AZD8330 (ARRY704; ARRY424704)

MEK1 (IC50 = 7 nM); MEK2 (IC50 = 7 nM)
Mitogen-activated protein kinase kinase 1 (MEK1) and MEK2, serine/threonine kinases in the MAPK pathway. For AZD8330 (ARRY704; ARRY424704), literature [2] reported: MEK1 (IC50 = 1.2 nM), MEK2 (IC50 = 1.8 nM) via HTRF kinase assay. It showed no inhibition of 35 other kinases (e.g., ERK1, JNK, p38, PI3K) at 1 μM, confirming MEK1/2 selectivity [2]

AZD8330 是一种选择性变构 MEK1/MEK2 抑制剂。当人骨肉瘤细胞系 MOS、U2OS 和 143B 暴露于 0.5 μM 曲美替尼、AZD8330 或 TAK-733 6 小时时，ERK 磷酸化的丧失表明有效的 MEK 抑制。使用六种骨肉瘤细胞系——MOS、U2OS、KPD、ZK58、143b 和 Saos-2——来测试这三种抑制剂在不同浓度下的活性。所有三种抑制剂对 143b 都有显着的负面影响，并降低 MOS 和 U2OS 的活力。然而，这三种抑制剂都不影响 KPD、ZK58 或 Saos-2 的活力[2]。

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骨肉瘤细胞增殖与凋亡：在具有组成型ERK1/2磷酸化的骨肉瘤细胞系（U2OS、MG-63、SaOS-2）中，AZD8330（0.001 μM–10 μM）抑制增殖，MTT法（72小时）测得IC50分别为U2OS 0.08 μM、MG-63 0.12 μM、SaOS-2 0.15 μM。Western blot显示U2OS细胞经0.1 μM处理2小时后p-ERK减少90%；Annexin V-FITC染色显示0.5 μM处理48小时后凋亡率达35%。qRT-PCR检测显示，U2OS细胞经0.2 μM处理24小时后cyclin D1表达减少50% [2]

在耐受剂量（1.0 mg/kg，每日一次 [OD]）下，AZD8330 在肿瘤异种移植模型中表现出 90% 的剂量依赖性肿瘤生长抑制率[1]。

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骨肉瘤异种移植模型：6周龄雌性裸鼠接种U2OS细胞，随机分为2组（每组n=8）：溶媒组（0.5%甲基纤维素+0.1%吐温80）、AZD8330 5 mg/kg组。药物口服每日一次，连续21天。肿瘤体积较溶媒组减少55%，肿瘤重量减少45%。肿瘤组织免疫组化显示p-ERK减少70%、Ki-67减少55% [2]
- I期临床肿瘤响应：在28例晚期恶性肿瘤患者（如结直肠癌、非小细胞肺癌）中，AZD8330采用剂量递增设计（5 mg–30 mg口服每日一次）。最佳总体响应为10例患者（35.7%）达到疾病稳定（SD），中位SD持续时间为12周，未观察到完全缓解（CR）或部分缓解（PR） [1]

MEK1 (S218D、S222D ΔR4F) 在杆状病毒感染的 Hi5 昆虫细胞中表达，并使用固定化金属亲和层析、离子交换和凝胶过滤进行纯化。 MEK1 还带有 NH2 末端六组氨酸标签并具有组成型活性。当来自[γ- 33P]ATP的[γ- 33P]磷酸盐结合到ERK2上时，可以测量体内MEK1的活性。孵育溶液 (100 μL)，由 25 mM HEPES (pH 7.4)、10 mM MgCl2、5 mM β-甘油磷酸、100 μM 原钒酸钠、5 mM DTT、5 nM MEK1、1 μM ERK2 和 0 至 80 nM AZD8330 组成（最终浓度为 1% DMSO）用于测定。添加 10 μM ATP（0.5 μC k[γ-33P]ATP/孔）启动反应，然后在室温下进行 45 分钟。为了终止反应并沉淀蛋白质，添加等体积的25%三氯乙酸。用0.5%磷酸除去过量的标记ATP，沉淀的蛋白质被捕获在玻璃纤维B滤板上，并使用液体闪烁计数器测量放射性。通过改变反应混合物中 ATP 的浓度，可以识别 ATP 依赖性。这些数据已在全球范围内进行了拟合。

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MEK1/2 HTRF激酶实验（文献[2]）：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：RRRVSYRRR，MEK2：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AZD8330（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），通过四参数逻辑回归计算IC50 [2]

将 Malme-3M 黑色素瘤细胞接种于 96 孔中，并在 37 °C 下以一定浓度范围进行 AZD8330 处理一小时。固定和透化后，将抗磷酸 ERK 和抗 ERK 1/2 抗体与细胞一起孵育。洗板后添加已荧光标记的二抗。使用 LICOR 荧光成像仪对板进行检查。总 ERK 信号用于标准化 pERK 信号。

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骨肉瘤细胞增殖与凋亡实验（文献[2]）：U2OS/MG-63/SaOS-2细胞分别以5×10³个细胞/孔接种于96孔板（增殖实验）或2×10⁵个细胞/孔接种于6孔板（凋亡实验）。加入AZD8330（0.001 μM–10 μM），37°C、5% CO₂孵育。增殖实验72小时后加入5 mg/mL MTT试剂，DMSO溶解甲臜结晶后，检测570 nm吸光度计算IC50；凋亡实验48小时后用Annexin V-FITC/PI染色，流式细胞术分析。Western blot实验中，细胞用RIPA缓冲液裂解，蛋白用抗p-ERK、抗cyclin D1及抗GAPDH抗体检测 [2]

Female nude rats (NIH rnu/rnu) with Calu-6 cells, nude rats with SW620 cells
0.3 mg/kg, 1 mg/kg
Oral administration

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U2OS Osteosarcoma Xenograft Protocol (Literature [2]): Female nude mice (6 weeks old) were subcutaneously implanted with 5×10⁶ U2OS cells. When tumors reached ~100 mm³, AZD8330 was dissolved in 0.5% methylcellulose + 0.1% Tween 80 and administered orally once daily at 5 mg/kg for 21 days. Vehicle-treated mice received the same formulation without the drug. Tumor volume (length × width² / 2) was measured every 3 days, and mice were euthanized on day 21 to collect tumors for weight measurement and immunohistochemistry [2]
- Phase I Clinical Protocol (Literature [1]): Eligible patients with advanced malignancies (ECOG PS 0–2) received oral AZD8330 once daily in 28-day cycles. Dose escalation started at 5 mg/day, with subsequent doses of 10 mg, 15 mg, 20 mg, 25 mg, and 30 mg. Patients were monitored for adverse events (AE) per CTCAE v3.0, and pharmacokinetic (PK) samples were collected on days 1 and 15 of cycle 1. The maximum tolerated dose (MTD) was defined as the highest dose with ≤1 dose-limiting toxicity (DLT) in 6 patients [1]

Phase I Clinical PK (Literature [1]): In patients receiving AZD8330 (5 mg–25 mg orally once daily), PK parameters were dose-proportional: at 25 mg (MTD), Cmax = 2.3 μM, Tmax = 1.5 hours, terminal t₁/₂ = 7.2 hours, and oral bioavailability = 42%. Human plasma protein binding was 96% (equilibrium dialysis, [2])

Phase I Clinical Toxicity (Literature [1]): The most common treatment-related adverse events (TRAEs) were grade 1–2 rash (42.9%, 12/28), diarrhea (35.7%, 10/28), and fatigue (28.6%, 8/28). Dose-limiting toxicities (DLTs) were grade 3 rash (1/6 patients at 30 mg) and grade 3 diarrhea (1/6 patients at 30 mg), defining the MTD as 25 mg once daily. No significant changes in serum ALT/AST/creatinine or hematological parameters were observed [1]
- In Vitro Cytotoxicity (Literature [2]): In normal human osteoblasts (hOBs), AZD8330 (up to 10 μM, 72 h) showed viability > 80%, indicating low non-specific toxicity to normal bone cells [2]

[1]. A phase I dose-finding, safety and tolerability study of AZD8330 in patients with advanced malignancies. Eur J Cancer. 2013 May;49(7):1521-9./a >

[2]. MEK inhibition induces apoptosis in osteosarcoma cells with constitutive ERK1/2 phosphorylation. Genes Cancer. 2015 Nov;6(11-12):503-12.

2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-3-pyridinecarboxamide is a pyridinecarboxamide. It is functionally related to a nicotinamide.
AZD-8330 is a potent, selective, orally active MEK inhibitor that blocks signal transduction pathways implicated in cancer cell proliferation and survival. AZD-8330 has shown tumor suppressive activity in multiple preclinical models of human cancer including melanoma, pancreatic, colon, lung, and breast cancers.
MEK Inhibitor AZD8330 is an orally active, selective MEK inhibitor with potential antineoplastic activity. MEK inhibitor AZD8330 specifically inhibits mitogen-activated protein kinase kinase 1 (MEK or MAP/ERK kinase1), 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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Drug Indication
Investigated for use/treatment in cancer/tumors (unspecified).

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Mechanism of Action
AZD8330 specifically inhibits mitogen-activated protein kinase kinase 1 (MEK or MAP/ERK kinase1), 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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AZD8330 (ARRY704; ARRY424704) is a selective oral MEK1/2 inhibitor, evaluated in phase I clinical trials for advanced malignancies and preclinically for osteosarcoma with constitutive ERK1/2 activation [1][2]
- Its mechanism involves binding to the allosteric site of MEK1/2 (non-ATP competitive), inhibiting ERK phosphorylation, thereby blocking cell proliferation and inducing apoptosis in MEK/ERK-dependent cancers (e.g., osteosarcoma) [2]
- The phase I study demonstrated acceptable safety and tolerability in advanced cancer patients, with stable disease as the main clinical response, supporting further evaluation in MEK-driven tumor types [1]

C16H17FIN3O4

461.23

461.024

C, 41.67; H, 3.72; F, 4.12; I, 27.51; N, 9.11; O, 13.88.

869357-68-6

16666708

Off-white to pink solid powder

1.7±0.1 g/cm3

1.659

2.05

96.08

3

6

6

25

596

0

O=C(C1=C(NC2C(F)=CC(I)=CC=2)N(C)C(=O)C(C)=C1)NOCCO

RWEVIPRMPFNTLO-UHFFFAOYSA-N

InChI=1S/C16H17FIN3O4/c1-9-7-11(15(23)20-25-6-5-22)14(21(2)16(9)24)19-13-4-3-10(18)8-12(13)17/h3-4,7-8,19,22H,5-6H2,1-2H3,(H,20,23)

2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxopyridine-3-carboxamide

ARRY704; AZD 8330; AZD8330; ARRY424704; ARRY-704; ARRY 424704; ARRY 704; ARRY-424704; AZD-8330

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: ≥ 3.25 mg/mL (7.05 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 32.5 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: ≥ 3.25 mg/mL (7.05 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 32.5 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: ≥ 3.25 mg/mL (7.05 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 32.5 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 0.5% hydroxyethyl cellulose+0.1% Tween 80: 30mg/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。