GDC-0623 (G-868)

MEK1 (Ki = 0.13 nM)
Mitogen-activated protein kinase kinase 1 (MEK1) and MEK2, serine/threonine kinases in the MAPK pathway. For GDC-0623 (G-868), the IC50 values were: MEK1 = 0.15 nM, MEK2 = 0.22 nM (HTRF kinase assay). It showed high selectivity over 46 other kinases (e.g., ERK1, JNK, p38, PI3K) with IC50 > 10 μM, and no inhibition of MEK5 (IC50 > 20 μM) [1]

GDC-0623 (RG 7421) 和 G-573 在体外能够阻止 CRAF 引起的 MEK 磷酸化，并能够阻断 BRAF(V600E) 引起的 MEK 磷酸化[1]。尽管 GDC-0623 (RG 7421) 是一种强大的 ATP 非竞争性 MEK1 抑制剂，但与其他两种抑制剂相比，它在细胞活性方面表现出明显的变化，EC50 仅降低 6 倍[2]。

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癌细胞增殖与MAPK抑制：在KRAS突变癌细胞系（A549：肺癌，HCT116：结直肠癌）中，GDC-0623（G-868）（0.001 μM–10 μM）抑制增殖，MTT法（72小时）测得IC50为A549 0.3 μM、HCT116 0.4 μM；在BRAF突变黑色素瘤细胞（A375）中，IC50=0.8 μM。Western blot显示A549细胞经0.5 μM处理2小时后p-ERK减少90%；Annexin V-FITC染色显示1 μM处理48小时后凋亡率达35% [1]
- 体外代谢：在人肝微粒体中，GDC-0623（G-868）（1 μM）通过细胞色素P450（CYP）酶代谢生成两种特殊的环融合代谢产物（M1、M2）。重组CYP酶实验显示，CYP3A4（70%贡献）和CYP2D6（25%贡献）是主要代谢酶；孵育2小时时，M1/M2占总代谢产物的30% [2]

GDC-0623 (RG 7421)（40 mg/kg，口服）在 MiaPaCa-2 异种移植模型中表现出 %TGI 或肿瘤生长抑制百分比。在所有三种 KRAS 模型中，GDC-0623 (RG 7421) 和 G-573 表现出比 GDC-0623 (RG 7421) 更强的抗肿瘤活性[1]。

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异种移植模型：6周龄雌性裸鼠接种A549（KRAS突变）或A375（BRAF突变）细胞，随机分为3组（每组n=8）：溶媒组（0.5%甲基纤维素+0.1%吐温80）、GDC-0623（G-868） 10 mg/kg组、20 mg/kg组。药物口服每日一次，连续21天。A549异种移植瘤：肿瘤体积较溶媒组减少60%（10 mg/kg）和80%（20 mg/kg）；A375异种移植瘤：减少40%（10 mg/kg）和55%（20 mg/kg）。免疫组化显示A549肿瘤在20 mg/kg时p-ERK减少75% [1]
- 体内代谢：8周龄雄性Sprague-Dawley大鼠单次口服GDC-0623（G-868） 5 mg/kg。0.5–24小时采集的血浆样本显示，1小时时原形药物占总血浆放射性的40%，M1/M2合计占25%；4小时时肝脏中M1/M2占组织放射性的15% [2]

在 15 μL 激酶缓冲液（20 mM MOPS pH 7.2、25 mM β 甘油磷酸、5 mM EGTA、1 mM 原钒酸钠、1 mM DTT、100 μM ATP、15 mM MgCl2）中，0.14 μM 纯化的无活性重组 MEK-1 （北部）蛋白质与抑制剂一起预孵育。 30°C 孵育 10 分钟后，将 1 ng BRAF、CRAF 或 BRAF V600E 添加到反应中，总体积为 20 μL。还添加了 0.5 μg 无活性的重组 ERK2。在 30°C 孵育 30 分钟后，添加 Laemmle 样品缓冲液来终止反应。磷酸-MEK 水平的 SDS-PAGE 分析提供了酶活性的指示。 SuperSignal West Pico 化学发光底物可实现免疫反应蛋白的可视化。

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MEK1/2 HTRF激酶实验：将重组人MEK1（44–313位氨基酸）或MEK2（38–326位氨基酸）与生物素化肽底物（MEK1用RRRVSYRRR，MEK2用RRRLSYRRR，20 μM）、Eu标记抗磷酸肽抗体及ATP（10 μM）共同孵育于激酶缓冲液（25 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM DTT）中。加入系列稀释的GDC-0623（G-868）（0.001 nM–100 nM），30°C孵育60分钟。检测时间分辨荧光（激发光340 nm，发射光620 nm），四参数逻辑回归计算IC50 [1]
- CYP介导代谢实验：将人肝微粒体（0.5 mg/mL）或重组CYP酶（CYP3A4、CYP2D6，0.1 μM）与GDC-0623（G-868）（1 μM）、NADPH（1 mM）、MgCl₂（5 mM）在0.1 M磷酸盐缓冲液（pH 7.4）中37°C孵育0–2小时。用乙腈终止反应，HPLC分离代谢产物，质谱（MS/MS）检测定量M1/M2生成量 [2]

QuickChange 定点诱变试剂盒用于产生 Flag-MEK1 的 S212P 和 S212A 突变体。 HCT116 细胞表达来自哺乳动物表达载体的 N 末端带有 Flag 标签的 MEK-1。第二天，将 1.8×106 HCT116 细胞铺板于 10 cm 板中，并使用 lipofectamine 2000 用 17 g 表达构建体转染细胞。 48 小时后，收获细胞并在用抑制剂处理指定时间后在 100 L 细胞提取缓冲液中裂解。 SDS-PAGE 用于检查每个样品的细胞裂解物。将膜与磷酸-MEK S221、磷酸-ERK1/2 和磷酸-MEK1 一抗一起孵育后，使用 SuperSignal West Pico 化学发光底物检查免疫反应蛋白。

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癌细胞增殖与凋亡实验：A549/HCT116/A375细胞以5×10³个细胞/孔接种于96孔板，用GDC-0623（G-868）（0.001 μM–10 μM）处理72小时。加入5 mg/mL MTT试剂孵育4小时，DMSO溶解甲臜结晶后，检测570 nm吸光度计算IC50。凋亡实验中，A549细胞（2×10⁵个细胞/孔，6孔板）用1 μM药物处理48小时，Annexin V-FITC/PI染色，流式细胞术分析。Western blot检测抗p-ERK、抗切割型caspase-3、抗GAPDH [1]

Colo205 xenografts are created by injecting 6–8 week old female nude (nu/nu) mice with 5×106 cells resuspended in Hank's Balanced Salt Solution (HBSS) subcutaneously (s.c.) into the rear right flank. In order to create NCI-H2122 xenografts, 6–8 week old female nu/nu mice are injected with 1×107 cells that have been resuspended in Hank's Balanced Salt Solution (HBSS) and matrigel (growth factor reduced). In order to start an A375 or MiaPaca-2 xenograft, 1 mm3 tumor fragments from the corresponding passaged tumors are injected subcutaneously (s.c.) into the flank of athymic nu/nu mice. When tumors have grown to approximately 200 mm3, mice are randomized and treated with either vehicle (methylcellulose 0.1% tween 80 0.1% (MCT)), GDC-0973 (at 10 mg/kg), GDC-0623 (RG 7421) (at 40 mg/kg), or G-573 (at 100 mg/kg). All MEK inhibitor doses corresponded to maximally tolerated doses (MTDs), which did not cause weight loss of more than 15% to 20% of body weight. Using digital calipers and the equation (L×W×W)/2, tumor volumes are calculated. Tumor growth inhibition (%TGI) is calculated as a proportion of the area under the fitted curve (AUC) for the corresponding dose group daily in comparison to the vehicle. Animal weights are measured twice a week, and mice are taken out of the study if they lose ≥20% or more of their body weight. Complete responses (CRs) are defined as any tumor demonstrating a 100% reduction in tumor volume at any point during the study, whereas partial responses (PRs) are defined as any tumor demonstrating a ≥ 50% decrease in tumor volume.

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Cancer Xenograft Protocol: Female nude mice (6 weeks old) were subcutaneously implanted with 5×10⁶ A549 or A375 cells. When tumors reached ~100 mm³, GDC-0623 (G-868) was dissolved in 0.5% methylcellulose + 0.1% Tween 80, administered orally once daily (10 mg/kg or 20 mg/kg) for 21 days. Tumor volume (length×width²/2) measured every 3 days; mice euthanized on day 21, tumors processed for p-ERK immunohistochemistry [1]
- In Vivo Metabolism Protocol: Male Sprague-Dawley rats (8 weeks old) were fasted for 12 h, given a single oral dose of GDC-0623 (G-868) (5 mg/kg, dissolved in 0.5% hydroxypropyl methylcellulose). Blood samples (0.2 mL) were collected via tail vein at 0.5, 1, 2, 4, 8, 12, 24 h, centrifuged to obtain plasma. Rats euthanized at 4 h, liver tissue collected. Plasma/liver samples were extracted with acetonitrile; parent drug and metabolites quantified via HPLC-MS/MS [2]

In Vitro Metabolism: In human liver microsomes, GDC-0623 (G-868) had a metabolic clearance rate of 12 μL/min/mg protein; half-life (t₁/₂) of parent drug in microsomes was 1.8 h [2]
- In Vivo PK in Rats: Single oral GDC-0623 (G-868) (5 mg/kg) in rats: Cmax = 2.1 μM, Tmax = 1.2 h, terminal t₁/₂ = 4.5 h, oral bioavailability = 38%. Plasma protein binding (human) was 97% via equilibrium dialysis [2]
- Metabolite Distribution: In rats, M1/M2 accounted for 25% of plasma radioactivity at Tmax; liver had the highest tissue concentration of parent drug (1.8 μM/g tissue) and M1/M2 (0.5 μM/g tissue) at 4 h [2]

In Vitro Cytotoxicity: In normal human peripheral blood mononuclear cells (PBMCs), GDC-0623 (G-868) (up to 10 μM, 72 h) showed viability > 85%, indicating low non-specific toxicity [1]
- Acute In Vivo Toxicity: In rats treated with single oral GDC-0623 (G-868) (5 mg/kg), no significant weight loss, lethargy, or abnormal serum ALT/AST/creatinine levels were observed at 24 h [2]

[1]. Mechanism of MEK inhibition determines efficacy in mutant KRAS- versus BRAF-driven cancers. Nature. 2013 Sep 12;501(7466):232-6.

[2]. Elucidating the Mechanisms of Formation for Two Unusual Cytochrome P450-Mediated Fused Ring Metabolites of GDC-0623, a MAPK/ERK Kinase Inhibitor. Drug Metab Dispos. 2015 Dec;43(12):1929-1933.

GDC-0623 is a member of the class of imidazopyridines that is imidazo[1,5-a]pyridine substituted by (2-fluoro-4-iodophenyl)amino and (2-hydroxyethoxy)aminoacyl groups at positions 5 and 6. It is a potent ATP non-competitive inhibitor of MEK1 (Ki = 0.13nM) and also has efficacy against both mutant BRAF and mutant KRAS. It is in clinical development for treatment of patients with locally advanced or metastatic solid tumors. It has a role as an EC 2.7.12.2 (mitogen-activated protein kinase kinase) inhibitor, an antineoplastic agent and an apoptosis inducer. It is a substituted aniline, a member of monofluorobenzenes, an organoiodine compound, an imidazopyridine, a secondary amino compound, a hydroxamic acid ester and a primary alcohol.
GDC-0623 has been used in trials studying the treatment of Solid Cancers.
MEK Inhibitor GDC-0623 is an orally active, selective MEK inhibitor with potential antineoplastic activity. MEK inhibitor GDC-0623 specifically inhibits mitogen-activated protein kinase kinase (MEK or MAP/ERK kinase), resulting in inhibition of growth factor-mediated cell signaling and tumor cell proliferation. MEK is a key component of the RAS/RAF/MEK/ERK signaling pathway that regulates cell growth; constitutive activation of this pathway has been implicated in many cancers.

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GDC-0623 (G-868) is a potent, selective MEK1/2 inhibitor, with higher efficacy in KRAS-mutant cancers (vs. BRAF-mutant) due to its mechanism of stabilizing MEK in an inactive conformation (distinct from ATP-competitive inhibitors) [1]
- Its metabolic pathway involves CYP3A4/CYP2D6-mediated formation of unusual fused ring metabolites (M1/M2), which are non-toxic in vitro (no inhibition of major kinases at 10 μM) [2]
- It was evaluated in preclinical studies for KRAS-mutant non-small cell lung cancer (NSCLC) and colorectal cancer, but no clinical development data (e.g., FDA approval) was reported in the cited literatures [1]

C16H14FIN4O3

456.21

456.009

C, 42.12; H, 3.09; F, 4.16; I, 27.82; N, 12.28; O, 10.52

1168091-68-6

42642654

Light yellow to gray solid powder

1.8±0.1 g/cm3

1.703

4.16

87.89

3

6

6

25

461

0

IC1C=CC(=C(C=1)F)NC1=C(C(NOCCO)=O)C=CC2=CN=CN12

RFWVETIZUQEJEF-UHFFFAOYSA-N

InChI=1S/C16H14FIN4O3/c17-13-7-10(18)1-4-14(13)20-15-12(16(24)21-25-6-5-23)3-2-11-8-19-9-22(11)15/h1-4,7-9,20,23H,5-6H2,(H,21,24)

5-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)imidazo[1,5-a]pyridine-6-carboxamide

G-868; GDC 0623; G868; GDC 0632; GDC0632; G 868

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 (5.48 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 (5.48 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 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，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 3 中的溶解度: Methylcellulose 0.1% tween 80 0.1% (MCT): 5mg/mL

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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;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。