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].

---

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).

---

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.

---

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.

---

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.

---

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.

---

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.

---

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.

---

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.

---

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 the multicenter, open-label clinical trial of lifirafenib in Chinese subjects with locally advanced or metastatic malignant solid tumor, this validated LCsingle bondMS/MS method was successfully applied in pharmacokinetic study. After a single oral dose of 10 mg or 15 mg, plasma concentration of lifirafenib increased rapidly and reached the maximum concentration (Cmax) around a median of 2–3 h. In most subjects, second peak concentrations were observed around 7˜10 h.. The plasma concentration-time profiles of lifirafenib in 10 mg and 15 mg groups are depicted in Fig. 4. The pharmacokinetic parameters from non-compartmental analysis using Pheonix WinNonlin (Version 8.1, Certara, MO, USA) are shown in Table 5. Lifirafenib exhibited the characteristics of large inter-individual variability and low elimination. After administration lifirafenib once daily for 22 consecutive days, the exposure within 24 h (AUC0-24 h) in day 25 was significantly higher than that in day 1, and the accumulation ratio was approximate 600%. In the most subjects, urinary concentrations of lifirafenib within 72 h after the first dose were below the limit of quantification, and the concentrations in day 25 were less than 10 ng/mL. The percentage of cumulative urine excretion of lifirafenib was less than 1%, which indicted that renal excretion might not be a main elimination route. [https://pubmed.ncbi.nlm.nih.gov/30599278/]

---

Stability: The stabilities for lifirafenib in human plasma and urine as well as in stock solution are summarized in Table 4. The validation results indicated that lifirafenib in human plasma and urine remained stable after being placed at ambient temperature for 18 h and 7 h, and also stable after being stored at −80 °C for 21 months and 15 months, respectively. Furthermore, the plasma and urine (after the addition of tween 80) samples could tolerate at least 3 freeze-thaw cycles by freezing at −80 °C for more than 24 h and thawing at room temperature. The post-preparation samples were placed in auto-sampler (4 °C) for at least 72 h, which had no impact on the accuracy of quantification. The stock solutions of lifirafenib and IS were stably preserved at −80 °C for at least 14 months.https://pubmed.ncbi.nlm.nih.gov/30599278/

---

PK Outcomes Systemic exposure increased from 5 to 50 mg on cycle 1, day 1, and cycle 2, day 1 (Data Supplement). Although not powered to assess proportionality, the log-log regression model accounted for > 80% of the observed variation from 10 to 60 mg for cycle 1, day 1. Lifirafenib was rapidly absorbed, with a median time to reach maximum plasma concentration of 3 hours. The accumulation ratio for maximum serum concentration (Cmax), area under the plasma concentration curve from 0-9 hours (AUC0-9), and AUC0-24 estimated from cycle 2, day 1/cycle 1, day 1, was similar from 10 to 50 mg (Data Supplement). Average accumulation ranged from 3.3- to 6.1-fold for Cmax and 3.6- to 7.6-fold for AUC0-9 and AUC0-24. Three patients had measurable terminal half-life (range, 15-59 hours); terminal half-life estimates should be interpreted with caution because samples were not collected beyond 72 hours after dosing.https://pmc.ncbi.nlm.nih.gov/articles/PMC7325368/#s7

Safety and Tolerability: During dose escalation, the most frequent TEAEs were fatigue (n = 24; 68.6%) and dermatitis acneiform (n = 15; 42.9%; Table 2). Five patients (14.3%) experienced TEAEs that led to discontinuation. Treatment-related TEAEs were predominantly grades 1-2. The most common grade ≥ 3 treatment-related TEAEs during dose escalation were thrombocytopenia (14.3%), hypertension (11.4%), and fatigue (11.4%; Data Supplement). Among patients eligible for DLT assessment (n = 31), 6 experienced reversible DLTs, 5 of which occurred at doses ≥ 40 mg/d (Data Supplement). Observed DLTs included grade 3 increased ALT (n = 1) and grade 4 thrombocytopenia (n = 5); thrombocytopenia typically occurred within 2-3 weeks of initial dosing. Comprehensive investigations (including bone marrow biopsies) in the first 2 patients with thrombocytopenia revealed normal bone marrow morphology and reserve, which suggested a peripheral cause. Patients were treated with platelet transfusion (n = 1) or prednisolone administration (n = 4) and withholding of lifirafenib; platelet counts recovered within 6-20 days. Grade ≥ 3 TEAEs (including DLTs) occurred in 26 patients (74.3%), with more patients reporting grade ≥ 3 TEAEs in the 40, 50, and 60 mg/d cohorts (61.5%) versus lower-dose cohorts (38.5%). Grade ≥ 3 TEAEs that occurred in patients who received ≥ 40 mg/d are listed in the Data Supplement. The MTD was established at 40 mg/d. Eighty percent (4 of 5 patients) of dose-limiting thrombocytopenia occurred in patients who received 40 and 60 mg/d (n = 2 each), and 70% of patients treated with 40 mg/d had dose interruptions/reductions as a result of drug toxicity, typically between days 13 and 28 of cycle 1. On the basis of these data, the RP2D was established at 30 mg/d.
Of 96 patients who received lifirafenib during dose expansion, the most commonly reported TEAEs were fatigue (n = 47; 49%) and decreased appetite (n = 35; 36.5%; Table 2). During the entire study, cutaneous SCC or keratoacanthoma was not reported. Grade ≥ 3 TEAEs occurred in 68 patients (70.8%), and serious TEAEs occurred in 56 patients (58.3%). TEAEs led to discontinuation in 19 patients (19.8%), most commonly fatigue (n = 5) and thrombocytopenia (n = 2; Data Supplement). Fifty patients (52%) experienced AEs that led to a dose adjustment, which resulted in a median relative dose intensity of 95.0%. Four patients (4.2%) experienced 6 TEAEs considered unrelated to treatment that led to death: pericardial effusion, sepsis, pleural effusion, intracranial hemorrhage, intestinal perforation as a result of disease progression, and small intestinal obstruction (n = 1 each). During dose expansion, the most common grade ≥ 3 treatment-related AEs were hypertension (8.3%) and fatigue (7.3%); 2 patients discontinued because of 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 under investigation in clinical trial NCT03641586 (The Study of BGB-283 in Chinese Subjects With Local Advanced or Metastatic Malignant Solid Tumor).
Lifirafenib is an inhibitor of the serine/threonine protein kinase B-raf (BRAF) and epidermal growth factor receptor (EGFR), with potential antineoplastic activity. Lifirafenib selectively binds to and inhibits the activity of BRAF and certain BRAF mutant forms, and EGFR. This prevents BRAF- and EGFR-mediated signaling and inhibits the proliferation of tumor cells that either contain a mutated BRAF gene or express over-activated EGFR. In addition, BGB-283 inhibits mutant forms of the Ras proteins K-RAS and N-RAS. BRAF and EGFR are mutated or upregulated in many tumor cell types.
LIFIRAFENIB is a small molecule drug with a maximum clinical trial phase of II (across all indications) and has 1 investigational indication.

---

Oncogenic BRAF, which drives cell transformation and proliferation, has been detected in approximately 50% of human malignant melanomas and 5% to 15% of colorectal cancers. Despite the remarkable clinical activities achieved by vemurafenib and dabrafenib in treating BRAF(V600E) metastatic melanoma, their clinical efficacy in BRAF(V600E) colorectal cancer is far less impressive. Prior studies suggested that feedback activation of EGFR and MAPK signaling upon BRAF inhibition might contribute to the relative unresponsiveness of colorectal cancer to the first-generation BRAF inhibitors. Here, we report characterization of a dual RAF kinase/EGFR inhibitor, BGB-283, which is currently under clinical investigation. In vitro, BGB-283 potently inhibits BRAF(V600E)-activated ERK phosphorylation and cell proliferation. It demonstrates selective cytotoxicity and preferentially inhibits proliferation of cancer cells harboring BRAF(V600E) and EGFR mutation/amplification. In BRAF(V600E) colorectal cancer cell lines, BGB-283 effectively inhibits the reactivation of EGFR and EGFR-mediated cell proliferation. In vivo, BGB-283 treatment leads to dose-dependent tumor growth inhibition accompanied by partial and complete tumor regressions in both cell line-derived and primary human colorectal tumor xenografts bearing BRAF(V600E) mutation. These findings support BGB-283 as a potent antitumor drug candidate with clinical potential for treating colorectal cancer harboring BRAF(V600E) mutation.[1]

---

In this article, we describe the activity of BGB-283, a second-generation BRAF inhibitor, with potential for the treatment of cancers with aberrations in the MAPK pathway. BGB-283 showed potent and reversible inhibitory activities against RAF family kinases, including wild-type A-RAF, BRAF, C-RAF, and BRAFV600E. In addition, BGB-283 also potently inhibited EGFR at both the biochemical and cellular level. BGB-283 demonstrated remarkable selectivity in a panel of 107 cancer cell lines for antiproliferation activity. BGB-283 potently inhibited the serum-induced cell proliferation of BRAFV600E-mutant cancer cell lines, with IC50 values ranging from 137 nmol/L to 580 nmol/L. It showed little or no inhibitory activity in cell lines lacking BRAFV600E mutation, with the exception of the HCC827 lung cancer cell line (EGFR E746-A750 deletion), ZR-75-30 (HER2 amplification), and the NCI-H322M lung cancer cell line (EGFR overexpression). These results suggested that RAF kinase and EGFR-inhibitory activities of BGB-283 contributed the most to its antiproliferative activities in the tested cancer cells. Despite the different kinase selectivity profile between BGB-283 and vemurafenib, both agents displayed noticeable selectivity toward cancer cells harboring BRAFV600E in a cell viability assay (Fig. 3A and B).
Despite the remarkable responses to vemurafenib and dabrafenib in melanoma, the clinical response of other BRAFV600E cancers to the first generation of BRAF inhibitors is much less impressive. The reported response of BRAFV600E colorectal cancer to vemurafenib is merely 5%. Two independent studies suggested that EGFR feedback activation could be one of the main mechanisms of the observed resistance to first-generation BRAF inhibitors. This article demonstrates that BGB-283 is a bona fide EGFR inhibitor and displays good EGFR inhibitory activity in in vitro and in vivo experiments. In WiDr colorectal cancer cells, BGB-283 was shown to be able to inhibit the feedback activation of EGFR signaling and achieves sustained inhibition of pERK. This sustained inhibition of pERK translates into remarkable antitumor activity in vivo. Notably, BGB-283 single-agent treatment at 10 mg/kg b.i.d. led to 50% partial regression in WiDr colorectal adenocarcinoma xenografts. In comparison, both PLX4720+cetuximab and vemurafenib+erlotinib combinations seemed to have achieved mostly TGI but not tumor regression in WiDr xenograft models.
BRAFV600E mutation is reported to occur in 5% to 15% of colorectal cancer patients. Among the 23 colorectal cancer primary tumor xenograft models established in this study, two of them were found to have the BRAFV600E mutation. BGB-283 demonstrated good efficacy in both models with the objective response rate ranging from 25% to 100%. We are carrying out more comprehensive characterizations of these models and trying to better understand the MAPK and EGFR pathways in these two primary tumor xenograft models. Currently, phase I clinical trials are in progress to test the safety, tolerability, pharmacokinetics, and pharmacodynamic activity of BGB-283 in human. To our knowledge, BGB-283 is the only small-molecule inhibitor in the clinic that simultaneously targets RAF kinases and EGFR. There have been strong interests from the community to test the hypothesis that EGFR feedback activation leads to lack of responses in colorectal cancer for BRAFV600E-selective inhibitors. A number of clinical trials that combines BRAF inhibitors with EGFR small-molecule inhibitors or monoclonal antibodies are currently under way (see www.clinicaltrials.gov). The preclinical results reported in this study warrant evaluation of BGB-283 as a single agent in BRAFV600E-mutated colorectal cancer patients.

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.

---

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.

---

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