EGFR (IC50 = 6 nM); ErbB2 (IC50 = 45.7 nM); ErbB4 (IC50 = 73.7 nM)

体外活性：PF299804 是 ERBB 激酶家族的特异性抑制剂。 PF299804 抑制 EGFR 信号传导并诱导含有 EGFR T790M 的 H3255 GR 细胞系凋亡。 PF299804 对吉非替尼敏感和吉非替尼耐药的 NSCLC 细胞系有效。 PF299804 抑制 H3255 和 HCC827 细胞的生长，这些细胞经过改造可表达 EGFR T790M。 PF299804 在存在 T790M 突变的情况下抑制 EGFR 磷酸化。据信，PF-299804 通过与 ATP 位点结合以及 ERBB 家族成员催化结构域中亲核半胱氨酸残基的共价修饰，不可逆地抑制 ERBB 酪氨酸激酶活性。 PF299804在HER2扩增的胃癌细胞（SNU216、N87）中表现出显着的生长抑制作用，与其他EGFR酪氨酸激酶抑制剂（包括吉非替尼、拉帕替尼、BIBW-2992和CI-1033）相比，其50%抑制浓度值更低。 PF299804 在 HER2 扩增的胃癌细胞中诱导细胞凋亡和 G1 期阻滞，并抑制 HER 家族受体和下游信号通路（包括 STAT3、AKT 和细胞外信号调节激酶 (ERK)）的磷酸化。 PF299804 还可阻断 SNU216 细胞中 EGFR/HER2、HER2/HER3 和 HER3/HER4 异二聚体的形成以及 HER3 与 p85α 的关联。最近的一项研究使用 47 个人类乳腺癌和永生化乳腺上皮细胞系来评估 PF299804 的抑制效果，结果表明 PF299804 比非扩增细胞系优先抑制 HER-2 扩增乳腺癌细胞系的生长（RR = 3.39，p < 0.0001）。 PF299804 降低大多数敏感细胞系中 HER2、EGFR、HER4、AKT 和 ERK 的磷酸化。 PF299804 通过联合 G0/G1 阻滞和诱导细胞凋亡来发挥其抗增殖作用。激酶测定：ERBB1、ERBB 2 和 ERBB4 细胞质融合蛋白是通过克隆 ERBB1 序列（Met-668 至 Ala-1211）、ERBB2（Ile-675 至 Val-1256）和 ERBB4 序列（Gly-259 至使用 PCR 将 Gly-690) 导入杆状病毒载体 pFastBac。蛋白质在杆状病毒感染的 Sf9 昆虫细胞中表达为 GST 融合蛋白。使用谷胱甘肽琼脂糖珠通过亲和层析纯化蛋白质。使用基于 ELISA 的受体酪氨酸激酶测定法评估 ERBB 酪氨酸激酶活性的抑制。激酶反应（50 mM HEPES，pH 7.4，125 mM NaCl，10 mM MgCl2，100 μM 原钒酸钠，2 mM 二硫苏糖醇，20 μM ATP，PF299804 或载体对照，以及每 50 μL 反应混合物 1-5 nM GST-erbB ）在涂有 0.25 mg/mL 聚-Glu-Tyr 的 96 孔板中运行。将反应物在室温下孵育 6 分钟，同时摇动。通过去除反应混合物来终止激酶反应，然后用洗涤缓冲液（0.1% Tween 20 的 PBS 溶液）洗涤孔。通过添加 0.2 μg/mL 抗磷酸酪氨酸抗体（Oncogene Ab-4；50 μL/孔）与稀释在含有 3% BSA 和 0.05% Tween 20 的 PBS 中的辣根过氧化物酶 (HRP) 偶联 25 分钟，同时在 30 ℃下摇动，检测磷酸化酪氨酸残基。室内温度。除去抗体，并用洗涤缓冲液洗涤板。添加 HRP 底物（SureBlue3,3,5,5-四甲基联苯胺或 TMB）（每孔 50 μL），并在室温下摇动的同时孵育 10-20 分钟。添加 50 μL 终止溶液 (0.09 N H2SO4) 终止 TMB 反应。通过测量 450 nm 处的吸光度来量化信号。使用中值效应法测定 PF299804 的 IC50 值。细胞测定：通过5-(3-羧基甲氧基苯基)-2-(4-磺基苯基)-2H-四唑(MTS)测定评估生长和生长抑制。该测定是一种用于确定活细胞数量的比色方法，基于细胞将 MTS 生物还原为可溶于细胞培养基的甲臜产物，可以通过分光光度法进行检测。细胞接受处理 72 小时，每次实验使用的细胞数量根据经验确定。所有实验点设置在6至12个孔中，并且所有实验至少重复三次。使用适用于 Windows 的 GraphPad Prism 3.00 版（GraphPad 软件）以图形方式显示数据。使用具有 S 形剂量响应的非线性回归模型来拟合曲线。

口服 PF299804 可有效抑制 HCC827 Del/T790M 异种移植物的生长。低剂量口服 PF-299804 (15mg/kg) 可引起显着的抗肿瘤活性，包括在表达和/或过度表达 ERBB 家族成员或包含 ERBB1 双突变 (L858R/T790M) 的各种人类肿瘤异种移植模型中显着的肿瘤消退(EGFR) 与吉非替尼和厄洛替尼耐药相关。

使用 PCR 将 ERBB1 序列（Met-668 至 Ala-1211）、ERBB2 序列（Ile-675 至 Val-1256）和 ERBB4 序列（Gly-259 至 Gly-690）克隆到杆状病毒载体 pFastBac 中以创建 ERBB1 、ERBB2 和 ERBB4 细胞质融合蛋白。在感染杆状病毒的Sf9昆虫细胞中，蛋白质表达为GST融合蛋白。谷胱甘肽琼脂糖珠用于亲和层析纯化蛋白质。基于 ELISA 的受体酪氨酸激酶测定用于测量 ERBB 酪氨酸激酶活性的抑制。在涂有 0.25 mg/mL 聚 Glu-Tyr 的 96 孔板中，在以下条件下进行激酶反应：50 mM HEPES，pH 7.4，125 mM NaCl，10 mM MgCl₂，100每 50 μL 反应混合物含有 μM 原钒酸钠、2 mM 二硫苏糖醇、20 μM ATP、PF299804 或载体对照，以及 1–5 nM GST-erbB。摇动反应物并在室温下孵育六分钟。除去反应混合物以停止激酶反应后，使用洗涤缓冲液（PBS 中的 0.1% Tween 20）清洁孔。磷酸化酪氨酸残基的检测涉及在含有 3% BSA 和 0.05% Tween 20 的 PBS 中添加 0.2 μg/mL 抗磷酸酪氨酸抗体（Oncogene Ab-4；50 μL/孔）以及稀释的辣根过氧化物酶 (HRP)。然后在室温下摇动25分钟。消除抗体后，使用洗涤缓冲液洗涤板。每孔中加入 50 μL HRP 底物（SureBlue3,3,5,5-四甲基联苯胺，或 TMB），并在室温下摇动孵育 10 至 20 分钟。将 50 μL 终止溶液 (0.09 N H₂SO₄) 添加到 TMB 反应中以终止反应。 450 nm 处的吸光度用于量化信号。使用中值效应法来确定 PF299804 的 IC50 值。

在 24 孔板中，以每孔 5×10³ 至 5×10⁴ 细胞接种重复细胞，并计算生长抑制数据。简而言之，通过添加 10 μM 达克替尼并在铺板后第二天在 12 个浓度范围内进行 2 倍稀释来生成剂量反应曲线。还接种了不含药物的对照孔。在第一天添加药物时对细胞进行计数，并在实验结束（即六天后）时再次对细胞进行计数。使用 Coulter Z1 粒子计数器，在胰蛋白酶消化后将细胞放入等酮溶液中后立即进行计数。使用 Coulter Vi-Cell 计数器对悬浮培养物进行计数 [2]。

Absorption, Distribution and Excretion
Dacomitinib has shown a linear kinetics after single and multiple dose range studies. The absorption and distribution do not seem to be affected by food or the consumption of antacids. The peak plasma concentration after a dosage of 45 mg for 4 days is of 104 ng/ml. The reported AUC0-24h and tmax are of 2213 ng.h/mL and 6 hours, respectively. As well, following oral administration, the absolute oral bioavailability is 80%.
From the administered dose, 79% is recovered in feces, from which 20% represents the unmodified form of dacomitinib, and 3% is recovered in urine, from which <1% is represented by the unchanged form.
The volume of distribution of dacomitinib was reported to be of 2415 L.
The geometric apparent clearance of dacomitinib is 27.06 L/h.

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Metabolism / Metabolites
Dacomitinib presents an oxidative and conjugative metabolism marked mainly by the activity of glutathione and cytochrome P450 enzymes. After metabolism, its major circulating metabolite is an O-desmethyl dacomitinib form named PF-05199265. This metabolite has been shown to be formed by an oxidative step by CYP2D6 and to a smaller extent by CYP2C9. The following steps of the metabolism are mainly mediated by CYP3A4 for the formation of smaller metabolites. From these metabolic studies, it was shown that dacomitinib inhibited strongly the activities of CYP2D6.

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Biological Half-Life
Dacomitinib is reported to have a very large half-life of 70 hours.

Hepatotoxicity
In large early clinical trials, elevations in serum aminotransferase levels were common during dacomitinib therapy, arising in 40% of patients treated with standard doses. However, most elevations were transient and asymptomatic, and they rarely led to dose modification or discontinuation. Serum ALT elevations above 5 times the ULN occurred in only 1.4% of patients, these rates being lower than with other EGRF inhibitors such as erlotinib and gefitinib. Serum alkaline phosphatase elevations also occurred but were not common. There were no instances of clinically apparent liver injury with jaundice. However, clinical experience with dacomitinib has been limited.
Likelihood score: E* (unproven but suspected rare 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 dacomitinib during breastfeeding. Because dacomitinib is 98% bound to plasma proteins, the amount in milk is likely to be low. However, because of its potential toxicity in the breastfed infant and its half-life of 70 hours, the manufacturer recommends that breastfeeding be discontinued during dacomitinib therapy and for at least 17 days 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
Dacomitinib is known to present a protein binding of 98%.

[1]. PF00299804, an irreversible pan-ERBB inhibitor, is effective in lung cancer models with EGFR and ERBB2 mutations that are resistant to ZD1839. Cancer Res. 2007 Dec 15;67(24):11924-32.

[2]. Dacomitinib (PF-00299804), an irreversible Pan-HER inhibitor, inhibits proliferation of HER2-amplified breast cancer cell lines resistant to Anti-Human HER2 and GW572016. Mol Cancer Ther. 2012 Sep;11(9):1978-87.

Dacomitinib is a member of the class of quinazolines that is 7-methoxyquinazoline-4,6-diamine in which the amino group at position 4 is substituted by a 3-chloro-4-fluorophenyl group and the amino group at position 6 is substituted by an (E)-4-(piperidin-1-yl)but-2-enoyl group. It has a role as an epidermal growth factor receptor antagonist and an antineoplastic agent. It is a member of quinazolines, a member of piperidines, an enamide, a member of monochlorobenzenes, a member of monofluorobenzenes, a tertiary amino compound, a secondary amino compound and a secondary carboxamide.
Dacomitinib, designed as (2E)-N-16-4-(piperidin-1-yl) but-2-enamide, is an oral highly selective quinazalone part of the second-generation tyrosine kinase inhibitors which are characterized by the irreversible binding at the ATP domain of the epidermal growth factor receptor family kinase domains. Dacomitinib was developed by Pfizer Inc and approved by the FDA on September 27, 2018. Some evidence in the literature suggests the therapeutic potential of dacomitinib in the epithelial ovarian cancer model, although further investigations are needed.
Dacomitinib is a multi-kinase receptor inhibitor used in the therapy of cases of non-small cell lung cancer that harbor activating mutations in the epidermal growth factor receptor gene (EGFR). Dacomitinib is associated with high rate of transient serum aminotransferase elevations during therapy but has not been linked to instances of clinically apparent acute liver injury.
Dacomitinib is a highly selective, orally bioavailable small-molecule inhibitor of the HER family of tyrosine kinases with potential antineoplastic activity. Dacomitinib specifically and irreversibly binds to and inhibits human Her-1, Her-2, and Her-4, resulting in the proliferation inhibition and apoptosis of tumor cells that overexpress these receptors.

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Drug Indication
Dacomitinib is indicated as the first-line treatment of patients with metastatic non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 19 deletion or exon 21 L858R substitution mutations as verified by an FDA-approved test. Lung cancer is the leading cause of cancer death and NSCLC accounts for 85% of lung cancer cases. From the cases of NSCLC, approximately 75% of the patients present a late diagnosis with metastatic and advanced disease which produces a survival rate of 5%. The presence of a mutation in EGFR accounts for more than the 60% of the NSCLC cases and the overexpression of EGFR is associated with frequent lymph node metastasis and poor chemosensitivity.
FDA Label
Vizimpro, as monotherapy, is indicated for the first-line treatment of adult patients with locally advanced or metastatic non small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) activating mutations.

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Mechanism of Action
Dacomitinib is an irreversible small molecule inhibitor of the activity of the human epidermal growth factor receptor (EGFR) family (EGFR/HER1, HER2, and HER4) tyrosine kinases. It achieves irreversible inhibition via covalent bonding to the cysteine residues in the catalytic domains of the HER receptors. The affinity of dacomitinib has been shown to have an IC50 of 6 nmol/L. The ErbB or epidermal growth factor (EGF) family plays a role in tumor growth, metastasis, and treatment resistance by activating downstream signal transduction pathways such as such as Ras-Raf-MAPK, PLCgamma-PKC-NFkB and PI3K/AKT through the tyrosine kinase-driven phosphorylation at the carboxy-terminus. Around 40% of cases show amplification of EGFR gene and 50% of the cases present the _EGFRvIII_ mutation which represents a deletion that produces a continuous activation of the tyrosine kinase domain of the receptor.

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Pharmacodynamics
Preclinical data suggested that dacomitinib increases the inhibition of the epidermal growth factor receptor kinase domain as well as the activity in cell lines harboring resistance mutations such as T790M. This activity further produced a significant reduction of EGFR phosphorylation and cell viability. In these studies, non-small cell lymphoma cancer cell lines with L858R/T790M mutations where used and an IC50 of about 280 nmol/L was observed. In clinical trials with patients with advanced non-small cell lung carcinoma who progressed after chemotherapy, there was an objective response rate of 5% with a progression-free survival of 2.8 months and an overall survival of 9.5 months. As well, phase I/II studies showed positive dacomitinib activity despite prior failure with tyrosine kinase inhibitors. Phase III clinical trials (ARCHER 1050), done in patients suffering from advanced or metastatic non-small cell lung carcinoma with EGFR-activating mutations, reported a significant improvement in progression-free survival when compared with gefitinib.

C24H25CLFN5O2

469.9390

469.17

C, 61.34; H, 5.36; Cl, 7.54; F, 4.04; N, 14.90; O, 6.81

1110813-31-4

Dacomitinib hydrate;1042385-75-0;Dacomitinib-d10 dihydrochloride;Dacomitinib-d10

11511120

White to off-white solid powder

184-187 ºC

4.4

79.4

2

7

7

33

665

0

COC1=C(C=C2C(=C1)N=CN=C2NC3=CC(=C(C=C3)F)Cl)NC(=O)/C=C/CN4CCCCC4

LVXJQMNHJWSHET-AATRIKPKSA-N

InChI=1S/C24H25ClFN5O2/c1-33-22-14-20-17(24(28-15-27-20)29-16-7-8-19(26)18(25)12-16)13-21(22)30-23(32)6-5-11-31-9-3-2-4-10-31/h5-8,12-15H,2-4,9-11H2,1H3,(H,30,32)(H,27,28,29)/b6-5+

(E)-N-[4-(3-chloro-4-fluoroanilino)-7-methoxyquinazolin-6-yl]-4-piperidin-1-ylbut-2-enamide

Vizimpro; PF-00299804; PF00299804; PF 00299804; PF-299; PF299804; PF-299804; PF 299804; PF299; PF 299; dacomitinib

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 (5.32 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 (5.32 mM) 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), 悬浊液; 超声助溶。
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: ≥ 2.5 mg/mL (5.32 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 4 中的溶解度: 1% DMSO+30% polyethylene glycol+1% Tween 80, pH 9: 10mg/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。