BRafV600E (IC50 = 0.6 nM); CRAF (IC50 = 5 nM); B-Raf (IC50 = 5.2 nM)

在 Raf 激酶选择性方面，达拉非尼对 B-Raf 的偏好程度超过 91% 的其他测试激酶的 400 倍。 Dabrafenib 通过最初将特异性编码 B-RafV600E 突变的癌细胞的细胞周期阻滞在 G1 期，减少 ERK 磷酸化并抑制细胞增殖。 [1]

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BRAF抑制剂GSK2118436（Dabrafenib）和PLX4032（vemurafenib）的临床试验结果显示了令人鼓舞的反应率；然而，响应的持续时间是有限的。为了确定对GSK2118436获得性耐药性的决定因素和克服耐药性的策略，我们从A375 BRAFV600E和YUSIT1 BRAFV600K黑色素瘤细胞系中分离出GSK2118443耐药克隆。这些克隆对变构丝裂原激活蛋白/细胞外信号调节激酶（MEK）抑制剂GSK1120212（曲美替尼）的敏感性也降低。这些克隆的遗传特征表明，在BRAFV600E背景中存在MEK1的框内缺失（MEK1K59del）或NRAS突变（NRASQ61K和/或NRASA146T），有和没有MEK1P387S，BRAFV600K背景中有NRASQ618K。用短发夹RNA稳定敲除NRAS部分恢复了突变NRAS克隆中GSK2118436的敏感性，而A375亲本细胞中NRASQ61K或NRASA146T的表达降低了对Dabrafenib/GSK2118436敏感性。同样，表达MEK1K59del而非MEK1P387S会降低A375细胞对GSK2118436的敏感性。GSK2118436和GSK1120212的组合有效地抑制了抗性克隆中的细胞生长，降低了ERK磷酸化，降低了细胞周期蛋白D1蛋白，增加了p27kip1蛋白。此外，GSK2118436或GSK1120212与磷酸肌醇3-激酶/mTOR抑制剂GSK2126458的组合增强了这些克隆中的细胞生长抑制作用，降低了S6核糖体蛋白磷酸化。我们的结果表明，NRAS和/或MEK突变在体外导致BRAF抑制剂耐药性，GSK2118436和GSK1120212的组合克服了这种耐药性。此外，这些抗性克隆对GSK2126458与GSK2118436或GSK1120212的组合有反应。临床试验正在进行或计划测试这些组合。[2]

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GSK2118436/Dabrafenib（12）在酶和细胞机制测定以及B-RafV600E驱动的黑色素瘤系SKMEL28和A375P F11（IC50=3和8 nM）和结直肠癌系Colo205（IC50=7 nM）的细胞增殖测定中显示出令人信服的抑制活性。令人欣慰的是，GSK2118436在体外对具有野生型B-Raf的细胞（HFF IC50=3.0μM）和不携带激活性B-RafV600E突变的肿瘤细胞的影响很小[3]。

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GSK2118436/Dabrafenib针对一组61种激酶进行了筛选，这些激酶代表了激酶组的广泛覆盖范围，被发现是B-RafV600E、野生型B-Raf和c-Raf的强效生化抑制剂，显示出亚纳米或纳摩尔的效力（见支持信息）。重要的是，还发现GSK2118436具有高度选择性，与筛选的大多数激酶相比，对B-RafV600E的选择性超过500倍。11在面板中观察到单个激酶Alk5的显著活性（<100倍选择性）。通过测量HepG2细胞中SMAD2/3的下游磷酸化水平，进一步研究了Alk5酶活性对细胞信号传导的影响。12在细胞环境中，与抑制ERK磷酸化（IC50=4 nM）相比，GSK2118436在抑制SMAD2/3磷酸化方面的效果明显较差（IC50=3.7μM）。这些数据强调了这种独特有效和选择性的B-RafV600E抑制剂所实现的显著选择性。[3]

Dabrafenib（口服）可抑制在免疫缺陷小鼠皮下生长的 B-RafV600E 突变结肠癌 (Colo205) 和黑色素瘤 (A375P) 人类肿瘤异种移植物的发育。 [1]

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还对GSK2118436/Dabrafenib进行了体内研究，发现它能显著降低携带B-RafV600E人类黑色素瘤的小鼠的肿瘤生长。在该模型中，携带A375P F11（B-RafV600E）肿瘤的CD1-nu/nu小鼠口服GSK2118436，剂量为0.1、1、10和100mg/kg，每日一次，持续14天（图3）。观察到肿瘤生长的剂量成比例减少。还测量了体重，在任何测试剂量下都没有观察到显著变化。值得注意的是，在100mg/kg组中，50%的受试动物肿瘤完全消退。[3]

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此外，GSK2118436/Dabrafenib在单次口服剂量后以剂量依赖的方式降低了A375P F11（B-RafV600E）肿瘤组织中pERK的水平。在给药后2、6和24小时收集肿瘤，测量每个肿瘤中的pERK水平，并将其归一化为存在的总ERK（tERK）。使用MEK抑制剂PD0325901作为对照。图4显示了pERK/tERK水平与载体处理动物的水平的比较，以及两项单独研究的药代动力学数据。给药后2小时和6小时，pERK/tERK水平显著降低，在这项单剂量研究中，当剂量≥3 mg/kg时，药效作用显著（抑制率>50%），当剂量≤30 mg/kg时，24小时后恢复到未经治疗的水平。研究过程中血液和肿瘤中测得的药物浓度与观察到的药效作用相关。这些数据表明，在测试条件下，单次口服剂量≥3 mg/kg的Dabrafenib/GSK2118436可以在至少6小时内保持≥50%的B-Raf抑制。[3]

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Raf激酶通过细胞外信号调节激酶1/2（ERK1/2）发出信号，以驱动细胞分裂。由于BRAF的激活突变（B-Raf原原癌基因，丝氨酸/苏氨酸激酶）是高度致癌的，因此已开发出用于癌症的BRAF抑制剂，包括Dabrafenib。用于癌症的ERK1/2信号抑制剂在一些患者中具有心脏毒性，这就提出了达巴芬尼是否具有心脏毒性的问题。在心脏中，ERK1/2信号传导不仅促进心肌细胞肥大，具有心脏保护作用，还促进纤维化。我们的假设是，ERK1/2信号传导在非应激心脏中不是必需的，但在心脏重塑中是必需的。因此，dabrafenib可能会在高血压等情况下影响心脏。在心肌细胞、心脏成纤维细胞和灌注大鼠心脏的实验中，Dabrafenib抑制ERK1/2信号传导。我们评估了Dabrafenib（3 mg/kg/d）对雄性C57BL/6J小鼠心脏的体内影响。Dabrafenib单独使用对心脏功能/尺寸（通过超声心动图评估）或心脏结构没有明显影响。在用0.8mg/kg/d血管紧张素II（AngII）治疗以诱导高血压的小鼠中，dabrafenib在急性（长达7d）和慢性（28d）情况下抑制ERK1/2信号传导并抑制心肌肥大，从而保持射血分数。在细胞水平上，dabrafenib抑制了AngII诱导的心肌细胞肥大，降低了肥大基因标志物的表达，几乎完全消除了间质和血管周围区域心脏纤维化的增加。Dabrafenib没有明显的心脏毒性。此外，它还能抑制由AngII诱导的高血压引起的适应不良肥大。因此，Raf是高血压心脏病的潜在治疗靶点，而针对癌症开发的药物如Dabrafenib可用于此目的[4]。

生化和细胞分析。[3]
B-Raf生化酶和细胞测定的详细信息已在参考文献6中报道。Dabrafenib对61种蛋白激酶的抑制作用在葛兰素史克公司进行了表征。一般来说，这些测定被配置为使IC50值接近Dabrafenib对每种酶的内在结合常数（Ki或Kd），因此可以比较对这些激酶的选择性。A375P-F11测定：通过有限稀释将A375P细胞铺在96孔板中，收获单细胞衍生的克隆，并测试其对B-Raf抑制剂的敏感性。F11克隆被选为未来研究的对象，命名为A375P-F11。测定抗TGF-β活性的细胞pSmad试验：在HepG2细胞中通过机械试验测试化合物的活性。将细胞以500000个细胞/孔的密度接种在12孔板中，并在37℃/5%CO2下粘附过夜。去除培养基（BME+10%血清），在37℃/5%CO2下将无血清培养基中的化合物加入细胞中45分钟。用1ng/ml TGF-β刺激细胞60分钟。将细胞在缓冲液（25 mM Tris-HCl ph:7.5，2 mM EDTA，2 mM EGTA，1%Triton X-100，0.1%SDS，50 mMβ-甘油磷酸钠，2 mM原钒酸钠，12.5 mM焦磷酸钠，蛋白酶和磷酸酶抑制剂混合物）中裂解30分钟，刮除，收集，离心澄清，并在LDS/还原试剂中制备蛋白质印迹。样品在4-12%的Bis-Tris凝胶上分离，转移到PVDF上，并使用来自Cell Signaling的抗体检测总和磷酸化-Smad2。凝胶使用奥德赛印迹扫描仪成像，并使用Licor软件定量。测定磷酸：总Smad2比值，IC50定义为使磷酸：总比值降低50%的化合物浓度。

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代谢物鉴定。[3]
犬肝微粒体代谢物鉴定研究：犬肝微粒体购自Xenotech。将1.5 mL Eppendorf管中含有50 mM磷酸钾缓冲液、肝微粒体部分（1.0 mg/mL蛋白质）和10µM研究化合物的孵育混合物（800µL）预热至37°C。辅因子在37°C下预孵育5分钟。在0分钟的时间点，取出孵育混合物（200µL）和辅因子溶液（50µL）的等分试样，并用终止溶液[250µL，（80/20/1，乙腈/乙醇/乙酸，v/v/v）]粉碎。通过加入150µL NADPH生成系统[2.2 mM NADP、28 mM葡萄糖-6-磷酸和葡萄糖-6-磷酸脱氢酶（6单位/mL）以及4.0 mM UDPGA的2%碳酸氢钠溶液]开始反应，并在37°C下孵育样品（最终体积750µL）。没有使用含有适量甲醇的空白培养基代替肝微粒体化合物进行药物对照培养。孵育30分钟后，通过加入一体积的终止溶液终止反应。样品以34000rpm离心5分钟，将50µL上清液注入LC/MS进行代谢物鉴定。犬肝细胞代谢产物鉴定研究：犬肝细胞（Db157）购自CellzDirect。将由William培养基E（pH=7.4）、肝细胞悬浮液（70万个细胞/mL）和10µM研究化合物组成的总体积为600µL的孵育混合物放入12孔培养板的单孔中，在37°C下孵育4小时。使用含有适量甲醇的空白培养基代替对肝细胞的积极治疗，没有同时进行药物对照培养。孵育后，从每个孔的底部刮下细胞，并与600µL的终止溶液混合。然后将混合物储存在-80°C的冰箱中。样品以34000rpm离心5分钟，将50µL上清液注入LC/MS进行代谢物鉴定。

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结合模式：[3]
GSK2118436/Dabrafenib是B-Raf11的ATP竞争性抑制剂，根据GSK2118436与最近报道的B-RafV600E晶体结构对接的模型，推测其与激酶的非活性样构象结合（图S1），该晶体结构与其他小分子ATP结合位点抑制剂结合。1在该模型中，αC螺旋相对于活性样构象“移位”，保守的赖氨酸和谷氨酸之间的盐桥被打破。虽然Phe595不处于“DFG out”构象，但它会旋转形成类似于拉帕替尼/EGFR共结构中观察到的口袋底部。2嘧啶N1与酶ATP结合口袋中的Cys532形成经典的铰链相互作用。叔丁基和噻唑核在P-环下方结合，只留下相对较小的一部分抑制剂作为溶剂暴露。预计GSK2118436的芳基磺酰胺头基会结合到亲脂性后囊中，使磺酰胺与Asp594和Phe595的骨架NH形成两个氢键。磺酰胺NH以脱质子化形式描述，留下氮作为氢键受体参与。在R2处带有氟取代的化合物中观察到的效力的显著提高被认为是调节磺酰胺NH的pKa的结果，这可以在细胞pH值下增加12的电离，并为头基结合提供更有利的构象。相反，在R1处含有氟取代的化合物（化合物4和9）可能会诱导结合袋中苯磺酰胺部分的不利构象，这可能是用这些类似物观察到的靶亲和力降低的原因。含苯胺尾部分子的类似模型（如1）表明，尾部基团中的芳香环主要位于B-Raf结合口袋内，而吗啉代环延伸到溶剂暴露区域。比较1和2，在将尾部完全截断为“裸”氨基嘧啶并损失13个重原子后，观察到效力下降了10倍。尽管效力略有下降，但与1相比，2实际上表现出更高的配体效率（2的LE=0.30比1的LE=0.25），因为2中所有剩余的重原子都完全包含在结合袋中。

对于长期增殖测定，将细胞置于含有 10% FBS 的 RMPI-1640 中 12 天，并用单一化合物或化合物组合进行处理。该测定涉及至少一种化合物治疗替代。 12 天后，使用 50% 乙醇中的 0.5% 亚甲蓝对细胞进行染色。使用平板扫描仪拍摄照片。

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A375PF11细胞（以下简称A375）暴露于浓度逐渐增加的Dabrafenib和GSK2118436中，并保持在1.2或1.6μmol/L的终浓度。同样，YUSIT1细胞暴露于0.1μmol/L的GSK2118443中。通过限制稀释从这些群体中分离出单细胞克隆。通过SNP芯片分析确定，A375克隆与亲本A375细胞有95%的相似性。通过外显子组测序确定，具有代表性的抗性YUSIT1克隆共享100%的亲本YUSIT1 SNP。在实验之前，所有细胞在含有10%FBS的RPMI-1640培养基中生长至少一代，不含GSK2118436。[2]

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Affymetrix表达分析： 选择16R6-4与A375进行比较，分别用Dabrafenib/GSK2118436和GSK1120212单独和联合处理24小时。数据分析按照补充方法中的描述进行。微阵列数据保存在NCBI的基因表达综合数据库（GEO，http://www.ncbi.nlm.nih.gov/geo/)可通过GEO系列登录号GSE35230访问。[2]

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细胞生长抑制和凋亡测定： 如前所述，使用CellTiter Glo 用化合物或化合物组合处理3天后，估计细胞生长的抑制作用。在用化合物进行类似处理24小时后，通过Caspase-Glo 3/7测定法 测定Caspase-3/7活性。在3天生长试验之前，用补充方法中所述的短干扰RNA（siRNA）、短发夹RNA（shRNA）或表达构建体转染或转导细胞。 对于长期增殖试验，细胞被铺板并用含有10%FBS的RMPI-1640中的化合物或化合物组合处理12天。在试验过程中，至少更换了一次化合物处理。12天后，用50%乙醇中的0.5%亚甲基蓝对细胞进行染色。使用平板扫描仪拍摄图像[2]。

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如前所述，从2至4天的Sprague-Dawley大鼠制备和培养新生大鼠心肌细胞。人心脏成纤维细胞在成纤维细胞生长培养基-3中生长。在实验前一天接种成纤维细胞（密度为24小时后达到90%融合），并在含有0.1%（v/v）胎牛血清和100U/ml青霉素和链霉素的M199培养基中同步过夜。细胞暴露于 Dabrafenib中，并在收获用于免疫印迹的时间内暴露[4]。

The 26 10-week-old, time-mated, virus-antibody-free SD (Crl:CD[SD]) female rats that were chosen as the test system gave birth to the rat pups. From Day 20 to Day 23 postpartum, mated females are monitored for spontaneous deliveries (the day parturition is complete is designated PND 0). When parturition is complete, on PNDs 3 and 6, litter examinations are carried out. These examinations include external morphologic examinations, gender determination, and individual pup weights. Clinical signs and body weights are used to select parturient dams and their litters for the study, and chosen dams and their litters are then randomly assigned to study groups based on clinical observations and PND 3 litter mean body weights. On PND 3 or 4, litters are reduced to four or five males and females, with only a small amount of fostering required to achieve the desired sex ratio. This helps to preserve natural litter sizes as much as possible. Records of the pups raised by the original and foster dams are kept. Paw tattoos are used to identify each puppy. Nonlittermates are placed in subsets to the greatest extent possible. Dabrafenibis administered to young male and female rats by oral gavage at a dose volume of 5 ml/kg, based on daily body weight, in a suspension of vehicle, 0.5% hydroxypropylmethylcellulose K15M, and 0.1% (v/v) Tween80 in purified water.

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A375P F11 Melanoma Xenograft Studies. [3]
Cells were implanted in nude mice and grown as tumor xenografts. Dosing began when tumors achieved ~150- 200mm3 volume. Dabrafenib/GSK2118436 was administered by oral gavage at a dose volume of 0.2 mL/20 gram body weight in 0.5% hydroxypropylmethylcellulose and 0.2% Tween-80 in distilled water pH 8.0. Dosing was daily for duration stipulated. Results are reported as mean tumor volume for n=7-8 mice/group. Tumors were measured twice weekly with Vernier calipers, and tumor volume was estimated from two-dimensional measurements using a prolate ellipsoid equation (Tumor volume mm3 = (length x width2 ) x 0.5). Complete tumor regression was defined as a >93% decrease in an individual tumor volume for at least 1 week.

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Pharmacokinetic (PK) Analysis. [3]
Blood was drawn and hemolyzed immediately with an equal volume of water. Concentrations of compound in tumor were determined on polytron homogenized tissues in 4 volumes of water per volume of frozen tissue. Aliquots of the homogenized tumor and blood were flash frozen and subsequently evaluated for compound concentration by HPLC/MS/MS analysis.

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Pharmacodyamic measurement of pERK levels in tissues. [3]
Tissues were harvested and homogenized using Medimachine with 1 mL of lysis buffer (25 mM Tris-HCl (pH 7.5), 2 mM EDTA (pH 8.0), 2 mM EGTA (pH 8.0), 1% Triton X-100, 0.1% SDS, 50 mM sodium glycerol phosphate, 2 mM Na3VO4, 4 mM Na-pyrophosphate, 2x phosphatase inhibitor cocktail), and kept on ice. Following homogenization of all samples, homogenates were centrifuged at 14,000 rpm for 15 min at 4°C and flash frozen for later analysis. All samples were analyzed with a duplex ELISA measuring total ERK1/2 and phospho-ERK1/2 according to manufacturer’s instructions. Plates were read on MSD.SI6000.

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In vivo mouse studies [4]
Wild-type male (7 wk) C57Bl/6J mice were allowed to acclimatize for 2 weeks before experimentation. Mice were randomly allocated to each treatment group; body weights are provided in Supplementary Table S1. Drug delivery used Alzet osmotic pumps (models 1007D or 1004), filled according to the manufacturer’s instructions. Mice received minipumps for delivery of 0.8 mg/kg/d angiotensin II (AngII) or vehicle (acidified PBS) without/with DMSO/PEG mix [50% (v/v) dimethyl sulphoxide (DMSO), 20% (v/v) polyethylene glycol 400, 5% (v/v) propylene glycol, 0.5% (v/v) Tween 80] or 3 mg/kg/d Dabrafenib dissolved in DMSO/PEG mix. Separate minipumps were used for AngII and Dabrafenib delivery. Minipumps were incubated overnight in sterile PBS (37°C), then implanted subcutaneously under continuous inhalation anaesthesia using isoflurane (induction at 5%, maintenance at 2–2.5%) mixed with 2 l/min O2, as previously described

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Histology and assessment of myocyte size and fibrosis [4]
Histological staining and analysis were performed as previously described, assessing general morphology by haematoxylin and eosin (H&E) and fibrosis by Masson’s trichrome and picrosirius red (PSR). Sections for the study of the effects of Dabrafenib on AngII-induced cardiac pathology over 28 d were prepared and stained by HistologiX Limited. Analysis was performed by independent assessors blinded to treatment groups.

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Adult rat heart perfusions [4]
Adult male (300–350 g) Sprague-Dawley rats were used for heart perfusions. Hearts were prepared and perfused in the Langendorff mode as described. Hearts were perfused for 15 min with Krebs-Henseleit bicarbonate-buffered saline (25 mM NaHCO3, 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, pH 7.4, containing 10 mM glucose and equilibrated with 95% O2/5% CO2) without or with Dabrafenib (5 µM) or trametinib (1 µM). Dabrafenib and trametinib were from Selleck Chemicals. Perfusions were continued for 10 min without/with addition of human FGF2 (0.5 µg/ml; Cell Guidance Systems Ltd., U.K.). Hearts were ‘freeze-clamped’ between aluminium tongs cooled in liquid nitrogen and pulverized under liquid N2. Heart powders were stored at −80°C.

Absorption, Distribution and Excretion
After oral administration, the median time to achieve peak plasma concentration (Tmax) is 2 hours. Mean absolute bioavailability of oral dabrafenib is 95%. Following a single dose, dabrafenib exposure (Cmax and AUC) increased in a dose-proportional manner across the dose range of 12 mg to 300 mg, but the increase was less than dose-proportional after repeat twice-daily dosing. After repeated twice-daily dosing of 150 mg, the mean accumulation ratio was 0.73, and the inter-subject variability (CV%) of AUC at steady-state was 38%.
Fecal excretion is the major route of elimination accounting for 71% of radioactive dose while urinary excretion accounted for 23% of total radioactivity as metabolites only.
The apparent volume of distribution (Vc/F) is 70.3 L.Distribution to the brain is restricted because dabrafenib is a substrate and undergoes efflux by P-glycoprotein and breast cancer resistance protein.
The clearance of dabrafenib is 17.0 L/h after single dosing and 34.4 L/h after 2 weeks of twice-daily dosing.

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Metabolism / Metabolites
The metabolism of dabrafenib is primarily mediated by CYP2C8 and CYP3A4 to form hydroxy-dabrafenib. Hydroxy-dabrafenib is further oxidized via CYP3A4 to form carboxy-dabrafenib and subsequently excreted in bile and urine. Carboxy-dabrafenib is decarboxylated to form desmethyl-dabrafenib; desmethyl-dabrafenib may be reabsorbed from the gut. Desmethyl-dabrafenib is further metabolized by CYP3A4 to oxidative metabolites.

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Biological Half-Life
The mean terminal half-life of dabrafenib is 8 hours after oral administration. Hydroxy-dabrafenib's terminal half-life (10 hours) parallels that of dabrafenib while the carboxy- and desmethyl-dabrafenib metabolites exhibit longer half-lives (21 to 22 hours).

Hepatotoxicity
Elevations in serum ALT levels were reported in 11% of patients treated with dabrafenib alone, but all elevations were above 5 times ULN. When dabrafenib was given in combination with trametinib, serum ALT elevations occurred in 35% to 42% of patients and were above 5 times ULN in 4%. Similarly, serum alkaline phosphatase elevations occurred in 26% of patients given dabrafenib alone, but in 60% to 67% given dabrafenib and trametinib. These abnormalities were largely asymptomatic and fully reversible. There were no instances of clinically apparent acute liver injury or hepatic failure reported in prelicensure studies of dabrafenib and, since its approval and more wide spread use, there have been no published reports of dabrafenib hepatotoxicity.
Likelihood score: E* (unproven but suspected cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of dabrafenib during breastfeeding. Because dabrafenib is more than 99% bound to plasma proteins, the amount in milk is likely to be low. The manufacturer recommends that breastfeeding be discontinued during dabrafenib therapy and for 2 weeks after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Dabrafenib is 99.7% bound to human plasma proteins.

[1]. 2009, EORTC International Conference. Abst B88.

[2]. Mol Cancer Ther. 2012 Apr;11(4):909-20.

[3]. ACS Med Chem Lett. 2013 Feb 7;4(3):358-62.

[4]. Clin Sci (Lond). 2021 Jul 30;135(14):1631-1647.

Pharmacodynamics
Dabrafenib is a kinase inhibitor that is mainly used to target BRAF V600E mutation in multiple types of cancer. Although dabrafenib and [trametinib] both inhibit the RAS/RAF/MEK/ERK pathway, they inhibit different effectors of the pathway, thus increasing response rate and mitigating resistance without cumulative toxicity. The melanoma approval for use with [trametinib] is based on results from COMBI-AD, a Phase III study of 870 patients with Stage III BRAF V600E/K mutation-positive melanoma treated with dabrafenib + trametinib after complete surgical resection. Patients received doses of dabrafenib (150 mg BID) + trametinib (2 mg QD) combination (n = 438) or matching placebos (n = 432). After a median follow-up of 2.8 years, the primary endpoint of relapse-free survival (RFS) was met. In the case of thyroid cancer, Dabrafenib plus Trametinib is the first regimen demonstrated to have potent clinical activity in BRAF V600E–mutated anaplastic thyroid cancer and is well tolerated. These findings represent a meaningful therapeutic advance for this orphan disease.

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Dabrafenib is an organofluorine compound and antineoplastic agent, used as its mesylate salt in treatment of metastatic melanoma. It has a role as an antineoplastic agent, a B-Raf inhibitor and an anticoronaviral agent. It is a sulfonamide, an organofluorine compound, a member of 1,3-thiazoles and an aminopyrimidine.
Dabrafenib mesylate (Tafinlar) is a reversible ATP-competitive kinase inhibitor and targets the MAPK pathway. It was approved on May 29, 2013, for the treatment of melanoma with V600E or V6000K mutation. It was also used for metastatic non-small cell lung cancer with the same mutation. In May 2018, Tafinlar (dabrafenib), in combination with Mekinist ([DB08911]), was approved to treat anaplastic thyroid cancer caused by an abnormal BRAF V600E gene.
Dabrafenib is a Kinase Inhibitor. The mechanism of action of dabrafenib is as a Protein Kinase Inhibitor, and Cytochrome P450 3A4 Inducer, and Cytochrome P450 2B6 Inducer, and Cytochrome P450 2C8 Inducer, and Cytochrome P450 2C9 Inducer, and Cytochrome P450 2C19 Inducer, and Organic Anion Transporting Polypeptide 1B1 Inhibitor, and Organic Anion Transporting Polypeptide 1B3 Inhibitor, and Organic Anion Transporter 1 Inhibitor, and Organic Anion Transporter 3 Inhibitor, and Breast Cancer Resistance Protein Inhibitor.

Dabrafenib is a selective inhibitor of mutated forms of BRAF kinase and is used alone or in combination with trametinib in the treatment of advanced malignant melanoma. Dabrafenib therapy is associated with transient elevations in serum aminotransferase during therapy, but has not been linked to instances of clinically apparent acute liver injury.
Dabrafenib is an orally bioavailable inhibitor of B-raf (BRAF) protein with potential antineoplastic activity. Dabrafenib selectively binds to and inhibits the activity of B-raf, which may inhibit the proliferation of tumor cells which contain a mutated BRAF gene. B-raf belongs to the raf/mil family of serine/threonine protein kinases and plays a role in regulating the MAP kinase/ERKs signaling pathway, which may be constitutively activated due to BRAF gene mutations.
DABRAFENIB is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 2013 and has 7 approved and 19 investigational indications.

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Activation of the Ras‐Raf‐MEK‐ERK pathway has been implicated in a large range of human cancers. Growth factor receptor stimulation by extracellular ligands activates Ras, which then sets in motion a signal transduction cascade through the Raf, MEK and ERK serine/threonine kinases. Mutation of the B‐Raf kinase constitutively activates MAPK signalling, thus bypassing the need for upstream stimuli. This has been genetically associated with several human cancers, especially occurrence of the B‐RafV600E mutant and its high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. The ability to selectively and potently inhibit B‐Raf should provide a potential therapy for patients with mutant B‐Raf tumors, for which addictive dependency on this pathway is observed. We have identified a novel, potent, and selective Raf kinase inhibitor that is capable of inhibiting the kinase activity of wild‐type B‐Raf, B‐RafV600E and c‐Raf with IC50 values of 3.2, 0.8, and 5.0 nM, respectively. Kinase panel screening for over 270 kinases has indicated that this inhibitor is selective for Raf kinase, with ∼400 fold selectivity towards B‐Raf over 91% of the other kinases tested. Specific cellular inhibition of B‐RafV600E kinase by this inhibitor leads to decreased ERK phosphorylation and inhibition of cell proliferation by an initial arrest in the G1 phase of the cell cycle, followed by cell death. This inhibition is selective for cancer cells that specifically encode the mutation for B‐RafV600E. Oral compound administration inhibits the growth of B‐RafV600E mutant melanoma (A375P) and colon cancer (Colo205) human tumor xenografts, growing subcutaneously in immuno‐compromised mice. This cell‐specific B‐RafV600E inhibitor is currently being evaluated in a human Phase I clinical trial. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B88.[1]

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Hyperactive signaling of the MAP kinase pathway resulting from the constitutively active B-RafV600E mutated enzyme has been observed in a number of human tumors, including melanomas. Herein we report the discovery and biological evaluation of Dabrafenib/GSK2118436, a selective inhibitor of Raf kinases with potent in vitro activity in oncogenic B-Raf-driven melanoma and colorectal carcinoma cells and robust in vivo antitumor and pharmacodynamic activity in mouse models of B-RafV600E human melanoma. GSK2118436 was identified as a development candidate, and early clinical results have shown significant activity in patients with B-Raf mutant melanoma. [3]

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In summary, lead molecule 1, with high potency and poor pharmacokinetic properties in higher species, was rapidly evolved into clinical development candidate Dabrafenib/GSK2118436 (12). Identification of the key B-RafV600E potency contributors (sulfonamide head, R2 fluorination, and tert-butyl thiazole core) through detailed and iterative SAR studies allowed for the elimination of nonessential portions of the lead structure and high selectivity to be achieved across the kinome. With high enzyme and cellular potency engineered into the original lead, pharmacokinetic issues were addressed by combining multiple changes in the template, resulting in the improved multispecies PK properties observed for 12. With high rodent exposure realized in GSK2118436, we were able to demonstrate target-related pharmacology in two in vivo models, gaining a high level of confidence in the progression of this molecule into clinical studies. Additionally, vast improvements in higher-species PK allowed for the achievement of exposures necessary to fully explore the safety of the molecule preclinically. Finally, a >20% decrease in molecular weight compared to that of 1 resulted in improved physicochemical properties, expediting the identification of a salt version (mesylate) and form suitable for early clinical studies.
The efforts described herein ultimately led to the discovery of GSK2118436 (Dabrafenib), which is currently in advanced clinical studies in B-Raf mutant tumors. GSK2118436 has shown remarkable efficacy in melanoma patients with activating B-Raf mutations, including patients with brain metastases, and has further shown enhanced clinical activity in combination with the MEK inhibitor, trametinib. Details of these studies will be reported in due course. [3]

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The final considerations for our work are the potential implications of using drugs such as Dabrafenib for cardiac diseases. Dabrafenib itself does not appear to be cardiotoxic and is generally well-tolerated. There are side effects in cancer patients which are generally managed by dose reduction. The most severe effect is probably an increase in cutaneous squamous cell carcinoma (∼12% of patients. Thus, if dabrafenib were to be used as a therapy for hypertensive heart disease, it may be important to consider dosage monitoring and whether patients have a predisposition for other diseases such as cancer (in the context, perhaps, of ‘onco-cardiology’). Nevertheless, reducing cardiac fibrosis with a drug such as dabrafenib may be such a powerful tool in treating cardiac diseases that the benefits could outweigh these costs.[4]

C23H20F3N5O2S2

519.56

519.101

C, 53.17; H, 3.88; F, 10.97; N, 13.48; O, 6.16; S, 12.34

1195765-45-7

Dabrafenib Mesylate;1195768-06-9;Dabrafenib-d9;1423119-98-5

44462760

White to off-white solid powder

1.4±0.1 g/cm3

653.7±65.0 °C at 760 mmHg

349.2±34.3 °C

0.0±2.0 mmHg at 25°C

1.626

3.54

148.21

2

11

6

35

817

0

S1C(C2C([H])=C([H])N=C(N([H])[H])N=2)=C(C2C([H])=C([H])C([H])=C(C=2F)N([H])S(C2C(=C([H])C([H])=C([H])C=2F)F)(=O)=O)N=C1C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H]

BFSMGDJOXZAERB-UHFFFAOYSA-N

InChI=1S/C23H20F3N5O2S2/c1-23(2,3)21-30-18(19(34-21)16-10-11-28-22(27)29-16)12-6-4-9-15(17(12)26)31-35(32,33)20-13(24)7-5-8-14(20)25/h4-11,31H,1-3H3,(H2,27,28,29)

N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1,3-thiazol-4-yl]-2-fluorophenyl]-2,6-difluorobenzenesulfonamide

GSK2118436A; GSK-2118436B; GSK 2118436A; Dabrafenib; 1195765-45-7; GSK2118436-A; UNII-QGP4HA4G1B; QGP4HA4G1B; GSK-2118436-A; CHEBI:75045; Benzenesulfonamide, N-[3-[5-(2-amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-4-thiazolyl]-2-fluorophenyl]-2,6-difluoro-; GSK2118436B (Dabrafenib Mesylate ); GSK-2118436A; GSK 2118436B. Trade name: Tafinlar

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: 5 mg/mL (9.62 mM) in 30% PEG400 0.5% Tween80 + 5% Propanediol 64.5%H2O (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。

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配方 2 中的溶解度: 2.5 mg/mL (4.81 mM) in 0.5%HPMC (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (4.81 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。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 4 中的溶解度: ≥ 2.5 mg/mL (4.81 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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100μL 25.0mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 5 中的溶解度: ≥ 2.5 mg/mL (4.81 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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配方 6 中的溶解度: 2.5 mg/mL (4.81 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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 7 中的溶解度: ≥ 2.5 mg/mL (4.81 mM)(饱和度未知) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加),澄清溶液。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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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));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。