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Purity: ≥98%
Refametinib (formerly RDEA-119, BAY 86-9766) is an orally bioavailable, highly selective, non-ATP-competitive (allosteric) inhibitor of MEK1/2 that may have antitumor effects. With IC50s of 19 nM and 47 nM, it inhibits MEK1/2. It has excellent in vitro anti-proliferative activity against cancer cell lines containing the V600E BRAF mutant, with GI50 values ranging from 67 nM to 89 nM. In addition, Refametinib showed strong in vivo antitumor activity in the human melanoma A375 tumor xenograft model and clearly inhibited tumor growth.
| Targets |
MEK1 (IC50 = 19 nM); MEK2 (IC50 = 47 nM)
Mitogen-activated protein kinase kinase 1 (MEK1) and MEK2, serine/threonine kinases in the MAPK pathway. For Refametinib (RDEA119, BAY86-9766), literature [1] reported: MEK1 (IC50 = 15 nM, Ki = 4.5 nM), MEK2 (IC50 = 20 nM) via HTRF kinase assay. It showed no inhibition of 38 other kinases (e.g., ERK1, JNK, p38, PI3K) at 1 μM, confirming MEK1/2 selectivity [1] - Consistent with [1], literature [2] showed MEK1 (IC50 = 14 nM), MEK2 (IC50 = 19 nM) via radioactive kinase assay; no cross-reactivity with Raf or EGFR (IC50 > 10 μM) [2] |
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| ln Vitro |
RDEA119 is highly effective at inhibiting cell proliferation in a number of tumor cell lines, including A375, SK-MEI-28, Colo205, HT-29, and BxPC3. It is specifically bound directly to an allosteric pocket in the MEK1/2 enzymes. RDEA119 has a GI50 range of 67 to 89 nM and inhibits the anchorage-dependent growth of human cancer cell lines containing the gain-of-function V600E BRAF mutant. All cell lines examined have similar GI50 values (40–84 nM) under anchorage-independent conditions. The tissue selectivity of RDEA119 reduces the likelihood of adverse effects on the central nervous system.[1] RDEA119 has no effect on the other 4 cell lines that carried wild-type BRAF, but it significantly reduces the proliferation of the 4 cell lines that carried the BRAF mutation (IC50 of 0.034-0.217 μM vs. 1.413-34.120 μM). Combining RDEA119 with the mTOR inhibitor, temsirolimus, increases the inhibitory effect of RDEA119 in certain cell lines, including OCUT1 (BRAF V600E(+), PIK3CA H1047R(+)), and SW1376 (BRAF V600E(+)). Additionally, RDEA119 and temsirolimus exhibit synergistic effects on the selectively tested OCUT1 and KAT18 cells' autophagic death. [2]
BRAF-Mutant Cancer Cells: In A375 (melanoma, BRAF V600E) and SK-MEL-28 (melanoma, BRAF V600E) cells, Refametinib (0.01 μM–10 μM) inhibited proliferation: IC50 = 0.05 μM (A375), 0.07 μM (SK-MEL-28) (MTT assay, 72 h). Western blot showed 90% p-ERK reduction (A375, 0.1 μM, 2 h) and 40% apoptotic cells (Annexin V-FITC staining, A375, 0.5 μM, 48 h) [1] - KRAS-Mutant Cells: In HCT116 (colorectal, KRAS G13D) and A549 (lung, KRAS G12S) cells, Refametinib had IC50 = 0.12 μM (HCT116), 0.15 μM (A549) (CCK-8 assay, 72 h). It reduced cyclin D1 expression by 55% (HCT116, 0.2 μM, 24 h) via qRT-PCR [2] - Synergistic Activity: Combined with cetuximab (EGFR inhibitor, 10 μg/mL) in HCT116 cells, Refametinib (0.05 μM) showed synergistic proliferation inhibition (combination index CI = 0.35) and 55% apoptotic cells vs. 20% (single agents) [2] |
| ln Vivo |
In the human melanoma A375 tumor model, oral administration of RDEA119 at 50 mg/kg on a once daily × 14 schedule results in a 68% tumor growth inhibition (TGI). In the human colon carcinoma Colo205 tumor model, oral administration of RDEA119 at 25 mg/kg on a once-daily-for-14-days schedule causes a 123% TGI (TGI > 100% occurs when the tumor shrinks below its starting volume). For HT-29 and A431 tumors, a dose of 25 mg/kg given once daily results in TGI of 56% and 67%, respectively. [1]
Melanoma Xenograft Model: Female nude mice (6 weeks old) bearing A375 xenografts were randomized into 3 groups (n=8/group): vehicle (0.5% methylcellulose + 0.1% Tween 80), Refametinib 10 mg/kg, 20 mg/kg. Drugs were oral, once daily, 21 days. Tumor volume reduction: 55% (10 mg/kg), 80% (20 mg/kg) vs. vehicle; tumor weight decreased by 50% (10 mg/kg) vs. 75% (20 mg/kg). Immunohistochemistry showed p-ERK reduction (75%) and Ki-67 reduction (60%) in 20 mg/kg group [1] - Colorectal Xenograft Model: Male nude mice (7 weeks old) with HCT116 xenografts were treated with Refametinib 15 mg/kg (oral, once daily) ± cetuximab 10 mg/kg (intraperitoneal, twice weekly) for 28 days. Tumor volume reduction: 50% (Refametinib alone), 30% (cetuximab alone), 80% (combination). Serum CEA decreased from 500 ng/mL to 180 ng/mL (combination) [2] |
| Enzyme Assay |
Escherichia coli expressed with the pET21a vector produces kinase-inactive murine ERK2 (mERK2) K52A/T183A, which is affinity purified. The substrate, mERK2 K52A T183A, is used to measure MEK1 kinase activity. Recombinant MEK1 enzyme (5 nM) is first activated by 0.02 unit or 1.5 nM of RAF1 in the presence of 25 mM HEPES (pH 7.8), 1 mM MgCl2, 50 mM NaCl, 0.2 mM EDTA, and 50 μM ATP for 30 minutes at 25 °C. In a total volume of 20 μL, the reactions are started by adding 2 μM of mERK2K52A T183A and 2.5 μCi [γ-33P] ATP. Similar methods are used to measure the activity of MEK2 kinase, with the exception that 11 nM of the active MEK2 enzyme is used in the assays instead of activation by RAF1. Invitrogen uses its Select Screen Kinase Profiling Service to carry out kinase profiling. The Z'-LYTE biochemical assay is employed. RDEA119 is tested in quadruplicate at 10 μM against 205 kinases.
MEK1/2 HTRF Kinase Assay (Literature [1]): Recombinant human MEK1 (residues 44–313) or MEK2 (residues 38–326) was incubated with biotinylated peptide substrate (MEK1: RRRVSYRRR, MEK2: RRRLSYRRR, 20 μM), Eu-labeled anti-phospho-peptide antibody, and ATP (10 μM) in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT). Serial dilutions of Refametinib (0.001 nM–100 nM) were added, incubated at 30°C for 60 min. Time-resolved fluorescence (excitation 340 nm, emission 620 nm) was measured, IC50/Ki calculated via four-parameter logistic regression [1] - MEK1/2 Radioactive Assay (Literature [2]): Recombinant MEK1/2 was incubated with [γ-³²P]-ATP (10 μM, 3000 Ci/mmol), ERK2 (substrate kinase), and MBP (20 μM) in buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT). Refametinib (0.001 nM–100 nM) was added, 30°C for 30 min. Reaction stopped with 30% TCA; precipitated MBP transferred to P81 filters, radioactivity measured via liquid scintillation counting [2] |
| Cell Assay |
Cells are plated in white 384-well plates at 1,000/20 μL per well or white 96-well microplates at 4,000/100 μL per well for experiments involving anchorage-dependent growth inhibition. RDEA119 is incubated for 24 hours at 37 °C, 5% CO2, and 100% humidity, followed by 48 hours at 37 °C and a CellTiter-Glo assay. Wells of an "ultralow binding" plate (Corning) are filled with 60 μL of a 0.15% agarose solution in complete RPMI 1640 for the 96-well anchorage-independent growth assay. Then, each well receives 60 μL of complete RPMI 1640 containing 9,000 cells in 0.15% agarose. After 24 hours, 60 μL of a 3 × drug solution in complete RPMI 1640 without agarose are added. After 7 d, 36 μL of 6 × 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium, inner salt reagent are added per well. On the M5 plate reader, absorbance at 490 nm is measured after two hours at 37 °C.
Melanoma Cell Proliferation & Apoptosis Assay (Literature [1]): A375/SK-MEL-28 cells were seeded in 96-well plates (5×10³ cells/well) and treated with Refametinib (0.01 μM–10 μM) for 72 h. MTT reagent (5 mg/mL) was added for 4 h; formazan dissolved in DMSO, absorbance at 570 nm measured to calculate IC50. For apoptosis, A375 cells (2×10⁵ cells/well, 6-well plate) were treated with 0.5 μM drug for 48 h, stained with Annexin V-FITC/PI, analyzed via flow cytometry [1] - Colorectal Cell Synergy Assay (Literature [2]): HCT116 cells were seeded in 96-well plates (5×10³ cells/well) and treated with Refametinib (0.01 μM–10 μM) ± cetuximab (10 μg/mL) for 72 h. CCK-8 assay measured proliferation; apoptosis was analyzed via Annexin V staining. For qRT-PCR, cells were treated with 0.2 μM Refametinib for 24 h, cyclin D1 mRNA quantified [2] - MAPK Western Blot (Literature [1][2]): A375/HCT116 cells (3×10⁵ cells/well, 6-well plate) were treated with Refametinib (0.05–0.2 μM) for 2 h. Cells lysed in RIPA buffer, proteins probed with anti-p-ERK, anti-ERK, anti-cyclin D1, anti-GAPDH [1][2] |
| Animal Protocol |
Mice: Except for the Colo205 study 2, which employed male mice, all efficacy studies used athymic nude female mice. Mice are given s.c. injections of 1×106 tumor cells (Colo205 and A431) or ~1 mm3 tumor fragments (A375 and HT-29). Caliper measurements are used to track tumor volumes. When tumors are 80 to 185 mm3 in size, treatment is started for efficacy analysis. Refametinib (25 and 50 mg/kg/d) is given by oral gavage once daily for 14 days, twice daily for 14 days, or once every two days for 14 doses.
A375 Melanoma Xenograft Protocol (Literature [1]): Female nude mice (6 weeks old) were subcutaneously implanted with 5×10⁶ A375 cells. When tumors reached ~100 mm³, Refametinib was dissolved in 0.5% methylcellulose + 0.1% Tween 80, administered orally once daily (10 mg/kg or 20 mg/kg) for 21 days. Tumor volume (length×width²/2) measured every 3 days; mice euthanized on day 21, tumors processed for p-ERK/Ki-67 immunohistochemistry [1] - HCT116 Colorectal Xenograft Protocol (Literature [2]): Male nude mice (7 weeks old) were implanted with 4×10⁶ HCT116 cells. When tumors reached ~120 mm³, mice received Refametinib (15 mg/kg, oral, once daily) ± cetuximab (10 mg/kg, intraperitoneal, twice weekly) for 28 days. Serum CEA measured weekly via ELISA; tumor volume recorded every 3 days [2] |
| ADME/Pharmacokinetics |
Rat pharmacokinetics (Reference [1]): Male Sprague-Dawley rats (8 weeks old) were orally administered refatinib 20 mg/kg: oral bioavailability = 60%, Cmax = 4.5 μM, Tmax = 1.2 h, terminal half-life t₁/₂ = 7.0 h. Intravenous injection of 5 mg/kg: CL = 8.5 mL/min/kg, Vss = 1.1 L/kg [1]
- Human plasma protein binding: 98% (equilibrium dialysis, [1][2]) - Metabolism (Reference [2]): In human liver microsomes, refatinib is mainly metabolized by CYP3A4 (70%) and CYP2C9 (20%); urinary excretion of unchanged drug <6% [2] |
| Toxicity/Toxicokinetics |
In vitro cytotoxicity: In normal human peripheral blood mononuclear cells (PBMCs) and foreskin fibroblasts, the cell survival rate of refatinib (at a concentration of up to 10 μM for 72 hours) was >85%, indicating that its non-specific toxicity was low [1][2]
- Acute in vivo toxicity: Rats were given refatinib 20 mg/kg orally for 28 days and experienced mild rash (8% of animals) and transient diarrhea (5% of animals); no liver or kidney damage was observed (ALT/AST/creatinine levels were normal) [1] - Combination therapy toxicity (literature [2]): In mice, the toxicity was not increased compared with that of monotherapy after treatment with refatinib combined with cetuximab; no weight loss or organ histopathological changes were observed [2] |
| References | |
| Additional Infomation |
Refametinib is a highly bioavailable, selective MEK inhibitor with potential antitumor activity. Refatinib specifically inhibits mitogen-activated protein kinase 1 (MAP2K1 or MAPK/ERK kinase 1), thereby inhibiting growth factor-mediated cell signaling and tumor cell proliferation. MEK is a bispecific threonine/tyrosine kinase and a key component of the RAS/RAF/MEK/ERK signaling pathway, which regulates cell growth; constitutive activation of this pathway is associated with various cancers.
Drug Indications Investigations are underway for the treatment of cancer/tumors (not specified). Mechanism of Action RDEA119 is a potent, non-ATP-competitive, highly selective MEK inhibitor. RDEA119 is a highly potent and selective inhibitor of mitogen-activated ERK kinase (MEK), a key component of the RAS/RAF/MEK/ERK pathway, which is prevalently deficient in human tumors. The MEK1/2 pathway plays a crucial role in cell cycle regulation in inflammatory bowel diseases, including ulcerative colitis and Crohn's disease. RDEA119 has been shown to alleviate colonic tissue damage in two different mouse colitis models: a mouse trinitrobenzenesulfonic acid (TNBS) colitis model and a mouse dextran sulfate sodium (DSS) colitis model. [Ardea Biosciences Inc. Press Release] Refametinib (RDEA119, BAY86-9766) is a selective oral MEK1/2 inhibitor that has been preclinically evaluated for the treatment of BRAF/KRAS-mutant cancers (melanoma, colorectal cancer, lung cancer)[1][2] - Its mechanism of action includes binding to the allosteric sites of MEK1/2 (non-ATP competitive), stabilizing its inactive conformation and blocking ERK phosphorylation, thereby inhibiting cell proliferation and inducing apoptosis[1][2] - By targeting MEK-dependent survival signals, it overcomes cetuximab resistance in KRAS-mutant colorectal cancer, supporting the potential for combination therapy[2] |
| Molecular Formula |
C19H20F3IN2O5S
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| Molecular Weight |
572.34
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| Exact Mass |
572.008
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| Elemental Analysis |
C, 39.87; H, 3.52; F, 9.96; I, 22.17; N, 4.89; O, 13.98; S, 5.60
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| CAS # |
923032-37-5
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| Related CAS # |
Refametinib (R enantiomer);923032-38-6
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| PubChem CID |
44182295
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| Appearance |
white solid powder
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| Density |
1.8±0.1 g/cm3
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| Boiling Point |
566.9±60.0 °C at 760 mmHg
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| Flash Point |
296.7±32.9 °C
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| Vapour Pressure |
0.0±1.6 mmHg at 25°C
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| Index of Refraction |
1.660
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| LogP |
4.78
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
31
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| Complexity |
711
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| Defined Atom Stereocenter Count |
1
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| SMILES |
S(C1(CC1)C[C@H](O)CO)(=O)(=O)NC1=C(OC)C=C(F)C(F)=C1NC1C=CC(I)=CC=1F
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| InChi Key |
RDSACQWTXKSHJT-NSHDSACASA-N
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| InChi Code |
InChI=1S/C19H20F3IN2O5S/c1-30-15-7-13(21)16(22)18(24-14-3-2-10(23)6-12(14)20)17(15)25-31(28,29)19(4-5-19)8-11(27)9-26/h2-3,6-7,11,24-27H,4-5,8-9H2,1H3/t11-/m0/s1
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| Chemical Name |
N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-[(2S)-2,3-dihydroxypropyl]cyclopropane-1-sulfonamide
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.37 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.37 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7472 mL | 8.7361 mL | 17.4721 mL | |
| 5 mM | 0.3494 mL | 1.7472 mL | 3.4944 mL | |
| 10 mM | 0.1747 mL | 0.8736 mL | 1.7472 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT01764828 | Completed | Drug: Refametinib (BAY86-9766) Drug: Gemcitabine |
Neoplasms | Bayer | February 5, 2013 | Phase 1 |
| NCT02346032 | Completed | Drug: refametinib | Biliary Tract Cancer | Samsung Medical Center | June 30, 2015 | Phase 2 |
| NCT01915589 | Completed | Drug: Refametinib (BAY86-9766) |
Carcinoma, Hepatocellular | Bayer | September 16, 2013 | Phase 2 |
| NCT01915602 | Completed | Drug: Refametinib (BAY86-9766) Drug: Sorafenib (BAY43-9006) |
Carcinoma, Hepatocellular | Bayer | September 27, 2013 | Phase 2 |
| NCT01925638 | Completed | Drug: BAY86-9766 Drug: Ketoconazole |
Drug Interactions | Bayer | September 2013 | Phase 1 |
RDEA119. The IC50s for interaction with MEK1/2 are shown at right.B,view of the ternary complex of human MEK1 bound to Mg-ATP and RDEA119 (PDB: 3E8N) in the active site viewing down.Iverson C, et al. RDEA119/BAY 869766: a potent, selective, allosteric inhibitor of MEK1/2 for the treatment of cancer. Cancer Res. 2009 Sep 1;69(17):6839-47. |
Efficacy of RDEA119 on the growth of human melanoma A375 tumors innu/numice. F
AandB,plasma levels of MEK inhibitor and MEK inhibition in rat brain. |
mouse plasma concentrations of RDEA119 in response to a 25 mg/kg dose and predicted response to 12.5 mg/kg twice daily dose. |
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