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

体外活性：BGB-283 在体外有效抑制 BRAFV600E 激活的 ERK 磷酸化和细胞增殖。它具有选择性细胞毒性，并优先抑制携带 BRAFV600E 和 EGFR 突变/扩增的癌细胞的增殖。在 BRAFV600E 结直肠癌细胞系中，BGB-283 有效抑制 EGFR 的再激活和 EGFR 介导的细胞增殖。它表现出对含有 BRAFV600E 或 EGFR 突变的细胞系的选择性细胞毒性。 BGB-283 以剂量依赖性方式抑制 A431 细胞中 EGF 诱导的 Tyr1068 上的 EGFR 自磷酸化。在WiDr结直肠癌细胞中，BGB-283被证明能够抑制EGFR信号传导的反馈激活，并实现对pERK的持续抑制。激酶测定：在生化测定中，Lifirafenib (BGB-283) 抑制 RAF 激酶和 EGFR 活性，对于重组 BRAFV600E 激酶结构域、EGFR 和 EGFR T790M/L858R 突变体的 IC50 值为 23、29 和 495 nM。在基于时间分辨荧光共振能量转移 (TR-FRET) 方法的测定中，测试了化合物对 RAF 和 WT EGFR 激酶活性的抑制作用。 MEK1 (K97R) 用作 RAF 激酶的底物，生物素化肽底物用作 EGFR (61TK0BLC, CisBio Bioassys)。将激酶与连续稀释的化合物在室温 (RT) 下孵育 60 至 120 分钟，添加 ATP（终浓度为 100 μmol/L）和激酶底物以引发反应。根据制造商说明（CisBio Bioassays）通过等体积的终止/检测溶液来终止反应。将板密封并在室温下孵育 2 小时，并在 PHERAstar FS 读板器 (BMG Labtech) 上记录 TR-FRET 信号（665 nm 处的荧光发射与 337 nm 波长激发下的 620 nm 处发射的比率）。 Life Technologies 使用其在 ATP 浓度 Km 浓度下的标准测定法，对 BGB-283 在 10 μmol/L 固定浓度下的一组 277 种激酶中的活性进行了筛选。然后测定在 10 μmol/L BGB-283 下显示 >80% 抑制作用的激酶的 IC50。细胞测定：使用基于 TR-FRET 的方法测量细胞磷酸-ERK 和磷酸-EGFR。将细胞按 3 × 104 个/孔接种到 96 孔板中，并贴壁 16 小时。然后用 100 μL 不含血清的 DMEM 替换生长培养基。然后用化合物的10点滴定处理细胞。化合物处理1小时后，向每孔添加50μL裂解缓冲液(Cisbio)。然后将板在室温下摇动孵育30分钟。将 96 孔板每孔总共 16 μL 的细胞裂解液转移至 384 孔小体积白色板。将每孔的裂解物与 2 μL Eu3+- 或 Tb3+- 穴状化合物（供体）标记的抗 ERK 或抗 EGFR 抗体 (Cisbio) 和 2 μL D2（受体）标记的抗磷酸-ERK 或抗磷酸-一起孵育。 EGFR 抗体 (Cisbio) 室温 2 小时。使用 PHERAstar FS 读数器 (BMG Labtech) 测量 FRET 信号。

BGB-283 治疗可导致剂量依赖性肿瘤生长抑制，并伴有 BRAFV600E 突变的细胞系来源和原代人结直肠肿瘤异种移植物的部分和完全肿瘤消退。 BGB-283 在 BRAF(V600E) 结直肠癌异种移植模型中非常有效，包括 HT29、Colo205 和两种带有 BRAFV600E 突变的原发肿瘤异种移植模型。此外，BGB-283 在 WiDr 异种移植模型中显示出引人注目的功效，其中 BRAF 抑制后可诱导 EGFR 再激活。 BGB-283 可诱导 HCC827 中的肿瘤消退，但不会诱导 A431 异种移植物中的肿瘤消退。 BGB-283 抑制 ERK1/2 和 EGFR 的磷酸化，并在 WiDr 肿瘤异种移植物中显示出有效的抗肿瘤活性。 BGB-283 不会诱导 EGFR 反馈激活，如维莫非尼报道的那样。重复给药时，BGB-283 可有效抑制体内 MEK 和 ERK 磷酸化以及 DUSP6 表达。 AKT 磷酸化没有可检测到的差异。

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BGB-283在癌症小鼠异种移植模型中表现出抗肿瘤活性[1]
在源自携带BRAFV600E突变的HT29和Colo205结直肠癌癌症细胞系的皮下异种移植物模型中评估BGB-283的体内功效。之前有报道称，维罗非尼对HT-29异种移植物的疗效有限，与EGFR抑制剂联合使用可提高其抗肿瘤活性。BGB-283在5mg/kg b.i.d.剂量下显著抑制了HT29异种移植物的肿瘤生长（P<0.001），动物对此具有良好的耐受性。在该异种移植物模型中，添加西妥昔单抗（一种靶向EGFR的单克隆抗体）并没有进一步增强BGB-283的治疗效果（P>0.05，BGB-283+西妥昔单抗与BGB-283相比；图4A），这表明单独使用BGB-283可能足以阻断EGFR的反馈激活。针对Colo205异种移植物，BGB-283产生了3至30mg/kg的剂量依赖性肿瘤抑制作用（图4B）。更重要的是，在10mg/kg（1/7只小鼠）的剂量下观察到部分回归。在30 mg/kg BGB-283剂量下，7只小鼠中有3只出现退化（2只PR和1只CR；补充表S6）

在人肿瘤组织来源的原发性癌症异种移植物模型中进一步评估了BGB-283的肿瘤抑制活性。在体内建立了23个患者来源的癌症大肠癌模型，其中两个BCCO002和BCCO-028被鉴定为携带BRAFV600E突变。这两种患者来源的癌症大肠癌模型对BGB-283治疗敏感（图4C和D）；>口服BGB-283（10mg/kg，b.i.d.；图4C和d以及补充表S6）后第24天观察到100%TGI。对于BCCO-002，用BGB-283（10mg/kg b.i.d.）治疗的8只小鼠中有2只（25%）观察到部分退化。添加西妥昔单抗并没有进一步增强BGB-283对BCCO-002的抗肿瘤活性（P>0.05，BGB-283+西妥昔玛vs.BGB-283），这与HT29异种移植物模型中观察到的结果一致（图4A和C）。BCCO-028似乎对BGB-283治疗更敏感；在用BGB-283（5mg/kg b.i.d.）治疗的8只小鼠中，有3只（38%）观察到部分退化。将BGB-283增加到10mg/kg，导致8只小鼠中有7只（88%）出现退化（5只部分退化，2只完全退化）。相比之下，dabrafenib（50mg/kg b.i.d.）治疗对BCCO-028的疗效较差，观察到86%的TGI，并且没有肿瘤消退（图4D补充表S6）。应该指出的是，对于50mg/kg b.i.d.的达比非尼，其在小鼠中的暴露量已经比150mg b.i.d.剂量的患者高出2至3倍。在任何测试的肿瘤模型中，剂量高达30mg/kg的BGB-283治疗对体重没有显著影响（补充图S2）

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BGB-283抑制ERK1/2和EGFR的磷酸化，并在WiDr肿瘤异种移植物中显示出强大的抗肿瘤活性[1]
对WiDr肿瘤异种移植物（BRAFV600E结直肠癌癌症模型）进一步评估了BGB-283，在两项独立研究中，报告了BRAF抑制后EGFR的强反馈激活。在这两份报告中，都表明BRAFV600E选择性抑制剂vemurafemib及其类似物PLX-4720作为单一药物在WiDr异种移植物中没有活性，当与厄洛替尼或西妥昔单抗联合使用时，它们的抗肿瘤活性显著增强。相比之下，BGB-283作为单一药物在WiDr异种移植物模型中诱导了明显的剂量依赖性肿瘤生长抑制。在这项研究中，BGB-283以5和10mg/kg的剂量口服给药于测试小鼠，每天两次（图4E）。在最低剂量为5mg/kg时观察到95%的TGI，在10mg/kg的剂量下，8只小鼠中有4只（50%）的TGI加部分回归达到>100%（补充表S6）。为了确定肿瘤抑制是否与有效抑制EGFR和MAPK信号传导相关，在不同剂量水平的BGB-283下，通过蛋白质印迹分析检测了肿瘤裂解物中磷酸化EGFR（pEGF）、磷酸化MEK（pMEK）和磷酸化ERK（pERK）及其下游DUSP6水平。BGB-283没有像维罗非尼报告的那样诱导EGFR反馈激活。此外，BGB-283在两种剂量的第一剂或第五剂后均能有效抑制pEGFR。相应地，当反复给药时，BGB-283在体内有效地抑制了MEK和ERK的磷酸化以及DUSP6的表达（图4F）。AKT磷酸化没有可检测的差异。总之，这些发现表明，抑制RAF家族激酶和EGFR的BGB-283可以持续抑制MAPK通路。其抑制EGFR的能力可能有助于其在WiDr异种移植物模型中的强效抗肿瘤活性。

在生化检测中，Lifafenib（BGB-283） 抑制 EGFR 和 RAF 激酶，对于 EGFR、EGFR T790M/L858R 突变体和重组 BRAFV600E 激酶结构域的 IC50 值为 23、29 和 495 nM。使用时间分辨荧光共振能量转移 (TR-FRET) 测定，检查化合物抑制 WT 和 RAF 中 EGFR 激酶活性的能力。对于 RAF 激酶，采用 MEK1 (K97R) 作为底物，对于 EGFR，采用生物素化肽底物。在室温 (RT) 下连续化合物稀释孵育 60 至 120 分钟后，将最终浓度为 100 mol/L 的 ATP 和激酶底物添加到激酶中。根据制造商的说明，用等体积的终止/检测溶液终止反应。将板密封并在室温下孵育两小时后，使用 PHERAstar FS 读板器记录 TR-FRET 信号，即 665 nm 处的荧光发射与激发时 620 nm 处的发射的比率波长为 337 nm。 Life Technologies 使用其在 Km ATP 浓度下的标准测定法来筛选 BGB-283，以筛选 BGB-283 在 10 μmol/L 固定浓度下在一组 277 种激酶中的活性。然后通过计算 IC50 来鉴定在 10 μmol/L BGB-283 下表现出 >80% 抑制的激酶。

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BRAFV600E激酶结构域/利法非尼（BGB-283）共结晶和结构测定[1]
BRAFV600E（444-723）的表达和纯化方法与之前报道的方法相似。为了使BRAFV600E与Lifafenib（BGB-283）共结晶，将蛋白质溶液与BGB-283以1:5的比例孵育1小时，并与等体积的储备溶液（pH 6.5的100 mmol/L Bis-Tris、23%PEG3350和200 mmol/L MgCl2）混合。在4°C下通过坐滴蒸汽扩散法生长钴晶体。晶体属于P212121空间群（a=49.395Å，b=101.601Å，c=109.786Å），在不对称单元中包含两个BRAFV600E分子。衍射图像用HKL2000进行处理和缩放。使用MOLREP软件，通过分子替换方法，利用之前发表的结构求解相。随后，在PHENIX中使用刚体精化和最大似然法对所得模型进行了精化（补充表S1）。BRAFV600E激酶结构域与BGB-283的复合结构以PDB ID代码4R5Y提交给蛋白质数据库（PDB）。

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体外激酶测定[1]
在基于时间分辨荧光共振能量转移（TR-FRET）方法的测定中测试化合物对RAF和WT EGFR激酶活性的抑制作用。MEK1（K97R）用作RAF激酶的底物，生物素化肽底物用于EGFR。在室温（RT）下，将激酶与一系列稀释的化合物一起孵育60至120分钟，加入ATP（终浓度为100μmol/L）和激酶底物以引发反应。根据制造商的说明，用等体积的停止/检测溶液停止反应。将板密封并在室温下孵育2小时，在PHERAstar FS板读数器上记录TR-FRET信号（665nm处的荧光发射与波长为337nm的激发下620nm处的发射之比）。 Life Technologies使用其ATP Km浓度的标准检测方法，在10μmol/L的固定浓度下，对277种激酶中的利法非尼/Lifafenib（BGB-283）进行了活性筛选。然后测定在10μmol/L BGB-283下显示>80%抑制的激酶的IC50。

对于每个细胞系，96 孔板每孔接种的细胞数量经过优化，以保证在三天的治疗期间实现对数生长。 16 小时贴壁期后，对重复细胞进行 10 点系列稀释。化合物暴露三天后，添加与每孔中细胞培养基体积相等的 CellTiter-Glo 试剂。将混合物在定轨摇床上混合两分钟以使细胞裂解后，将混合物在室温下放置十分钟以产生并稳定发光信号。光度信号被量化。

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细胞培养[1]
A375、Sk-Mel-28、HT29、Colo205、WiDr、Ba/F3、A431、HCC827、SW620、HCT116和用于细胞面板分析的细胞系购自ATCC。在购买之前，使用形态学、核型分析和基于PCR的方法在ATCC对细胞系进行了测试和鉴定。所有细胞系均在添加了10%FBS、100 U/mL青霉素、0.1 mg/mL链霉素的指定培养基中培养，并在含有5%CO2的37°C加湿环境中培养。细胞系是从购买的原始细胞经过三次传代后的冷冻储备中恢复的，传代次数不超过30次。细胞面板分析的培养条件列于补充表S2中。通过在无血清DMEM中加入EGF并孵育10分钟，实现了A431细胞中EGFR磷酸化的刺激。通过向补充有10%FBS的DMEM或RPMI中添加EGF来实现EGF刺激的细胞生长。

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蛋白质印迹分析[1]
对于体外研究，在37°C下处理1小时后收获细胞，并如前所述立即裂解。对于体内研究，在指定的时间点收获肿瘤，在液氮中快速冷冻，并储存在-80°C下。肿瘤在MP匀浆装置的500μL裂解缓冲液中匀浆，然后将裂解物在4°C下以13000 rpm离心10分钟，以去除不溶性碎片。使用Pierce BCA蛋白检测试剂盒测定裂解物的蛋白质浓度。蛋白质通过10%SDS-PAGE凝胶或NuPAGE Novex 4%至12%Bis-Tris蛋白质凝胶分离，并使用iBlot干吸系统转移到硝化纤维膜上。在室温下用TBSTM[50 mmol/L Tris（pH 7.5）、150 mmol/L NaCl、0.1%吐温20和5%脱脂乳]阻断印迹1小时，并用稀释在TBSTM中的指定抗体进行检测。对膜进行磷酸化蛋白探测，然后剥离以探测总蛋白。为了重新制作，在室温下将膜在剥离缓冲液（25 mmol/L甘氨酸，pH 2.0，1%SDS）中剥离30至60分钟，用TBST冲洗两次10分钟，并探测其他蛋白质。抗原-抗体复合物使用化学发光底物进行可视化，并使用Image-Quant LAS4000迷你数字成像系统进行检测。

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基于细胞的磷酸化ERK和磷酸化EGFR检测方法[1]
使用基于TR-FRET的方法测量细胞磷酸化ERK和磷酸化EGFR。在96孔板上以每孔3×104接种细胞，并放置16小时。然后用100μL不含血清的DMEM代替生长培养基。然后用化合物的10点滴定法处理细胞。化合物处理1小时后，向每个孔中加入50μL裂解缓冲液。然后在室温下摇动培养板30分钟。将96孔板的每个孔中总共16μL的细胞裂解物转移到384孔的小体积白色板上。在室温下，将来自每个孔的裂解物与2μL Eu3+或Tb3+隐窝（供体）标记的抗ERK或抗EGFR抗体和2μL D2（受体）标记的反磷酸化ERK或反磷酸化EGFR抗体一起孵育2小时。使用PHERAstar FS阅读器测量FRET信号。

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增殖试验[1]
使用CellTiter-Glo发光细胞活力测定法测定化合物在一组黑色素瘤、结肠癌、乳腺癌和肺癌细胞中的生长抑制活性。针对每种细胞系优化96孔板的每孔接种的细胞数量，以确保3天处理期内的对数生长（补充表S2）。让细胞附着16小时，然后用10点稀释系列处理两次。在暴露于该化合物3天后，加入与每个孔中存在的细胞培养基体积相等的CellTiter-Glo试剂。将混合物在轨道振荡器上混合2分钟以允许细胞裂解，然后在室温下孵育10分钟以允许发光信号的产生和稳定。

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 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/]

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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/

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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.

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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]

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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

89.13

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.23 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.23 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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