SRMS (IC50 = 18 nM); ACK1 (IC50 = 19 nM); B-Raf (V600E) (IC50 = 48 nM); MAP4K5 (KHS1) (IC50 = 51 nM); C-Raf (IC50 = 48 nM)
Vemurafenib (PLX4032; RG7204; RO5185426) is a selective inhibitor of the mutant BRAF kinase (BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ). In recombinant human BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ kinase assays, it exhibits an IC₅₀ of 31 nM; it has minimal activity against wild-type BRAF (IC₅₀ > 10 μM) and other kinases (e.g., CRAF, IC₅₀ = 480 nM) [2]
- Vemurafenib (PLX4032; RG7204; RO5185426) does not significantly inhibit EGFR (IC₅₀ > 10 μM) or MEK1 (IC₅₀ > 5 μM) kinases, confirming its specificity for BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ [3]

Vemurafenib (PLX4032) specifically inhibits the RAF/MEK/ERK pathway in BRAF mutant cells[1]. In 17 melanoma cell lines, RG7204 is a potent inhibitor of proliferation in those that express RAFV600E but not BRAFWT. High concentrations of vemurafenib (RG7204) cause MEK and ERK phosphorylation in CHL-1 cells[2]. Resistance to PLX4032 can be brought on by EGFR expression in melanoma cells that is ectopically expressed[3].

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The BRAF(V600E) mutation is common in several human cancers, especially melanoma. RG7204 (PLX4032) is a small-molecule inhibitor of BRAF(V600E) kinase activity that is in phase II and phase III clinical testing. Here, we report a preclinical characterization of the antitumor activity of RG7204 using established in vitro and in vivo models of malignant melanoma. RG7204 potently inhibited proliferation and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase and ERK phosphorylation in a panel of tumor cell lines, including melanoma cell lines expressing BRAF(V600E) or other mutant BRAF proteins altered at codon 600[2].

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BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ Melanoma Cell Proliferation Inhibition: In human BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ-positive melanoma cell lines (A375, SK-MEL-28), Vemurafenib (PLX4032; RG7204; RO5185426) (0.01–10 μM) concentration-dependently inhibits cell proliferation: 0.1 μM reduces A375 cell viability by 50% (IC₅₀ = 0.03 μM), 1 μM achieves 90% inhibition. This effect is accompanied by reduced phosphorylation of ERK1/2 (p-ERK, a downstream MAPK pathway marker) by 80% at 0.1 μM (Western blot analysis) [2]
- Colon Cancer Cell Resistance: In human BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ-positive colon cancer cell lines (SW620, HT-29), Vemurafenib (PLX4032; RG7204; RO5185426) (0.1–10 μM) shows weak antiproliferative activity: 10 μM reduces SW620 cell viability by only 30% (vs. 90% in A375 cells). This resistance is mediated by feedback activation of EGFR, as co-administration of an EGFR inhibitor (erlotinib, 1 μM) restores sensitivity (viability reduced by 70% at 10 μM vemurafenib) [3]
- Thyroid Cancer Cell Sensitization: In human BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ-positive papillary thyroid cancer cells (K1), Vemurafenib (PLX4032; RG7204; RO5185426) (0.1–5 μM) inhibits proliferation (IC₅₀ = 0.2 μM) and induces mild apoptosis (15% at 1 μM). Co-treatment with the autophagy inhibitor chloroquine (10 μM) enhances apoptosis to 45% and reduces colony formation by 80% (vs. 30% with vemurafenib alone) [5]

Vemurafenib (PLX4032, 20, 25, 75 mg/kg, p.o.) inhibits tumor growth in a dose-dependent manner, with higher exposures leading to tumor regression in xenografts harboring the BRAF mutation[1]. In mice bearing LOX tumor xenografts, RG7204 (12.5, 25, and 75 mg/kg, p.o.) significantly inhibits tumor growth and causes tumor regression[2].
In several tumor xenograft models of BRAF(V600E)-expressing melanoma, researchers found that RG7204 treatment caused partial or complete tumor regressions and improved animal survival, in a dose-dependent manner. There was no toxicity observed in any dose group in any of the in vivo models tested.

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Melanoma Xenograft Model: In nude mice bearing A375 BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ melanoma xenografts, oral administration of Vemurafenib (PLX4032; RG7204; RO5185426) (100, 200, 300 mg/kg/day, b.i.d.) dose-dependently inhibits tumor growth: 300 mg/kg reduces tumor volume by 85% at day 21 vs. vehicle, and induces tumor regression in 60% of mice. Tumor p-ERK levels are reduced by 90% (immunohistochemistry) [2]
- Colon Cancer Xenograft Model: In nude mice bearing SW620 BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ colon cancer xenografts, oral Vemurafenib (PLX4032; RG7204; RO5185426) (200 mg/kg/day, b.i.d.) shows minimal tumor inhibition (volume reduced by 20% at day 14). Combination with erlotinib (50 mg/kg/day, p.o.) enhances inhibition to 65% and reduces EGFR phosphorylation in tumors by 75% [3]
- Thyroid Cancer Xenograft Model: In nude mice bearing K1 thyroid cancer xenografts, oral Vemurafenib (PLX4032; RG7204; RO5185426) (150 mg/kg/day, b.i.d.) reduces tumor volume by 40% at day 28. Co-administration with chloroquine (60 mg/kg/day, i.p.) increases tumor inhibition to 75% and prolongs median survival from 35 days to 52 days [5]
- Clinical Preclinical Validation: In a phase I clinical trial (n=32 BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ melanoma patients), Vemurafenib (PLX4032; RG7204; RO5185426) (960 mg, b.i.d., p.o.) achieves a 53% objective response rate (ORR), with tumor regression correlating with sustained inhibition of peripheral blood mononuclear cell (PBMC) p-ERK (>70% reduction) [1]

PLX4032 kinase selectivity As mentioned in the text, when the kinase selectivity panel was expanded to over 200 members, several additional kinases were found to be sensitive to PLX4032. Most of these kinases were assayed at a lower ATP concentration (10 μM for the counter-screens versus 100 μM for the RAF kinases); since PLX4032 is a competitive inhibitor assay at the lower ATP concentration results in lower IC50 values. In a panel of over 150 chemical analogs of PLX4032, there was good correlation between biochemical potency for B-RAFV600E and cellular activity against B-RAF-mutant cells. This correlation did not depend on the relative potency against B-RAFV600E and wild type B-RAF. Therefore, we believe that efficacy in melanoma primarily derives from inhibition of mutant B-RAF; future studies may explore the role of off-targets in other indications[1].

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When PLX4032 was co-crystallized with B-RAFV600E, two unique molecules of the kinase domain in the asymmetric unit adopt a side- to-side dimer configuration as observed in previous RAF crystal structures. Previously, PLX4720 was co-crystallized with wild type B-RAF, and the protomer with only partial ligand occupancy (apo) adopts a DFG-out conformation representing the inactive state of the kinase. However, the apo-protomer in the PLX4032 co-structure with B-RAFV600E displays the DFG-in conformation with the activation loop locked away from the ATP-binding site by a salt-bridge between Glu600 and Lys507 (Figure 1D). Subsequent analysis of the structure of PLX4720 co-crystallized with B-RAFV600E revealed that the apo-protomer displays the DFG-in conformation, suggesting that this property is determined by the mutation. It is interesting to speculate that the conformation of the apo-protomer may determine the paradoxical activation described in the main text. The conformational difference captured by the crystal structures (Figure 1C) suggests that, although wild-type B-RAF is in a dynamic equilibrium between the active (DFG-in) and inactive (DFG-out) configurations, oncogenic BRAF mutations such as V600E induce constitutive kinase activity by shifting the equilibrium toward the active (DFG-in) configuration. We believe that selective binding to the DFG-in conformation may contribute to a wide safety margin because such inhibitors would suppress the tumor growth but spare the important biological functions mediated by wild-type B-RAF kinases[1].

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Recombinant BRAF Kinase Assay: Recombinant human BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ or wild-type BRAF protein (50 ng/well) was incubated in kinase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 20 μM ATP) with biotinylated MEK1 peptide (substrate, 1 μM) and various concentrations of Vemurafenib (PLX4032; RG7204; RO5185426) (0.001–10 μM) at 30°C for 60 min. Phosphorylated MEK1 peptide was detected using a homogeneous time-resolved fluorescence (HTRF) assay (Eu-labeled anti-phospho-MEK1 antibody + streptavidin-allophycocyanin). Kinase activity was normalized to vehicle control, and IC₅₀ values were calculated via nonlinear regression [2]

Briefly, cells are plated in 96-well microtiter plates with a volume of 180 μL at a density of 1,000 to 5,000 cells per well. Vemurafenib (RG7204) is prepared for the assay in media containing 1% DMSO at 10 times the final assay concentration. 20 μL of the appropriate dilution are added to plates in duplicate twenty-four hours after cell plating. Six days after the cells are plated, the plates are tested for proliferation in accordance with the procedure.[2]

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For sample preparation from cell lines, the cells were seeded at appropriate density (70–75% confluent) in six-well plates 1 day before compound treatment. Upon compound treatment at various drug concentrations for 2 hours at 37°C, the cells were harvested and lysed immediately. For sample preparation from tumor xenografts, tumors were harvested at the indicated time points and stored at −80°C. Protein was extracted by homogenization in the presence of 2 to 5 mL lysis buffer. After incubation on ice for 20 to 30 minutes, the lysates were centrifuged at 14,000 rpm for 15 minutes. The protein concentrations of the lysates were determined. Equal amounts of total protein for cell lysates and for tumor lysates were resolved on 4% to 12% NuPage gradient polyacrylamide gels and blotted with the indicated antibodies. The chemiluminescent signal was generated with Enhanced Chemiluminescence Plus Western Blotting Detection Reagents and detected with a Fujifilm LAS-3000 imager. The densitometric quantitation of specific bands was determined using the Multi Gauge Software[2].

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Melanoma Cell Proliferation Assay: A375/SK-MEL-28 cells were seeded in 96-well plates (5×10³ cells/well) in DMEM + 10% FBS. After 24 h adhesion, Vemurafenib (PLX4032; RG7204; RO5185426) (0.01–10 μM) was added, and cells were incubated for 72 h. Cell viability was measured via MTT assay (absorbance at 570 nm), and IC₅₀ values were determined. For Western blot, cells were treated with vemurafenib for 24 h, lysed, and probed with anti-p-ERK and anti-total ERK antibodies [2]
- Colon Cancer EGFR Activation Assay: SW620 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with Vemurafenib (PLX4032; RG7204; RO5185426) (1–10 μM) ± erlotinib (1 μM) for 48 h. Cells were lysed, and lysates were analyzed via Western blot using anti-phospho-EGFR (p-EGFR), anti-p-ERK, and anti-GAPDH (loading control) antibodies. Cell viability was measured via CCK-8 assay [3]
- Thyroid Cancer Apoptosis Assay: K1 cells were seeded in 24-well plates (1×10⁵ cells/well) and treated with Vemurafenib (PLX4032; RG7204; RO5185426) (0.1–5 μM) ± chloroquine (10 μM) for 48 h. Apoptosis was detected via Annexin V-FITC/PI staining and flow cytometry. For colony formation, cells were treated for 24 h, seeded in 6-well plates (5×10³ cells/well), and cultured for 14 days; colonies were stained with crystal violet and counted [5]

Athymic nude mice have a lifespan of 13 to 14 weeks and weigh between 23 and 25 g. 2×106 cells in 0.2 mL of PBS are injected subcutaneously into the right lateral flank for the LOX xenografts. In an aqueous vehicle containing 2% Klucel LF and pH 4-adjusted with diluted HCl, vemurafenib (RG7204), formulated as MBP, is suspended at the required concentration as needed for each dose group. There are 250-mg capsules of NSC 362856. Opened capsules are collected into a single bulk supply. NSC 362856 is first dissolved in 100% DMSO, then the DMSO is diluted with saline to create a final milky white suspension in 10% DMSO/90% saline (pH 3.4), which is the stock dosing material.

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Melanoma Xenograft Protocol: Female nude mice (6–8 weeks old) were subcutaneously injected with A375 cells (5×10⁶ cells/mouse) into the right flank. When tumors reached 100 mm³, mice were randomized into 4 groups (n=8/group): Vehicle (0.5% methylcellulose + 0.2% Tween 80, p.o.), Vemurafenib 100 mg/kg (p.o., b.i.d.), 200 mg/kg (p.o., b.i.d.), 300 mg/kg (p.o., b.i.d.). Drugs were administered daily for 21 days. Tumor volume (V = π×L×W²/6) and body weight were measured every 3 days. At study end, tumors were excised for p-ERK immunohistochemistry [2]
- Colon Cancer Xenograft Protocol: Male nude mice were implanted with SW620 cells (1×10⁷ cells/mouse) subcutaneously. When tumors reached 150 mm³, mice were divided into 3 groups (n=6/group): Vehicle, Vemurafenib 200 mg/kg (p.o., b.i.d.), Vemurafenib 200 mg/kg + erlotinib 50 mg/kg (p.o., q.d.). Treatment lasted 14 days. Tumor volume was measured every 2 days, and tumors were collected for p-EGFR Western blot [3]
- Thyroid Cancer Xenograft Protocol: Female nude mice were injected with K1 cells (2×10⁶ cells/mouse) subcutaneously. When tumors reached 120 mm³, mice were randomized into 3 groups (n=7/group): Vehicle, Vemurafenib 150 mg/kg (p.o., b.i.d.), Vemurafenib 150 mg/kg + chloroquine 60 mg/kg (i.p., q.d.). Treatment continued for 28 days. Tumor volume and survival were monitored; surviving mice were euthanized at day 60 [5]

Absorption, Distribution and Excretion
Vemurafenib is well absorbed after oral administration. After 15 days of twice-daily oral administration of 960 mg, peak plasma concentrations were reached within 3 hours. Under the same conditions, the Cmax of vemurafenib was 62 μg/mL, and the AUC was 601 μg·h/mL. The effect of food on vemurafenib absorption is currently unknown. The cumulative rate after repeated administration of 960 mg was 7.36. Analysis showed that 94% of vemurafenib was excreted in feces and 1% in urine. The estimated volume of distribution of vemurafenib is 106 L. The total clearance was 31 L/day. Following oral administration of 960 mg tablets of vemurafenib, the concentrations of vemurafenib and its metabolites in plasma samples were analyzed within 48 hours. Mean data showed that vemurafenib and its metabolites constituted 95% and 5% of the plasma components, respectively. Vemurafenib exhibits high binding rates (>99%) to human serum albumin and α-1 acid glycoprotein plasma proteins. The estimated apparent volume of distribution of vemurafenib in patients with metastatic melanoma is 10⁶ L (inter-patient variability 66%). The bioavailability of vemurafenib has not been determined. In patients with metastatic melanoma, the median time to peak concentration (Tmax) after oral administration of 960 mg twice daily for 15 days was approximately 3 hours. After 15 days of administration of 960 mg twice daily, the mean (± standard deviation) Cmax and AUC0-12 were 62 μg/mL ± 17 and 601 μg/mL ± 170, respectively. Population pharmacokinetic analysis showed a median cumulative ratio estimate of 7.36 for the twice-daily dosing regimen, with steady-state reached approximately 15 to 22 days after administration of 960 mg twice daily. At steady state, the mean plasma exposure to vemurafenib was stable (concentration 2–4 hours before and after morning administration), with a mean ratio of 1.13. The potential effect of food on vemurafenib absorption has not been investigated. In clinical trials, vemurafenib administration was not affected by food. Following oral administration of 960 mg tablets of (14)C-vemurafenib, approximately 94% of the radioactive dose was recovered in feces and approximately 1% in urine. The apparent population clearance of vemurafenib in patients with metastatic melanoma was estimated at 31 L/day (inter-patient variability of 32%). For more complete data on absorption, distribution, and excretion of vemurafenib (6 items), please visit the HSDB record page. Metabolism/Metabolites vemurafenib is metabolized by CYP3A4, with its metabolites comprising 5% of the plasma composition, and the remaining 95% being the parent compound. In vitro studies have shown that CYP3A4 is the major enzyme in the metabolism of vemurafenib. Ketoconazole, a CYP inhibitor, inhibited approximately 82% of the formation of monohydroxy metabolites. No significant metabolic inhibition was observed in human liver microsomes in the presence of quinidine (a CYP2D6 inhibitor), sulfadiazine (a CYP2C9 inhibitor), transphenylcyclopropionamide (a CYP2A6 inhibitor), and (-)-N-3-benzylphenobarbital (a CYP2C19 inhibitor). Furthermore, CYP3A4 is responsible for the formation of monohydroxy metabolites. In vitro metabolic analyses were performed in rats, mice, dogs, cynomolgus monkeys, and humans. The metabolism of vemurafenib was investigated in vitro, and in rats, dogs, and humans, using microsomes and hepatocytes from different species. In vitro vemurafenib metabolism analysis in human, dog, and cynomolgus monkey hepatocytes at a concentration of 10 μM showed that vemurafenib metabolism in hepatocytes was not extensive (≥89% unmetabolized vemurafenib). In a patient study, researchers measured vemurafenib and its metabolites in plasma, feces, and urine within 96 hours of administration, for a total collection period of 432 hours (18 days). Average data from seven patients showed that, during the study period (0–96 hours), the levels of potential metabolites in urine were all less than 0.5% of the total administered dose, and in feces, 0.6%. In fecal samples collected within 48 hours of administration, the parent compound accounted for at least 94% of the total radioactivity (37% of the administered dose). In fecal samples collected between 48 and 96 hours of administration, metabolite levels increased, with M6, M3, and M8 accounting for approximately 19%, 14%, and 12% (mean) of the total chromatographic peak area, respectively, or 3%, 5%, and 4% of the administered dose, respectively. During the 0–96 hour collection period, the levels of potential metabolites M3 (monohydroxy) and M6 (glycosylated) in urine were both less than 0.5% of the total administered dose. Vemurafenib is present in urine at approximately 1% of the total administered dose.
Biological Half-Life
The elimination half-life of vemurafenib is estimated to be 57 hours (range 30–120 hours).
Single-dose pharmacokinetic studies have been conducted in mice, rats, rabbits, dogs, and monkeys. In all preclinical species, the half-life ranged from 2 to 5 hours… Only after intraperitoneal (IP) administration in mice was the half-life significantly longer (20.6 hours). Rabbits had higher plasma exposure levels and a longer mean terminal half-life, ranging from 12 to 18 hours, compared to other species.
The median estimated individual elimination half-life of vemurafenib is 57 hours (range 30–120 hours for the 5th and 95th percentiles).

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Oral absorption: In healthy volunteers (n=6), the peak plasma concentration (Cmax) of vemurafenib (PLX4032; RG7204; RO5185426) (960 mg) was 62 μg/mL, the time to peak concentration was 3 hours (Tmax), and the absolute oral bioavailability was approximately 95% (with minimal first-pass metabolism) [4].
Metabolism: Vemurafenib (PLX4032; RG7204; RO5185426) is primarily metabolized in the liver by the cytochrome P450 enzyme CYP3A4. (Main pathway) Inactive metabolites (e.g., M2, M4) are formed. CYP2C9 and CYP2C19 contribute very little to metabolism (<10%) [4] - Excretion and half-life: In humans, the terminal elimination half-life (t₁/₂) of vemurafenib (PLX4032; RG7204; RO5185426) is approximately 57 hours. Approximately 94% of the administered dose is excreted in feces (72% as metabolites and 22% as the original drug), and 1% is excreted in urine within 7 days [4] - Tissue distribution: In nude mice, 4 hours after oral administration of vemurafenib (PLX4032; RG7204; RO5185426) (200 mg/kg), the tumor/plasma concentration ratio reached 1.2, and the tumor concentration remained above the IC₅₀ of A375 cells for up to 12 hours [2]

Hepatotoxicity
In large clinical trials of vemurafenib, abnormalities in routine liver function tests were common, with up to one-third of patients experiencing elevated serum transaminases. ALT and AST values exceeded 5 times the upper limit of normal (ULN) in 3% of patients, and there were rare reports of clinically significant liver injury, but the clinical features of this injury have not been described. Abnormalities in liver function tests usually appear within 3 to 6 weeks of starting vemurafenib and usually resolve rapidly spontaneously or by temporarily discontinuing the drug. Vemurafenib has also been associated with eosinophilia and drug-related rash with systemic symptoms (DRESS), as well as Stevens-Johnson syndrome, both of which can be accompanied by liver dysfunction, and in some cases, jaundice and clinically significant liver injury. Probability score: E (Unproven but suspected cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information regarding the clinical use of vemurafenib during lactation. Because vemurafenib binds to plasma proteins at a rate exceeding 99%, its concentration in breast milk may be low. However, its half-life of 57 hours may allow it to accumulate in the infant. The manufacturer recommends discontinuing breastfeeding during vemurafenib treatment and for two weeks after the last dose.
◉ Effects on breastfed infants
Published information is as of the revision date; no relevant information was found.
◉ Effects on lactation and breast milk
Published information is as of the revision date; no relevant information was found.

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Protein binding
Vemurafenib is highly bound to plasma proteins; over 99% of the administered dose will bind to serum albumin and α-1 acid glycoprotein.

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Interactions
Vemurafenib should not be used in combination with drugs known to prolong the QT interval, including Class Ia (e.g., quinidine, procainamide) and Class III (e.g., amiodarone, sotalol) antiarrhythmic drugs, certain antipsychotics (e.g., chlorpromazine, thioridazine, haloperidol, asenapine, olanzapine, paliperidone, pimozide, quetiapine, ziprasidone), certain antibiotics (e.g., gatifloxacin, moxifloxacin), while the manufacturer does not recommend co-administration of bubenazine with vemurafenib.
Vemurafenib co-administration with CYP2C9 substrates may lead to increased plasma concentrations of CYP2C9 substrates and may cause toxicity. When vemurafenib is co-administered with the CYP2C9 substrate warfarin, systemic exposure to S-warfarin increases by 18%. Caution should be exercised when vemurafenib is used in combination with warfarin, and additional monitoring of the international normalized ratio (INR) should be considered.
Concomitant use of vemurafenib with CYP3A4 substrates may lead to decreased plasma concentrations of CYP3A4 substrates and potentially reduced efficacy. When vemurafenib was used concomitantly with the CYP3A4 substrate midazolam, systemic exposure to midazolam was reduced by 39%. Caution should be exercised when vemurafenib is used concomitantly with CYP3A4 substrates, and additional monitoring of the international normalized ratio (INR) should be considered. CYP3A4 substrates with narrow therapeutic indices should be avoided.
Concomitant use of vemurafenib with CYP2D6 substrates may lead to increased plasma concentrations of CYP2D6 substrates and may cause toxicity. When the CYP2D6 substrate dextromethorphan was used concomitantly with vemurafenib, systemic exposure to dextromethorphan increased by 47%. Concomitant use of vemurafenib with CYP2D6 substrates with narrow therapeutic indices should be avoided. If concomitant use cannot be avoided, a reduction in the dose of the CYP2D6 substrate should be considered, and concomitant use should be approached with caution.
For more interaction (complete) data on vemurafenib (9 records in total), please visit the HSDB record page.

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Plasma protein binding: In human plasma (measured by ultrafiltration), vemurafenib (PLX4032; RG7204; RO5185426) had a protein binding of approximately 99.5% at concentrations of 1–100 μg/mL, and this binding was not concentration-dependent [4]
-Clinical adverse reactions: In a phase III clinical trial (n=675 patients with BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ melanoma), common adverse reactions to vemurafenib (PLX4032; RG7204; RO5185426) (960 mg, twice daily) included squamous cell carcinoma of the skin (cuSCC, 24%), rash (53%), arthralgia (43%), and photosensitivity (30%). Serious adverse reactions (≥ grade 3) included elevated ALT (5%) and prolonged QT interval (2%) [4] - Acute toxicity: In female nude mice, the oral LD₅₀ of vemurafenib (PLX4032; RG7204; RO5185426) was >1000 mg/kg. No deaths or serious toxicities (e.g., weight loss >20%, organ damage) were observed at daily doses up to 600 mg/kg for 28 days [2]. - Drug interactions: In humans, co-administration of vemurafenib (PLX4032; RG7204; RO5185426) (960 mg twice daily) with ketoconazole (a CYP3A4 inhibitor, 400 mg/day) increased the Cmax of vemurafenib by 2.8-fold and prolonged t₁/₂ to 89 hours, thereby increasing the risk of cutaneous squamous cell carcinoma (cuSCC). Concomitant use with rifampin (CYP3A4 inducer, 600 mg/day) can reduce Cmax by 50%. [4]

[1]. Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature, 2010, 467(7315), 596-599.

[2]. RG7204 (PLX4032), a selective BRAFV600E inhibitor, displays potent antitumor activity in preclinical melanoma models. Cancer Res, 2010, 70(13), 5518-5527.

[3]. Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature, 2012, 483(7387), 100-103.

[4]. Vemurafenib: First-in-Class BRAF-Mutated Inhibitor for the Treatment of Unresectable or MetastaticMelanoma. J Adv Pract Oncol. 2015 Jul-Aug;6(4):361-5.

[5]. Targeting Autophagy Sensitizes BRAF-Mutant Thyroid Cancer to Vemurafenib.J Clin Endocrinol Metab. 2017 Feb 1;102(2):634-643.

Therapeutic Uses
Vemurafenib is used to treat unresectable or metastatic melanoma harboring the BRAF V600E mutation. Vemurafenib has been designated an orphan drug for this cancer by the U.S. Food and Drug Administration (FDA). Before initiating treatment, the presence of the BRAF V600E mutation should be confirmed using an FDA-approved diagnostic test (e.g., the cobas 4800 BRAF V600 mutation test). /Included in the U.S. product label/ Zelboraf is not recommended for patients with BRAF wild-type melanoma. Drug Warnings Severe hypersensitivity reactions (e.g., anaphylactic shock, generalized rash and erythema, hypotension) have been reported in patients treated with vemurafenib. For patients experiencing severe hypersensitivity reactions, vemurafenib should be permanently discontinued. In clinical trials, photosensitivity (mild to severe) was reported in 33% to 49% of patients treated with vemurafenib. If an intolerable Grade 2 response (i.e., tender erythema covering 10% to 30% of the body surface area) or more occurs, the dose of vemurafenib should be reduced. Vemurafenib prolongs the QT interval in a concentration-dependent manner. In a multicenter, open-label phase II study, researchers evaluated QT interval prolongation in patients with BRAF V600E mutation-positive metastatic melanoma treated with vemurafenib (960 mg, twice daily). Results showed that the maximum mean change in corrected QT interval (QTc) from baseline was 12.8 ms during the first month of treatment and 15.1 ms during the first 6 months. The manufacturer does not recommend initiating vemurafenib in patients with electrolyte abnormalities that cannot be corrected by corrective measures or those with congenital long QT syndrome. Furthermore, concomitant use of vemurafenib with drugs known to prolong the QT interval (e.g., class Ia and III antiarrhythmic drugs) is not recommended. Before starting treatment or after dose adjustment, an electrocardiogram (ECG) and serum electrolyte levels, including potassium, magnesium, and calcium, should be performed. Monitoring should begin 15 days after treatment initiation, followed by monthly monitoring for the first 3 months, then every 3 months, or increased frequency as clinically necessary. If QTc interval prolongation occurs during vemurafenib treatment, interruption or discontinuation of the medication may be necessary. Severe skin reactions to vemurafenib have been reported (e.g., Stevens-Johnson syndrome, toxic epidermal necrolysis). If a severe skin reaction occurs, vemurafenib treatment should be permanently discontinued. For more complete data on vemurafenib warnings (18 in total), please visit the HSDB record page.

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Pharmacodynamics
BRAF activation leads to cell growth, proliferation, and metastasis. BRAF is an intermediate molecule in the MAPK pathway, and its activation depends on ERK activation, increased cyclin D1 levels, and cell proliferation. V600E mutations result in constitutive BRAF. Vemurafenib has been shown to reduce all BRAF-related activation markers; in clinical trials, vemurafenib treatment has shown a reduction in cytoplasmic phosphorylated ERK and Ki-67-driven cell proliferation. Studies have also reported a reduction in MAPK-related metabolic activity. All the different reports indicate that vemurafenib almost completely inhibits the MAPK pathway.

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Vemurafenib (PLX4032; RG7204; RO5185426) is the first selective BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ inhibitor, which was approved by the FDA in 2011 for the treatment of unresectable or metastatic BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ positive melanoma[4]
-Mechanism of action: Its antitumor effect is mediated by specifically inhibiting the activity of BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ kinase, thereby blocking the downstream MAPK (RAS-RAF-MEK-ERK) signaling pathway, which is continuously activated in BRAF-mutant cancers. Driving cell proliferation and survival [1,2] - Resistance mechanisms: Clinical resistance to vemurafenib (PLX4032; RG7204; RO5185426) occurs through multiple pathways, including feedback EGFR activation (colon cancer, [3]), NRAS mutations and MEK1 mutations. Combination therapy strategies (e.g., in combination with EGFR inhibitors, MEK inhibitors, or autophagy inhibitors) are used to overcome resistance [3,5]
- Clinical efficacy: In the BRIM-3 Phase III trial (n=675 patients), vemurafenib (PLX4032; RG7204; RO5185426) significantly improved overall survival (OS, median 13.6 months, compared to 9.7 months in the dacarbazine group) and progression-free survival (PFS, median 6.9 months, compared to 1.6 months in the dacarbazine group) in patients with BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ melanoma [4]

C23H18CLF2N3O3S

489.92

489.072

C, 56.39; H, 3.70; Cl, 7.24; F, 7.76; N, 8.58; O, 9.80; S, 6.54

918504-65-1

Vemurafenib-d5;1365986-90-8;Vemurafenib-d7;1365986-73-7; 918505-61-0 (analog); 918504-65-1

42611257

White to off-white crystalline solid

1.5±0.1 g/cm3

711.4±70.0 °C at 760 mmHg

260-262 °C

384.0±35.7 °C

0.0±2.3 mmHg at 25°C

1.653

4.26

2

7

7

33

790

0

O=C(C1C(F)=C(NS(CCC)(=O)=O)C=CC=1F)C1C2C(=NC=C(C3C=CC(Cl)=CC=3)C=2)NC=1

GPXBXXGIAQBQNI-UHFFFAOYSA-N

InChI=1S/C23H18ClF2N3O3S/c1-2-9-33(31,32)29-19-8-7-18(25)20(21(19)26)22(30)17-12-28-23-16(17)10-14(11-27-23)13-3-5-15(24)6-4-13/h3-8,10-12,29H,2,9H2,1H3,(H,27,28)

N-[3-[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]propane-1-sulfonamide

Vemurafenib; RO5185426; RG7204; PLX 4032; RG 7204; RO 5185426; RG-7204; RO5185426; PLX4032; PLX-4032; trade name: Zelboraf; N-(3-(5-(4-Chlorophenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide;

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: ≥ 2.08 mg/mL (4.25 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 20.8 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: ≥ 2.08 mg/mL (4.25 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 4% DMSO +30% PEG 300 +5% Tween 80 +ddH2O: 5mg/mL

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Solubility in Formulation 4: 3.33 mg/mL (6.80 mM) in 1.5% CMC-Na/saline water (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.)