dabrafenib is used to treat hypertensive heart disease, dose monitoring and the patient's susceptibility to other conditions (such as cancer) may need to be considered (perhaps from the perspective of "oncocardiology"). Nevertheless, using drugs such as dabrafenib to reduce myocardial fibrosis may be an effective means of treating heart disease, and the benefits may outweigh these risks. [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.)