EGFR (IC50 = 29 nM); BRaf^V600E (IC50 = 23 nM); EGFR^L858R/T790M (IC50 = 495 nM)

BGB-283 strongly suppresses ERK phosphorylation and cell proliferation that is triggered by BRAFV600E in vitro. It exhibits preferentially inhibiting the growth of cancer cells with EGFR mutation/amplification and BRAFV600E mutation/amplification, as well as selective cytotoxicity. BGB-283 efficiently suppresses the reactivation of EGFR and EGFR-mediated cell proliferation in colorectal cancer cell lines BRAFV600E. It exhibits specific cytotoxicity against cell lines with EGFR or BRAFV600E mutations. In A431 cells, BGB-283 dose-dependently blocks the EGFR autophosphorylation on Tyr1068 caused by EGF. It has been demonstrated that BGB-283 can inhibit the feedback activation of EGFR signaling and achieve sustained inhibition of pERK in WiDr colorectal cancer cells[1].

In vivo, BGB-283 treatment causes both primary human colorectal tumor xenografts and cell line-derived tumors with the BRAFV600E mutation to exhibit dose-dependent inhibition of tumor growth along with partial and total tumor regressions. In xenograft models of colorectal cancer with the BRAF(V600E) mutation, such as HT29, Colo205, and two primary tumor xenografts, BGB-283 exhibits a high degree of efficacy. Furthermore, BGB-283 exhibits remarkable effectiveness in a WiDr xenograft model, demonstrating that BRAF inhibition triggers EGFR reactivation. In HCC827, but not in A431 xenograft, BGB-283 causes tumor regression. In WiDr tumor xenografts, BGB-283 exhibits strong antitumor activity and inhibits the phosphorylation of both EGFR and ERK1/2. Unlike vemurafenib, BGB-283 does not cause EGFR feedback activation. BGB-283 potently suppresses DUSP6 expression and the phosphorylation of MEK and ERK in vivo at repeated doses. Regarding AKT phosphorylation, there is no discernible difference[1].

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BGB-283 exhibits antitumor activity in mouse xenograft models of colorectal cancer [1]
The in vivo efficacy of BGB-283 was assessed in subcutaneous xenograft models derived from HT29 and Colo205 colorectal cancer cell lines harboring the BRAFV600E mutation. It was previously reported that vemurafenib had limited efficacy against HT-29 xenograft and combination with EGFR inhibitor improved its antitumor activity. BGB-283 significantly inhibited tumor growth of HT29 xenograft (P < 0.001) at 5 mg/kg b.i.d., which was well tolerated by animals. Addition of cetuximab, an EGFR-targeting monoclonal antibody, did not further enhance the therapeutic effect of BGB-283 in this xenograft model (P > 0.05, BGB-283 + cetuximab vs. BGB-283; Fig. 4A), suggesting that BGB-283 alone might be sufficient in blocking the feedback activation of EGFR. Against Colo205 xenograft, BGB-283 produced dose-dependent tumor inhibition from 3 to 30 mg/kg (Fig. 4B). More significantly, partial regression was observed at 10 mg/kg (1/7 mice). At 30 mg/kg BGB-283, regressions were observed in 3 of 7 mice (2 PR and 1 CR; Supplementary Table S6).

The tumor inhibitory activity of BGB-283 was further evaluated in human tumor tissue derived primary colorectal cancer xenograft models. A total of 23 patient-derived colorectal cancer models were established in vivo and two of them, BCCO-002 and BCCO-028, were identified to harbor BRAFV600E mutation. Both of these patient-derived colorectal cancer models were sensitive to treatment with BGB-283 (Fig. 4C and D); >100% TGI was observed on day 24 following oral treatment with BGB-283 (10 mg/kg, b.i.d.; Fig. 4C and D and Supplementary Table S6). For BCCO-002, partial regressions were observed in 2 of 8 (25%) mice treated with BGB-283 (10 mg/kg b.i.d.). Addition of cetuximab did not further enhance the antitumor activity of BGB-283 (P > 0.05, BGB-283 + cetuximab vs. BGB-283) against BCCO-002, which is consistent with the results observed in the HT29 xenograft model (Fig. 4A and C). BCCO-028 appeared to be more sensitive to treatment with BGB-283; partial regressions were observed in 3 of 8 (38%) mice treated with BGB-283 (5 mg/kg b.i.d.). Increasing the BGB-283 to 10 mg/kg, resulted in regressions in 7 of 8 (88%) mice (5 partial and 2 complete regressions). In contrast, dabrafenib (50 mg/kg b.i.d.) treatment was less effective against BCCO-028 with an observed 86% TGI, and no tumor regression (Fig. 4D Supplementary Table S6). It should be noted that for dabrafenib at 50 mg/kg b.i.d., its exposure in mouse is already 2- to 3-fold higher than the exposure it has achieved in patients at 150 mg b.i.d. dosing. Treatment with BGB-283 at doses up to 30 mg/kg had no significant effect on body weight in any of the tumor models tested (Supplementary Fig. S2).

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BGB-283 inhibits phosphorylation of both ERK1/2 and EGFR and displays potent antitumor activity in WiDr tumor xenografts [1]
BGB-283 was further evaluated against WiDr tumor xenografts, a BRAFV600E colorectal cancer model where strong feedback activation of EGFR was reported upon BRAF inhibition in two independent studies. In both reports, it was shown that BRAFV600E-selective inhibitors vemurafemib and its close analogue PLX-4720 were inactive as single agent in WiDr xenografts, and their antitumor activities were markedly enhanced when combined with erlotinib or cetuximab. In contrast, BGB-283 induced clear dose-dependent inhibition of tumor growth in the WiDr xenograft model as a single agent. In this study, BGB-283 was orally administrated to testing mice at 5 and 10 mg/kg twice daily (Fig. 4E). Ninety-five percent TGI was observed at lowest dosage of 5 mg/kg and >100% TGI plus partial regression in 4 of 8 (50%) mice was achieved at dosage of 10 mg/kg (Supplementary Table S6). In order to determine whether the tumor suppression was correlated to effective inhibition of EGFR and MAPK signaling, phospho-EGFR (pEGFR), phospho-MEK (pMEK), and phospho-ERK (pERK) and its downstream DUSP6 levels in tumor lysate were examined by Western blot analysis at various dose levels of BGB-283. BGB-283 did not induce EGFR feedback activation as reported for vemurafenib. In addition, BGB-283 potently inhibited pEGFR after either the first or the fifth dose at both dosages. Correspondingly, BGB-283 potently inhibited MEK and ERK phosphorylation and DUSP6 expression in vivo when dosed repeatedly (Fig. 4F). There is no detectable difference on AKT phosphorylation. In sum, these findings showed that BGB-283, which inhibits both RAF family kinases and EGFR, could have sustained inhibition of the MAPK pathway. Its ability to inhibit EGFR may contribute to its potent antitumor activity in this WiDr xenograft model.

In biochemical assays, Lifafenib (BGB-283) inhibits EGFR and RAF kinases with IC50 values of 23, 29, and 495 nM for EGFR, EGFR T790M/L858R mutant, and recombinant BRAFV600E kinase domain. Using time-resolved fluorescence-resonance energy transfer (TR-FRET) assays, compounds were examined for their ability to inhibit the activity of EGFR kinase in WT and RAF. For RAF kinases, MEK1 (K97R) was employed as a substrate, and for EGFR, a biotinylated peptide substrate was utilized. A final concentration of 100 mol/L of ATP and kinase substrates were added to the kinase after 60 to 120 minutes of serial compound dilution incubation at room temperature (RT). According to the manufacturer's instructions, the reaction was stopped with an equal volume of stop/detection solution. Once the plates were sealed and incubated at room temperature for two hours, the PHERAstar FS plate reader was used to record the TR-FRET signals, which are the ratio of fluorescence emission at 665 nm over emission at 620 nm with excitation at 337 nm wavelength. Life Technologies used their standard assays at Km concentration of ATP for perspective kinases to screen BGB-283 for activity in a panel of 277 kinases at a fixed concentration of 10 μmol/L. The kinases exhibiting >80% inhibition at 10 μmol/L BGB-283 were then identified by calculating their IC50.

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BRAFV600E kinase domain/Lifafenib (BGB-283) co-crystallization and structure determination [1]
BRAFV600E (444–723) was expressed and purified using methods similar to those previously reported. To co-crystallize BRAFV600E with Lifafenib (BGB-283), protein solution was incubated with BGB-283 at a ratio of 1:5 for 1 hour, and mixed with reservoir solution (100 mmol/L Bis Tris at pH 6.5, 23% PEG3350, and 200 mmol/L MgCl2) at equal volume. Co-crystals grew by sitting drop vapor diffusion method at 4°C. Crystals belonged to the space group P212121 (a = 49.395 Å, b = 101.601 Å, c = 109.786 Å) and contain two BRAFV600E molecules in an asymmetric unit. Diffraction images were processed and scaled with HKL2000. Phase was solved using software MOLREP by molecular replacement method with previously published structure. The resultant model was subsequently refined in PHENIX using rigid-body refinement and maximum likelihood method (Supplementary Table S1). Complex structure of the BRAFV600E kinase domain with BGB-283 was submitted to the Protein Data Bank (PDB) with the PDB ID code 4R5Y.

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In vitro kinase assay [1]
Compounds were tested for inhibition of RAF and WT EGFR kinase activity in assays based on time-resolved fluorescence-resonance energy transfer (TR-FRET) methodology. MEK1 (K97R) was used as a substrate for RAF kinases and a biotinylated peptide substrate was used for EGFR. The kinase was incubated with a serial dilution of compounds for 60 to 120 minutes at room temperature (RT), ATP (final concentration at 100 μmol/L) and kinase substrates were added to initiate the reaction. The reaction was stopped by an equal volume of stop/detection solution according to the manufacture's instruction. Plates were sealed and incubated at RT for 2 hours, and the TR-FRET signals (ratio of fluorescence emission at 665 nm over emission at 620 nm with excitation at 337 nm wavelength) were recorded on a PHERAstar FS plate reader. Lifafenib (BGB-283)was screened for activity in a panel of 277 kinases at a fixed concentration of 10 μmol/L by Life Technologies using their standard assays at Km concentration of ATP for perspective kinases. The IC50 was then determined for kinases showing >80% inhibition at 10 μmol/L BGB-283.

For each cell line, the number of cells seeded per well of a 96-well plate is optimized to guarantee logarithmic growth throughout the three-day treatment period. Following a 16-hour attachment period, duplicate cells are subjected to a 10-point dilution series. A volume of CellTiter-Glo reagent equal to the volume of cell culture medium in each well is added after the compound has been exposed for three days. After mixing the mixture for two minutes on an orbital shaker to allow the cells to lyse, the mixture is left to develop and stabilize the luminescent signal for ten minutes at room temperature. The luminosity signal is quantified.

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Cell culture [1]
A375, Sk-Mel-28, HT29, Colo205, WiDr, Ba/F3, A431, HCC827, SW620, HCT116, and cell lines used in cell panel profiling were purchased from ATCC. Cell lines were tested and authenticated at ATCC before purchase using morphology, karyotyping, and PCR-based approaches. All the cell lines were cultured in the designated medium supplemented with 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and in a humidified 37°C environment with 5% CO2. Cell lines were reinstated from frozen stocks laid down within three passages from the original cells purchased and passaged no more than 30 times. Culturing condition for cell panel profiling is listed in Supplementary Table S2. Stimulation of EGFR phosphorylation in A431 cells was achieved by addition of EGF to serum-free DMEM with 10-minute incubation. EGF-stimulated cell growth was achieved by addition of EGF to DMEM or RPMI supplemented with 10% FBS.

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Western blotting analysis [1]
For in vitro studies, cells were harvested after 1 hour treatment at 37°C and lysed immediately as previously described. For in vivo studies, tumors were harvested at the indicated time points, snap-frozen in liquid nitrogen, and stored at −80°C. Tumors were homogenized in 500 μL lysis buffer in MP homogenization unit and lysates were then centrifuged at 13,000 rpm for 10 minutes at 4°C to remove insoluble debris. The protein concentration of lysates was determined using the Pierce BCA protein assay kit. Proteins were separated by 10% SDS-PAGE gel or NuPAGE Novex 4% to 12% Bis-Tris protein gels and transferred to nitrocellulose membranes using iBlot Dry Blotting System. Blots were blocked with TBSTM [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 0.1% Tween 20, and 5% non-fat milk] at RT for 1 hour and probed with indicated antibodies diluted in TBSTM. The membranes were probed for phospho-proteins and then stripped to probe for total proteins. For reprobing, the membranes were stripped in stripping buffer (25 mmol/L glycine, pH 2.0, 1% SDS) for 30 to 60 minutes at RT, rinsed twice with TBST for 10 minutes, and probed for other proteins. Antigen–antibody complexes were visualized using the chemiluminescent substrate and detected with Image Quant LAS4000 mini digital imaging system.

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Cell-based phospho-ERK and phospho-EGFR detection assay [1]
Cellular phospho-ERK and phospho-EGFR were measured using a TR-FRET–based method. Cells were seeded at 3 × 104 per well of a 96-well plate and left to attach for 16 hours. Growth medium was then replaced with 100 μL of DMEM containing no serum. Cells were then treated with a 10-point titration of compound. After 1 hour of compound treatment, 50 μL of lysis buffer was added to each well. Plates were then incubated at room temperature with shaking for 30 minutes. A total of 16 μL of cell lysate from each well of a 96-well plate was transferred to a 384-well small volume white plate. Lysate from each well was incubated with 2 μL of Eu3+- or Tb3+- cryptate (donor) labeled anti-ERK or anti-EGFR antibody (Cisbio) and 2 μL of D2 (acceptor) labeled anti-phospho-ERK or anti-phospho-EGFR antibody for 2 hours at room temperature. FRET signals were measured using a PHERAstar FS reader.

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Proliferation assay [1]
The growth-inhibitory activity of compounds in a panel of melanoma, colon, breast, and lung cancer cells was determined using CellTiter-Glo luminescent cell viability assay. The number of cells seeded per well of a 96-well plate was optimized for each cell line to ensure logarithmic growth over the 3 days treatment period (Supplementary Table S2). Cells were left to attach for 16 hours and then treated with a 10-point dilution series in duplicate. Following a 3-day exposure to the compound, a volume of CellTiter-Glo reagent equal to the volume of cell culture medium present in each well was added. Mixture was mixed on an orbital shaker for 2 minutes to allow cell lysing, followed by 10 minutes incubation at room temperature to allow development and stabilization of luminescent signal.

Mice: Mice are randomized to treatment groups when the average tumor size reaches 110 to 200 mm3. Treatments consist of oral gavage (p.o.) with vehicle alone or 2.5 to 30 mg/kg of Lifafenib (BGB-283) administered twice daily or once daily. Mice given either cetuximab (40 mg/kg twice weekly) or erlotinib (100 mg/kg qd) are used as controls. Erlotinib and ligofenib (BGB-283) are combined to form a homogenous suspension at the required concentration in 0.5% (w/v) methylcellulose in purified water. Before administering, the injection solution for cetuximab is diluted with saline.

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Female NOD/SCID and BALB/c nude mice, ages 4 to 6 weeks, and weighing approximately 18 g, were used.
For HCC827, A431, HT29, Colo205, and WiDr xenografts, each mouse was injected subcutaneously with 2.5 to 5 × 106 cells in 200 μL PBS in the right front flank via a 26-gauge needle. When the average tumor size reached 110 to 200 mm3, animals were randomized to treatment groups (7–9 mice per group) and treated twice per day (b.i.d.) or once daily (qd) by oral gavage (p.o.) with vehicle alone or 2.5 to 30 mg/kg of Lifafenib (BGB-283). As control, mice were treated with erlotinib (100 mg/kg qd) or cetuximab (40 mg/kg twice weekly). Lifafenib (BGB-283) and erlotinib were formulated at the desired concentration as a homogenous suspension in 0.5% (w/v) methylcellulose in purified water. Cetuximab was formulated by diluting the injection solution with saline before dosing.
For BCCO-002 and BCCO-028 primary human tumor xenografts (PDX), colorectal cancer samples were collected from Beijing Cancer Hospital after patient's informed consent and immediately transferred in DMEM culture medium contained 200 U/mL penicillin and 200 mg/mL streptomycin. Within 2 to 4 hours of surgery, small fragments (3 mm × 3 mm × 3 mm) were subcutaneously engrafted into the scapular area or flank of anesthetized NOD/SCID mice. After three successful passages on NOD/SCID, tumors were subsequently passaged in BALB/c nude mice. Efficacy studies were conducted within six passages of the patient tumors. When the average tumor size reaches 100 to 200 mm3, animals were randomized to treatment groups (8 mice per group) and treated orally with vehicle (0.5% MC) alone, Lifafenib (BGB-283) (5–10 mg/kg, b.i.d.) or dabrafenib (50 mg/kg, b.i.d.). A further group received intravenous cetuximab (40 mg/kg every 3 days). Lifafenib (BGB-283) was formulated as described above. Dabrafenib was formulated in 10% DMSO + 90% HP-β-CD (Hydroxypropyl-β-cyclodextrin)/PBS.
In both cell line and primary tumor xenograft studies, individual body weights and tumor volumes were determined twice weekly, with mice being monitored daily for clinical signs of toxicity during the study [1].

In a multicenter, open-label clinical trial of riferafenib in Chinese patients with locally advanced or metastatic malignant solid tumors, this study successfully employed a validated liquid chromatography-single bond mass spectrometry (LC-Single Bond MS/MS) method for pharmacokinetic studies. Following a single oral dose of 10 mg or 15 mg riferafenib, plasma concentrations rapidly increased, reaching peak concentration (Cmax) approximately 2–3 hours later. A second peak concentration was observed in most subjects approximately 7–10 hours later. Figure 4 shows the plasma concentration-time curves for riferafenib in the 10 mg and 15 mg groups. Table 5 lists the pharmacokinetic parameters obtained from non-compartmental model analysis using Pheonix WinNonlin (version 8.1, Certara, MO, USA). Riferafenib exhibited significant inter-individual variability and low clearance. After 22 consecutive days of once-daily administration of riferafenib, the drug exposure (AUC0–24 h) over 24 hours on day 25 was significantly higher than that on day 1, with a cumulative rate of approximately 600%. In most subjects, urinary riferafenib concentrations were below the limit of quantitation within 72 hours of the first dose, and below 10 ng/mL on day 25. The cumulative urinary excretion percentage of riferafenib was less than 1%, indicating that renal excretion may not be its primary clearance route. [https://pubmed.ncbi.nlm.nih.gov/30599278/]

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Stability: Table 4 summarizes the stability of riferafenib in human plasma, urine, and the undiluted solution. Validation results showed that riferafenib remained stable in human plasma and urine after 18 hours and 7 hours at room temperature, respectively, and after 21 months and 15 months of storage at -80°C, respectively. Furthermore, plasma and urine samples (with Tween 80 added) withstood at least three freeze-thaw cycles after being frozen at -80°C for more than 24 hours and thawed at room temperature. Placement of prepared samples in an autosampler (4°C) for at least 72 hours did not affect the accuracy of quantification. Stock solutions of rifenib and IS are stable at −80 °C for at least 14 months. https://pubmed.ncbi.nlm.nih.gov/30599278/

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Pharmacokinetic Results: Systemic exposure increased from 5 mg to 50 mg on Day 1 of Cycle 1 and Day 1 of Cycle 2 (see Supplementary Data). Although this study did not perform an adequate statistical power assessment to validate proportionality, a log-log regression model explained more than 80% of the variability observed in the 10 mg to 60 mg dose range on Day 1 of Cycle 1. Rifenib is rapidly absorbed, with a median time to reach maximum plasma concentration of 3 hours. The cumulative ratios of maximum serum concentration (Cmax), area under the curve (AUC0-9), and AUC0-24 estimated based on Day 1 of Cycle 2/Day 1 of Cycle 1 were similar across the 10 mg to 50 mg dose range (see Supplementary Data). The mean cumulative fold increase for Cmax was 3.3 to 6.1 times, and the mean cumulative fold increases for AUC0-9 and AUC0-24 were 3.6 to 7.6 times. The terminal half-life was measurable in three patients (range 15 to 59 hours); however, due to the lack of sampling within 72 hours post-dose, the terminal half-life estimates should be interpreted with caution. https://pmc.ncbi.nlm.nih.gov/articles/PMC7325368/#s7

Safety and Tolerability: During dose escalation, the most common treatment-related adverse events (TEAEs) were fatigue (n = 24; 68.6%) and acneiform dermatitis (n = 15; 42.9%; Table 2). Five patients (14.3%) discontinued treatment due to TEAEs. The majority of treatment-related TEAEs were grade 1–2. The most common ≥ grade 3 treatment-related TEAEs during dose escalation were thrombocytopenia (14.3%), hypertension (11.4%), and fatigue (11.4%; Supplementary Data). Among patients who met the criteria for dose-limiting toxicity (DLT) assessment (n = 31), six experienced reversible DLTs, five of which occurred at doses ≥ 40 mg/day (Supplementary Data). Observed DLTs included grade 3 ALT elevation (n = 1) and grade 4 thrombocytopenia (n = 5). Thrombocytopenia typically occurs within 2–3 weeks of the first dose. The first two patients with thrombocytopenia underwent comprehensive examination (including bone marrow biopsy), which revealed normal bone marrow morphology and reserve function, suggesting a peripheral etiology. Each patient received either a platelet transfusion (n = 1) or prednisolone treatment (n = 4), and riferafenib was discontinued; platelet counts returned to normal within 6–20 days. 26 patients (74.3%) experienced ≥ Grade 3 treatment-emergent adverse events (TEAEs, including dose-limiting toxicities (DLTs), with a higher proportion of patients reporting ≥ Grade 3 TEAEs in the 40, 50, and 60 mg/day dose groups (61.5%) compared to the low-dose group (38.5%). ≥ Grade 3 TEAEs occurring in patients receiving ≥ 40 mg/day are listed in the supplemental data. The maximum tolerated dose (MTD) was set at 40 mg/day. Dose-limiting thrombocytopenic purpura occurred in 80% (4 out of 5 patients) of patients receiving 40 mg/day and 60 mg/day doses (n = 2 in each group), with 70% of patients in the 40 mg/day dose group discontinuing/reducing their dose due to drug toxicity, typically between day 13 and 28 of the first treatment cycle. Based on these data, the RP2D was determined to be 30 mg/day. Among the 96 patients treated with riferafenib during the dose extension period, the most common treatment-examined adverse events (TEAEs) were fatigue (n = 47; 49%) and decreased appetite (n = 35; 36.5%; Table 2). No squamous cell carcinoma or keratoacanthoma of the skin was reported throughout the study. 68 patients (70.8%) experienced ≥ grade 3 TEAEs, and 56 patients (58.3%) experienced serious TEAEs. Treatment-related adverse events (TEAEs) occurred during treatment, leading to discontinuation in 19 patients (19.8%). The most common TEAEs were fatigue (n = 5) and thrombocytopenia (n = 2; see Supplementary Data). Adverse events (AEs) occurred in 50 patients (52%), resulting in dose adjustments, with a median relative dose intensity of 95.0%. Six treatment-unrelated TEAEs occurred in 4 patients (4.2%), ultimately leading to death: pericardial effusion, sepsis, pleural effusion, intracranial hemorrhage, intestinal perforation due to disease progression, and small bowel obstruction (n = 1 each). During dose expansion, the most common ≥ grade 3 treatment-related adverse events were hypertension (8.3%) and fatigue (7.3%). Two patients discontinued treatment due to treatment-related ≥ grade 3 thrombocytopenia. https://pmc.ncbi.nlm.nih.gov/articles/PMC7325368/#s7

[1]. BGB-283, a Novel RAF Kinase and EGFR Inhibitor, Displays Potent Antitumor Activity in BRAF-Mutated Colorectal Cancers. Mol Cancer Ther. 2015 Oct;14(10):2187-97.

Lifirafenib is being investigated in the clinical trial NCT03641586 (BGB-283 study in Chinese patients with locally advanced or metastatic malignant solid tumors). Lifirafenib is an inhibitor of the serine/threonine protein kinase B-raf (BRAF) and epidermal growth factor receptor (EGFR), possessing potential antitumor activity. Lifirafenib selectively binds to and inhibits the activity of BRAF and certain mutants of it, as well as EGFR. This blocks BRAF and EGFR-mediated signaling and inhibits the proliferation of tumor cells containing mutated BRAF genes or overactivated EGFR. Furthermore, BGB-283 also inhibits mutants of the Ras protein K-RAS and N-RAS. BRAF and EGFR are mutated or upregulated in various tumor cell types. Lifirafenib is a small molecule drug that has completed Phase II clinical trials (covering all indications) and has one investigational indication. The oncogene BRAF drives cell transformation and proliferation, and is detected in approximately 50% of human malignant melanomas and 5% to 15% of colorectal cancers. While vemurafenib and dabrafenib have shown significant clinical efficacy in treating BRAF(V600E) metastatic melanoma, their efficacy in BRAF(V600E) colorectal cancer is far less satisfactory. Previous studies have suggested that feedback activation of the EGFR and MAPK signaling pathways following BRAF inhibition may be one reason for the relative insensitivity of colorectal cancer to first-generation BRAF inhibitors. This article reports the properties of a dual RAF kinase/EGFR inhibitor, BGB-283, which is currently undergoing clinical trials. In vitro experiments showed that BGB-283 effectively inhibits BRAF(V600E)-activated ERK phosphorylation and cell proliferation. It exhibits selective cytotoxicity, preferentially inhibiting the proliferation of cancer cells carrying BRAF(V600E) and EGFR mutations/amplifications. In BRAF(V600E) colorectal cancer cell lines, BGB-283 effectively inhibited EGFR reactivation and EGFR-mediated cell proliferation. In vivo experiments showed that BGB-283 treatment dose-dependently inhibited tumor growth and partial or complete tumor regression was observed in cell lines carrying BRAF(V600E) mutations and in primary human colorectal cancer xenograft models. These results support the clinical potential of BGB-283 as a potent anti-tumor candidate for the treatment of colorectal cancers carrying BRAF(V600E) mutations. [1]

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This article describes the activity of the second-generation BRAF inhibitor BGB-283, which has the potential to treat cancers with abnormal MAPK pathways. BGB-283 exhibits potent and reversible inhibitory activity against RAF family kinases, including wild-type A-RAF, BRAF, C-RAF, and BRAFV600E. In addition, BGB-283 effectively inhibits EGFR at both the biochemical and cellular levels. BGB-283 exhibited significant antiproliferative selectivity in 107 cancer cell lines. BGB-283 effectively inhibited serum-induced proliferation of BRAFV600E mutant cancer cell lines, with IC50 values ranging from 137 nmol/L to 580 nmol/L. Except for the HCC827 lung cancer cell line (EGFR E746-A750 deletion), ZR-75-30 lung cancer cell line (HER2 amplification), and NCI-H322M lung cancer cell line (EGFR overexpression), BGB-283 showed almost no inhibitory activity against cell lines lacking the BRAFV600E mutation. These results suggest that the RAF kinase and EGFR inhibitory activities of BGB-283 are the main mechanisms by which it exerts its antiproliferative effect in the tested cancer cells. Although BGB-283 and vemurafenib have different kinase selectivity profiles, both drugs showed significant selectivity against cancer cells carrying the BRAFV600E mutation in cell viability assays (Figures 3A and 3B). While vemurafenib and dabrafenib have achieved significant efficacy in melanoma, clinical responses to first-generation BRAF inhibitors in other BRAFV600E-mutant cancers have been far less satisfactory. It has been reported that the response rate to vemurafenib in BRAFV600E-mutant colorectal cancer is only 5%. Two independent studies have shown that EGFR feedback activation may be one of the main mechanisms leading to resistance to first-generation BRAF inhibitors. This article demonstrates that BGB-283 is a true EGFR inhibitor, exhibiting good EGFR inhibitory activity in both in vitro and in vivo experiments. In WiDr colorectal cancer cells, BGB-283 has been shown to inhibit EGFR signaling feedback activation and achieve sustained inhibition of pERK. This sustained inhibition of pERK translates into significant in vivo antitumor activity. Notably, BGB-283 monotherapy (10 mg/kg, twice daily) achieved a 50% partial regression in WiDr colorectal adenocarcinoma xenografts. In contrast, PLX4720 combined with cetuximab and vemurafenib combined with erlotinib appeared to primarily achieve tumor growth inhibition (TGI), but did not achieve tumor regression in the WiDr xenograft model. BRAFV600E mutations have been reported in 5% to 15% of colorectal cancer patients. In the 23 primary colorectal cancer xenograft models established in this study, two models were found to have BRAFV600E mutations. BGB-283 showed good efficacy in both models, with objective response rates ranging from 25% to 100%. We are currently conducting a more comprehensive characterization of these models and attempting to better understand the MAPK and EGFR pathways in these two primary tumor xenograft models. Currently, BGB-283 is undergoing a Phase I clinical trial to evaluate its safety, tolerability, pharmacokinetics, and pharmacodynamic activity in humans. To our knowledge, BGB-283 is currently the only small molecule inhibitor in clinical use that simultaneously targets RAF kinase and EGFR. There is significant interest in validating the hypothesis that EGFR feedback activation leads to poor efficacy of selective BRAFV600E inhibitors in colorectal cancer. Several clinical trials combining BRAF inhibitors with EGFR small molecule inhibitors or monoclonal antibodies are currently underway (see www.clinicaltrials.gov). The preclinical results reported in this study suggest the need to evaluate BGB-283 as a single agent in patients with BRAFV600E-mutant colorectal cancer.

C25H17F3N4O3

478.42

478.125

C, 62.76; H, 3.58; F, 11.91; N, 11.71; O, 10.03

1446090-79-4

rel-Lifirafenib;1446090-77-2; 2025321-07-5 (mesylate); 1446090-79-4; 2025321-56-4; 2025320-97-0 (HCl); 1854985-74-2

89670174

White to off-white solid powder

5.433

2

8

3

35

845

3

FC(C1C([H])=C([H])C2=C(C=1[H])N([H])C([C@]1([H])[C@]3([H])[C@@]1([H])C1C([H])=C(C([H])=C([H])C=1O3)OC1C([H])=C([H])N=C3C=1C([H])([H])C([H])([H])C(N3[H])=O)=N2)(F)F

NGFFVZQXSRKHBM-FKBYEOEOSA-N

InChI=1S/C25H17F3N4O3/c26-25(27,28)11-1-4-15-16(9-11)31-24(30-15)21-20-14-10-12(2-5-17(14)35-22(20)21)34-18-7-8-29-23-13(18)3-6-19(33)32-23/h1-2,4-5,7-10,20-22H,3,6H2,(H,30,31)(H,29,32,33)/t20-,21-,22-/m0/s1

5-[[(1R,1aS,6bR)-1-[6-(trifluoromethyl)-1H-benzimidazol-2-yl]-1a,6b-dihydro-1H-cyclopropa[b][1]benzofuran-5-yl]oxy]-3,4-dihydro-1H-1,8-naphthyridin-2-one

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 (5.23 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 (5.23 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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 (Please use freshly prepared in vivo formulations for optimal results.)