B-Raf (V600E) (IC50 = 0.3 nM)

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].

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]

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).

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.

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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6 mg/kg; oral

Rats

Absorption, Distribution and Excretion
The pharmacokinetics of encorafenib were studied in healthy subjects and patients with solid tumors, including advanced and unresectable or metastatic cutaneous melanoma harboring a BRAF V600E or V600K mutation, BRAF V600E mutation-positive metastatic CRC. After a single dose, systemic exposure of encorafenib was dose-proportional over the dose range of 50 mg to 700 mg (0.1 to 1.6 times the maximum recommended dose of 450 mg). After once-daily dosing, systemic exposure of encorafenib was less than dose-proportional over the dose range of 50 mg to 800 mg (0.1 to 1.8 times the maximum recommended dose of 450 mg). Steady-state was reached within 15 days, with exposure being 50% lower compared to Day 1; intersubject variability (CV%) of AUC ranged from 12% to 69%. After oral administration, the median Tmax of encorafenib is 2 hours. At least 86% of the dose is absorbed. Following administration of a single dose of encorafenib 100 mg (0.2 times the maximum recommended dose of 450 mg) with a high-fat, high-calorie meal (consisting of approximately 150 calories from protein, 350 calories from carbohydrates, and 500 calories from fat) the mean maximum encorafenib concentration (Cmax) decreased by 36% and there was no effect on AUC.
Following a single oral dose of 100 mg radiolabeled encorafenib, 47% (5% unchanged) of the administered dose was recovered in the feces and 47% (2% unchanged) was recovered in the urine.
The blood-to-plasma concentration ratio is 0.58. The geometric mean (CV%) of apparent volume of distribution is 164 L (70%).
The apparent clearance is 14 L/h (54%) at day 1, increasing to 32 L/h (59%) at steady-state.

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Metabolism / Metabolites
Encorafenib is primarily metabolized by CYP3A4 (83%) and to a lesser extent by CYP2C19 (16%) and CYP2D6 (1%).

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Biological Half-Life
The mean (CV%) terminal half-life (t_1/2) of encorafenib is 3.5 hours (17%).

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of encorafenib during breastfeeding. The manufacturer recommends that breastfeeding be discontinued during encorafenib therapy and for at least 2 weeks after the final 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
Encorafenib is 86% bound to human plasma proteins in vitro.

[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 BRAF gene, which encodes for B-raf protein, which is a proto-oncogene involved in various genetic mutations. This protein plays a role in regulating the MAP kinase/ERK signaling pathway, which impacts cell division, differentiation, and secretion. Mutations in this gene, most frequently the V600E mutation, are the most commonly identified cancer-causing mutations in melanoma, and have been isolated in various other cancers as well, including non-Hodgkin lymphoma, colorectal cancer, thyroid carcinoma, non-small cell lung carcinoma, hairy cell leukemia and adenocarcinoma of the lung. On June 27, 2018, the Food and Drug Administration approved encorafenib and [binimetinib] (BRAFTOVI and MEKTOVI, Array BioPharma Inc.) in combination for patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, as detected by an FDA-approved test.
Encorafenib is an orally available Raf kinase inhibitor with potential antineoplastic activity. Encorafenib specifically inhibits Raf kinase, a serine/threonine enzyme in the RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-related kinase (ERK) signaling pathway. By inhibiting the activation of the RAF/MEK/ERK signaling pathway, the administration of LGX818 may result in a decrease in proliferation of tumor cells. The Raf mutation BRAF V600E is frequently upregulated in a variety of human tumors and results in the constitutive activation of the RAF/MEK/ERK signaling pathway that regulates cellular proliferation and survival.

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Drug Indication
Encorafenib is indicated in combination with [binimetinib] for the treatment of adult patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation and metastatic non-small cell lung cancer (NSCLC) with a BRAF V600E mutation. It is also indicated in combination with [cetuximab] for the treatment of adult patients with metastatic colorectal cancer with a BRAF V600E mutation.
Encorafenib is indicated: in combination with binimetinib is indicated for the treatment of adult patients with unresectable or metastatic melanoma with a BRAF V600 mutationin combination with cetuximab, for the treatment of adult patients with metastatic colorectal cancer (CRC) with a BRAF V600E mutation, who have received prior systemic therapy
Treatment of melanoma
Treatment of colorectal carcinoma

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Mechanism of Action
Encorafenib is a kinase inhibitor that targets BRAF V600E, as well as wild-type BRAF and CRAF in in vitro cell-free assays with IC50 values of 0.35, 0.47, and 0.3 nM, respectively. Mutations in the BRAF gene, such as BRAF V600E, can result in constitutively activated BRAF kinases that may stimulate tumor cell growth. Encorafenib was also able to bind to other kinases in vitro including JNK1, JNK2, JNK3, LIMK1, LIMK2, MEK4, and STK36, and reduce ligand binding to these kinases at clinically achievable concentrations (≤0.9 µM).

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Pharmacodynamics
Encorafenib has a pharmacologic profile that is distinct from that of other clinically active BRAF inhibitors and has shown improved efficacy in the treatment of metastatic melanoma. Once-daily dosing of single-agent encorafenib has a distinct tolerability profile and shows varying antitumor activity across BRAFi-pretreated and BRAFi-naïve patients with advanced/metastatic stage melanoma. Encorafenib inhibited in vitro growth of tumor cell lines expressing BRAF V600 E, D, and K mutations. In mice implanted with tumor cells expressing BRAF V600E, encorafenib induced tumor regressions associated with RAF/MEK/ERK pathway suppression. Encorafenib and binimetinib target two different kinases in the RAS/RAF/MEK/ERK pathway. Compared with either drug alone, the co-administration of encorafenib and binimetinib resulted in greater anti-proliferative activity in vitro in BRAF mutation-positive cell lines and greater anti-tumor activity with respect to tumor growth inhibition in BRAF V600E mutant human melanoma xenograft studies in mice. Additionally, the combination of encorafenib and binimetinib delayed the emergence of resistance in BRAF V600E mutant human melanoma xenografts in mice compared to either drug alone. In a BRAF V600E mutant NSCLC patient-derived xenograft model in mice, coadministration of encorafenib and binimetinib resulted in greater anti-tumor activity compared to binimetinib alone, with respect to tumor growth inhibition. Increased tumor growth delay after dosing cessation was also observed with the co-administration compared to either drug alone. In the setting of BRAF-mutant CRC, induction of EGFR-mediated MAPK pathway activation has been identified as a mechanism of resistance to BRAF inhibitors. Combinations of a BRAF inhibitor and agents targeting EGFR have been shown to overcome this resistance mechanism in nonclinical models. The co-administration of encorafenib and cetuximab had an anti-tumor effect greater than either drug alone, in a mouse model of colorectal cancer with mutated BRAF V600E.

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.)