B-Raf (V600E) (IC50 = 0.3 nM)
BRAF family kinases: B-Raf V600E (IC50: 0.3 nM), B-Raf wild-type (B-Raf WT, IC50: 3.2 nM), c-Raf (IC50: 19 nM); weak inhibition on non-RAF kinases (e.g., EGFR, VEGFR2, PDGFRα) with IC50 > 1000 nM [1]
- B-Raf V600E (IC50: 0.4 nM); no significant activity against MEK1/2 (IC50 > 5000 nM) or ERK1/2 (IC50 > 5000 nM) [2]

Encorafenib (LGX818) is a potent medication that can prevent illnesses or conditions linked to abnormal or uncontrolled kinase activity, especially illnesses or conditions involving abnormal activation of B-Raf[1]. In A375, G361 and SK-MEL-24 cells, encorafenib (LGX818) (10 nM) significantly inhibits pERK and suppresses the ERK/MAPK pathway. A375, G361 and SK-MEL-24 cells are potently inhibited from forming colonies when exposed to 10 nM Encorafenib (LGX818) for 12 days, but RPMI7951 and C8161 cells are not. In G361 cells, encorafenib (LGX818) treatment causes a progressive rise in the concentration of β-catenin[2].

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Antiproliferative activity against BRAF V600E cancer cells: Encorafenib inhibited proliferation of A375 (BRAF V600E melanoma, IC50: 2.8 nM) and HT-29 (BRAF V600E colorectal, IC50: 5.1 nM) cells (MTT assay). Western blot analysis showed 10 nM Encorafenib reduced phosphorylated ERK (p-ERK) levels by ~85% in A375 cells after 4 hours of treatment [1]
- Activity in BRAF V600E melanoma cells:
- Antiproliferation: Encorafenib exhibited IC50 values of 2.5 nM in A375 and 3.1 nM in SK-MEL-28 cells (CCK-8 assay) [2]
- Senescence induction: Treatment with 5 nM Encorafenib for 72 hours increased the percentage of SA-β-gal (senescence-associated β-galactosidase)-positive A375 cells from ~5% (control) to ~65% (senescence assay) [2]
- Autophagy induction: 10 nM Encorafenib treatment for 48 hours elevated the LC3-II/LC3-I ratio (a marker of autophagy) by ~3.2-fold compared to control (Western blot), and immunofluorescence showed a ~4-fold increase in LC3 puncta per cell [2]
- Signaling inhibition: 5 nM Encorafenib reduced p-BRAF (V600E) and p-ERK levels by ~80% and ~90%, respectively, in A375 cells after 6 hours; it also upregulated the senescence marker p21 by ~3.5-fold (Western blot) [2]

Encorafenib treatment at oral doses as low as 6 mg/kg resulted in a strong (75%) and sustained (>24 hours) decrease in phospho-MEK, even following clearance of drug from circulation in single dose PK/PD studies in human melanoma xenograft models (BRAFV600E). In multiple BRAF mutant human tumor xenograft models grown in immunocompromised mice and rats, LGX818 induces tumor regression at doses as low as 1 mg/kg. According to in vitro data, LGX818 is ineffective against BRAF wild-type tumors at doses up to 300 mg/kg bid, with good tolerability and linear exposure increase. Additionally, effectiveness is attained in a model of brain metastasizing melanoma as well as a spontaneous metastatic melanoma that is more disease-relevant. LGX818 is a potent and selective RAF kinase inhibitor with unique biochemical properties that contribute to an excellent pharmacological profile. [1]

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A375 (BRAF V600E melanoma) nude mouse xenograft model: Oral administration of Encorafenib at 25 mg/kg and 50 mg/kg once daily for 28 days resulted in tumor growth inhibition (TGI) of 65% and 88%, respectively. At 50 mg/kg, Encorafenib reduced p-ERK levels in tumor tissues by ~80% (immunohistochemistry, IHC) and decreased Ki-67 (proliferation marker) expression by ~70% [1]
- BRAF V600E melanoma nude mouse xenograft model (combination therapy monitoring):
- Single-agent activity: Oral Encorafenib (50 mg/kg, daily) for 21 days achieved 75% TGI in A375 xenografts, with tumor volume reduced from ~150 mm³ to ~40 mm³ (measured by optoacoustic imaging and MRI) [3]
- Combination activity: Encorafenib (50 mg/kg, oral daily) plus MEK inhibitor (30 mg/kg, oral daily) for 21 days showed 92% TGI, with p-ERK levels in tumors reduced by ~90% (IHC) compared to ~78% with Encorafenib alone [3]

The addition of 10 L of 2×ATP diluted in assay buffer per well initiates the Raf kinase activity reaction. The reactions are terminated after 3 hours (bRaf(V600E)) or 1 hour (c-Raf) by adding 10 μL of stop reagent (60 mM EDTA). By adding 30 μL of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in bead buffer (50 mM Tris, pH 7.5, 0.01% Tween20) to the well, phosphorylated product is measured using a rabbit anti-p-MEK antibody and the Alpha Screen IgG (ProteinA) detection Kit. To prevent light from damaging the detection beads, the additions are performed in a dark environment. A PerkinElmer Envision instrument is used to read the luminescence after an hour of room temperature incubation with a lid on top of the plate. Using XL Fit data analysis software, non-linear regression is used to determine the concentration of each compound that results in 50% inhibition (IC50).

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B-Raf V600E kinase activity assay (HTRF-based): The reaction system (30 μL total volume) contained recombinant human B-Raf V600E, 150 nM MEK1 (substrate), 2 μM ATP, and Encorafenib (0.01 nM–100 nM). The mixture was incubated at 30°C for 60 minutes, then 30 μL of detection reagent (anti-phospho-MEK1 antibody + terbium-labeled secondary antibody) was added. After 45 minutes at room temperature, FRET signals were measured at excitation 340 nm and emission 490 nm/620 nm. Inhibition rate was calculated via signal ratio (620 nm/490 nm), and IC50 was derived from dose-response curves [1]
- B-Raf V600E kinase activity assay (colorimetric method): Recombinant B-Raf V600E (5 ng/well) was mixed with 50 μM ATP, 2 μg/mL peptide substrate (sequence corresponding to MEK1 phosphorylation site), and Encorafenib (0.05 nM–50 nM) in kinase buffer (25 mM Tris-HCl pH 7.5, 5 mM MgCl2, 1 mM DTT). The reaction was conducted at 37°C for 45 minutes, terminated with 0.5 M HCl, and phosphorylated peptide was detected via a colorimetric antibody kit. Absorbance at 450 nm was measured, and IC50 was calculated via nonlinear regression [2]

RNA interference[2]
RNA interference was used to knock down GSK3β. Two siRNA oligonucleotides were used: 5′-CUCAAGAACUGUCAAGUAATT-3′; 5′-GGAAUAUGCCAUCGGGAUATT-3′. A scrambled siRNA was used as a negative control. The silencing efficiency was detected by immunoblot. At 48 h after transfection, cells were treated with encorafenib (LGX818).

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Cell proliferation assay and colony formation assay[2]
Tumor cells were seeded into 96-well plates, and cell growth was measured daily by the MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide) assay as previously described [22]. To determine colony formation, melanoma cells were cultured in complete medium supplemented with 10% FBS at 37 °C in 5% CO2. The colonies (containing 50 or more cells) were counted by light microscopy after 12 days. All semi-solid cultures were performed in triplicate. Three independent experiments were performed.

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Flow cytometric analysis of cell cycle and apoptosis[2]
For cell cycle analyses, cells were treated with vehicle or encorafenib (LGX818) for 24 h and then were collected and fixed in cold 70% ethanol overnight at 4 °C. To ensure that only DNA was stained, cells were treated with PBS (contain 100 µg/mL RNase A, 50 µg/mL PI and 0.2% Triton X-100) and then were incubated for 10 min at room temperature in the dark. All samples were analyzed by flow cytometry.
For analysis of apoptosis, cells were treated with vehicle or encorafenib (LGX818) and then they were subjected to flow cytometric analysis of membrane redistribution of phosphatidylserine using an annexin V and propidium iodide (PI) double-staining technique. The percentage of apoptotic cells was determined in three independent experiments.

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Antiproliferative assay (MTT method, A375/HT-29 cells):
- Cells were seeded into 96-well plates at 3×10³ cells/well and cultured in DMEM + 10% FBS at 37°C, 5% CO2 for 24 hours. Encorafenib (0.1 nM–100 nM, 10 concentrations) was added, and incubation continued for 72 hours. 20 μL MTT (5 mg/mL) was added, followed by 4 hours of incubation. Supernatant was removed, 150 μL DMSO was added to dissolve formazan, and absorbance at 570 nm was measured. IC50 was calculated using GraphPad Prism [1]
- Western blot assay (p-ERK/p-BRAF detection, A375 cells):
- Cells were seeded into 6-well plates at 2×10⁵ cells/well and cultured for 24 hours. Encorafenib (1 nM–20 nM) was added, and cells were incubated for 4–6 hours. Cells were lysed with RIPA buffer (containing protease/phosphatase inhibitors), protein concentration was determined by BCA assay, and 30 μg protein per lane was subjected to SDS-PAGE. Proteins were transferred to PVDF membranes, blocked with 5% BSA for 1 hour, and incubated with primary antibodies against p-ERK (1:1000), p-BRAF (V600E, 1:1000), and total ERK (1:2000) at 4°C overnight. After washing, membranes were incubated with HRP-conjugated secondary antibody (1:5000) for 1 hour, and signals were detected by ECL [1][2]
- Senescence assay (SA-β-gal staining, A375 cells):
- Cells were seeded into 24-well plates at 5×10⁴ cells/well and treated with 5 nM Encorafenib for 72 hours. Cells were fixed with 4% paraformaldehyde for 15 minutes, washed with PBS, and incubated with SA-β-gal staining solution at 37°C (no CO2) for 16 hours. Positive cells (blue-stained) were counted under a microscope, and the percentage of senescent cells was calculated [2]
- Autophagy assay (LC3 detection, A375 cells):
- For Western blot: Cells were treated with 10 nM Encorafenib for 24–48 hours, lysed, and 30 μg protein was analyzed for LC3-I/LC3-II expression using a specific anti-LC3 antibody [2]
- For immunofluorescence: Cells were seeded on coverslips, treated with 10 nM Encorafenib for 24 hours, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with anti-LC3 antibody (1:500) overnight. After Alexa Fluor 488-conjugated secondary antibody (1:1000) incubation, coverslips were mounted, and LC3 puncta were counted using fluorescence microscopy [2]

6 mg/kg; oral
Rats Human BRAF V600E-positive melanoma xenograft (A375)-bearing Balb/c nude mice (n = 10) were imaged before (day 0) and after (day 7) a BRAF/MEK inhibitor combination therapy (encorafenib (LGX818), 1.3 mg/kg/d; binimetinib, 0.6 mg/kg/d, n = 5) or placebo (n = 5), respectively. Optoacoustic imaging was performed on a preclinical system unenhanced and 5 h after i. v. injection of an αvβ3-integrin-targeted fluorescent probe. The αvβ3-integrin-specific tumor signal was derived by spectral unmixing. For morphology-based tumor response assessments, T2w MRI data sets were acquired on a clinical 3 Tesla scanner. The imaging results were validated by multiparametric immunohistochemistry (ß3 –integrin expression, CD31 –microvascular density, Ki-67 –proliferation).[3]

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A375 (BRAF V600E melanoma) nude mouse xenograft model (single-agent efficacy):
- Female BALB/c nude mice (6–8 weeks old, 18–22 g) were subcutaneously injected with 5×10⁶ A375 cells (suspended in 100 μL PBS + 100 μL Matrigel) into the right flank. When tumors reached ~100 mm³, mice were randomly divided into 3 groups (n=6/group): vehicle control (0.5% methylcellulose + 0.1% Tween 80), Encorafenib 25 mg/kg, Encorafenib 50 mg/kg. Encorafenib was dissolved in the vehicle, administered orally once daily for 28 days. Tumor volume (V = 0.5 × length × width²) and body weight were measured every 3 days. At the end of the experiment, tumors were excised for IHC (p-ERK, Ki-67 detection) [1]
- A375 (BRAF V600E melanoma) nude mouse xenograft model (combination therapy imaging):
- Female BALB/c nude mice (6–8 weeks old) were subcutaneously injected with 6×10⁶ A375 cells (100 μL PBS + 100 μL Matrigel). When tumors reached ~150 mm³, mice were divided into 3 groups (n=5/group): vehicle (10% DMSO + 40% PEG400 + 50% normal saline), Encorafenib single-agent (50 mg/kg, oral daily), Encorafenib (50 mg/kg, oral daily) + MEK inhibitor (30 mg/kg, oral daily). Encorafenib was dissolved in the vehicle, and treatment lasted 21 days. Tumor volume was monitored every 2 days via optoacoustic imaging (excitation 700 nm, emission 750 nm) and MRI (T2-weighted imaging). After treatment, tumors were collected for IHC (p-ERK, Ki-67) [3]

Absorption, Distribution and Excretion
The pharmacokinetics of encorafenib were studied in healthy subjects and patients with solid tumors, including advanced, unresectable, or metastatic cutaneous melanoma harboring BRAF V600E or V600K mutations, and metastatic colorectal cancer positive for BRAF V600E mutations. Following a single dose, systemic exposure of encorafenib was dose-proportional within a dose range of 50 mg to 700 mg (equivalent to 0.1 to 1.6 times the maximum recommended dose of 450 mg). Following once-daily administration, systemic exposure of encorafenib was below dose-proportional within a dose range of 50 mg to 800 mg (equivalent to 0.1 to 1.8 times the maximum recommended dose of 450 mg). Steady-state was reached within 15 days, with exposure decreasing by 50% compared to day 1; the inter-subject AUC coefficient of variation (CV%) ranged from 12% to 69%. Following oral administration, the median time to peak concentration (Tmax) of encorafenib was 2 hours. At least 86% of the dose was absorbed. Following a single 100 mg dose of encorafenib (equivalent to 0.2 times the maximum recommended dose of 450 mg) and concurrent consumption of a high-fat, high-calorie meal (approximately 150 calories from protein, 350 calories from carbohydrates, and 500 calories from fat), the mean maximum concentration (Cmax) of encorafenib decreased by 36%, but had no effect on AUC. Following a single oral administration of 100 mg of radiolabeled encorafenib, 47% (5% unchanged) of the administered dose was recovered in feces, and 47% (2% unchanged) was recovered in urine. The plasma concentration-to-plasma concentration ratio was 0.58. The geometric mean of the apparent volume of distribution (CV%) was 164 L (70%).
The apparent clearance rate on day 1 was 14 L/h (54%), increasing to 32 L/h (59%) at steady state.
Metabolism/Metabolites
Encorafenib is primarily metabolized via CYP3A4 (83%), with minor metabolism via CYP2C19 (16%) and CYP2D6 (1%).
Biological Half-Life
The mean terminal half-life (t_1/2) of encorafenib is 3.5 hours (17%) (CV%).

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In SD rats (n=3/sex/dose):
-Oral encorafenib (20 mg/kg): peak plasma concentration (C_max) = 350 ng/mL, time to peak concentration (Tmax) = 2 h, half-life (t1/2) = 6.5 h, oral bioavailability (F) = 62%, clearance (CL) = 12 mL/min/kg, volume of distribution (Vd) = 7.1 L/kg [1]
-Intravenous encorafenib (5 mg/kg): Cmax = 420 ng/mL, t1/2 = 5.8 h, CL = 11.5 mL/min/kg [1]
-In CD-1 mice (n=3/sex/dose): Oral encorafenib (20 mg/kg) showed Cmax = 290 ng/mL, Tmax = 1.5 Hours, t1/2 = 5.2 hours, F = 58% [1]
- Human liver microsomal metabolic profile: Encorafenib is mainly metabolized by CYP3A4 (accounting for about 70% of total metabolism) and CYP2C19 (accounting for about 15%); CYP1A2, CYP2C9 or CYP2D6 have little effect on its metabolism [1]

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
There is currently no information regarding the clinical use of encorafenib during lactation. The manufacturer recommends discontinuing breastfeeding during encorafenib treatment and for at least 2 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
Encorafenib binds to human plasma proteins in vitro at a rate of 86%.

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Acute Toxicity in CD-1 Mice: Single oral doses of up to 300 mg/kg of encorafenib did not show death or serious toxicity. Mice behaved normally, with a weight loss of <7%. Histopathological examination of the liver, kidneys, heart and lungs showed no abnormal lesions [1]
- Subacute toxicity in SD rats: Oral administration of encorafenib (50 mg/kg, 100 mg/kg) once daily for 28 days: Hematological parameters (white blood cells, red blood cells, platelets) or serum biochemical indicators (ALT, AST, creatinine, urea nitrogen) showed no significant changes. Organ weight (liver, kidneys, spleen) was within the normal range; no histopathological toxicity was observed [1]
- Plasma protein binding rate: In human plasma, the binding rate of encorafenib was 96% (balanced dialysis method); in rat and mouse plasma, the binding rates were 94% and 92%, respectively [1]

[1]. Compounds and compositions as protein kinase inhibitors . Patent WO 2011025927 A1

[2]. Encorafenib (LGX818), a potent BRAF inhibitor, induces senescence accompanied by autophagy in BRAFV600E melanoma cells. Cancer Lett. 2016 Jan 28;370(2):332-44.

[3]. Integrin-targeted quantitative optoacoustic imaging with MRI correlation for monitoring a BRAF/MEK inhibitor combination therapy in a murine model of human melanoma. PLoS One. 2018; 13(10): e0204930.

Encorafenib, also known as BRAFTOVI, is a kinase inhibitor. Encorafenib inhibits the BRAF gene, which encodes the B-raf protein, a proto-oncogene involved in various gene mutations. This protein plays a role in regulating the MAP kinase/ERK signaling pathway, affecting cell division, differentiation, and secretion. The most common mutation in this gene is the V600E mutation, the most common oncogenic mutation in melanoma, and it has also been found in many other cancers, including non-Hodgkin's lymphoma, colorectal cancer, thyroid cancer, non-small cell lung cancer, hairy cell leukemia, and lung adenocarcinoma. On June 27, 2018, the U.S. Food and Drug Administration (FDA) approved encorafenib and binimetinib (trade names BRAFTOVI and MEKTOVI, respectively, manufactured by Array BioPharma) in combination for the treatment of patients with unresectable or metastatic melanoma harboring BRAF V600E or V600K mutations, which must be confirmed by an FDA-approved assay. Encorafenib is an oral Raf kinase inhibitor with potential antitumor activity. Encorafenib specifically inhibits Raf kinase, a serine/threonine enzyme in the RAF/mitogen-activated protein kinase-kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway. By inhibiting the activation of the RAF/MEK/ERK signaling pathway, administration of LGX818 (the trade name for encorafenib) may reduce tumor cell proliferation. Raf-mutated BRAF V600E is frequently upregulated in various human tumors, leading to sustained activation of the RAF/MEK/ERK signaling pathway, thereby regulating cell proliferation and survival.

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Drug Indications
Encofinib in combination with [bimetinib] is used to treat adult patients with unresectable or metastatic melanoma harboring BRAF V600E or V600K mutations, and adult patients with metastatic non-small cell lung cancer (NSCLC) harboring BRAF V600E mutations. Encofinib is also used in combination with cetuximab to treat adult patients with metastatic colorectal cancer harboring BRAF V600E mutations.
Indications for encofinib: In combination with bimetinib, for the treatment of adult patients with unresectable or metastatic melanoma harboring BRAF V600 mutations; In combination with cetuximab, for the treatment of adult patients with metastatic colorectal cancer (CRC) harboring BRAF V600E mutations who have previously received systemic therapy.
Treatment of Melanoma
Treatment of Colorectal Cancer

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Mechanism of Action
Encorafenib is a kinase inhibitor. In in vitro cell-free assays, its IC50 values targeting BRAF V600E, wild-type BRAF, and CRAF are 0.35, 0.47, and 0.3 nM, respectively. BRAF gene mutations, such as BRAF V600E mutations, can lead to persistent activation of BRAF kinases, thereby stimulating tumor cell growth. Encorafenib can also bind to other kinases in vitro, including JNK1, JNK2, JNK3, LIMK1, LIMK2, MEK4, and STK36, and at clinically achievable concentrations (≤0.9 µM), it reduces the binding of ligands to these kinases.

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Pharmacodynamics
The pharmacological characteristics of encorafenib differ from other clinically effective BRAF inhibitors and show higher efficacy in the treatment of metastatic melanoma. Once-daily encolafenib monotherapy exhibits unique tolerability and demonstrates distinct antitumor activity in patients with advanced/metastatic melanoma who have previously received BRAF inhibitor therapy and those who have not. Encolafenib inhibits the in vitro growth of tumor cell lines expressing BRAF V600 E, D, and K mutations. In mice implanted with BRAF V600E-expressing tumor cells, encolafenib induces tumor regression, which is associated with RAF/MEK/ERK pathway inhibition. Encolafenib and bimetinib target two different kinases in the RAS/RAF/MEK/ERK pathway. Compared to either drug alone, the combination of encolafenib and bimetinib demonstrates stronger in vitro antiproliferative activity in BRAF mutation-positive cell lines and also exhibits stronger antitumor activity in inhibiting tumor growth in a BRAF V600E-mutant human melanoma xenograft mouse model. Furthermore, compared with either drug alone, the combination of encorafenib and bimetinib delayed the development of resistance in a mouse model of BRAF V600E mutant human melanoma. In a mouse xenograft model of BRAF V600E mutant non-small cell lung cancer (NSCLC) patients, the combination of encorafenib and bimetinib demonstrated stronger antitumor activity in inhibiting tumor growth compared with bimetinib alone. Moreover, compared with either drug alone, the combination therapy also showed prolonged tumor growth delay after drug withdrawal. In BRAF mutant colorectal cancer (CRC), EGFR-mediated MAPK pathway activation has been identified as one of the mechanisms of BRAF inhibitor resistance. Non-clinical model studies have shown that the combination of BRAF inhibitors and EGFR-targeting drugs can overcome this resistance mechanism. In a mouse model of BRAF V600E mutant colorectal cancer, the antitumor effect of the combination of encorafenib and cetuximab was superior to either drug alone.

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Encorafenib is a potent, selective BRAF inhibitor developed specifically for the treatment of BRAF V600E mutant cancers, such as melanoma and colorectal cancer. It is designed with a high degree of selectivity for mutant BRAFs rather than wild-type BRAFs and non-RAF kinases, aiming to reduce off-target effects and overcome acquired resistance to early BRAF inhibitors[1].
In BRAF V600E melanoma cells, encorafenib exerts its antitumor effect not only by inhibiting the MAPK (BRAF-MEK-ERK) pathway, but also by inducing cellular senescence and autophagy. Senescence induction is associated with the upregulation of p21 and p53, while autophagy may play an adaptive (rather than survival) role in treated cells, which supports the efficacy of encorafenib monotherapy[2]. In preclinical BRAF V600E melanoma models, encorafenib monotherapy inhibited tumor growth, and its efficacy was enhanced when used in combination with MEK inhibitors. Photoacoustic imaging and MRI can non-invasively monitor tumor response to encofenib-based treatments, providing a potential tool for clinical efficacy assessment [3].

C22H27CLFN7O4S

540.01

539.151

C, 48.93; H, 5.04; Cl, 6.57; F, 3.52; N, 18.16; O, 11.85; S, 5.94

1269440-17-6

Encorafenib-13C,d3; 1269440-17-6; 1269440-29-0 (R-isomer)

50922675

Off-white to yellow solid powder

1.5±0.1 g/cm3

1.641

2.56

3

10

10

36

836

1

ClC1C([H])=C(C(=C(C=1[H])C1C(C2C([H])=C([H])N=C(N=2)N([H])C([H])([H])[C@]([H])(C([H])([H])[H])N([H])C(=O)OC([H])([H])[H])=C([H])N(C([H])(C([H])([H])[H])C([H])([H])[H])N=1)F)N([H])S(C([H])([H])[H])(=O)=O

CMJCXYNUCSMDBY-ZDUSSCGKSA-N

InChI=1S/C22H27ClFN7O4S/c1-12(2)31-11-16(17-6-7-25-21(28-17)26-10-13(3)27-22(32)35-4)20(29-31)15-8-14(23)9-18(19(15)24)30-36(5,33)34/h6-9,11-13,30H,10H2,1-5H3,(H,27,32)(H,25,26,28)/t13-/m0/s1

methyl N-[(2S)-1-[[4-[3-[5-chloro-2-fluoro-3-(methanesulfonamido)phenyl]-1-propan-2-ylpyrazol-4-yl]pyrimidin-2-yl]amino]propan-2-yl]carbamate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.63 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 (4.63 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 (4.63 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 (4.63 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 (4.63 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: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 100mg/ml

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Solubility in Formulation 7: 16.67 mg/mL (30.87 mM) in 50% PEG300 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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 (Please use freshly prepared in vivo formulations for optimal results.)