PF-03814735

PF-03814735 is a potent and orally bioavailable small-molecule inhibitor of Aurora kinases, with high selectivity for Aurora A and Aurora B kinases. The IC50 values are as follows: Aurora A (1.8 nM), Aurora B (4.6 nM). It shows weak inhibitory activity against other kinases (e.g., IC50 >1000 nM for Abl, EGFR, and VEGFR2), confirming its specificity for the Aurora kinase family [1]

由于 PF-03814735 对 Aurora1 和 Aurora2 激酶的抑制作用，完整细胞中磷酸化 Aurora1、磷酸化组蛋白 H3 和磷酸化 Aurora2 的含量均较低。 PF-03814735 会抑制多倍体多核细胞的发育和细胞增殖，因为它会抑制胞质分裂 [1]。小细胞肺癌 (SCLC) 细胞系和结肠癌细胞系均显示出对 PF-03814735 的高敏感性。 PF-03814735 的有效性与视网膜母细胞瘤途径和 Myc 基因家族成员的状态之间存在显着相关性。

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抗增殖活性：PF-03814735可抑制多种人类肿瘤细胞系的增殖，包括血液系统恶性肿瘤细胞系（K562、MV4-11、Raji）和实体瘤细胞系（HCT116、A549、MCF-7、PC-3），IC50值范围为9-68 nM。其中，对血液系统肿瘤细胞系的抑制活性更强（IC50：9-22 nM），对部分实体瘤细胞系的活性相对较低[1]
- 抑制组蛋白H3磷酸化：Western blot分析显示，用PF-03814735（10-100 nM）处理HCT116细胞4小时，可浓度依赖性降低磷酸化组蛋白H3（p-Histone H3，Ser10，Aurora B激酶的特异性底物）水平，在50 nM浓度下可完全抑制p-Histone H3的表达[1]
- 诱导细胞有丝分裂停滞与凋亡：流式细胞术分析表明，25 nM PF-03814735处理HCT116细胞24小时可导致G2/M期停滞（细胞比例从15%升至65%），处理48小时后通过Annexin V-FITC/PI染色检测到凋亡细胞比例达30%[1]
- 基于基因组分析的预测生物标志物筛选：在54种人类肿瘤细胞系中，PF-03814735的响应（IC50 <30 nM vs. >30 nM）与特定基因表达特征相关。RNA测序和qPCR验证显示，有丝分裂进程相关基因（如CCNB1、CDK1、AURKA）高表达与细胞对PF-03814735的敏感性增加相关；反之，DNA修复基因（如BRCA1、RAD51）高表达与耐药性相关[2]
- Aurora A表达与药物敏感性的相关性：免疫印迹和qPCR实验证实，内源性Aurora A mRNA和蛋白水平高的肿瘤细胞系（如MV4-11、K562）对PF-03814735的IC50值更低（9-12 nM），而Aurora A表达低的细胞系（如MCF-7）IC50值更高（45-68 nM）[2]

当每天一次给患有人类异种移植肿瘤的小鼠口服 PF-03814735 时，它通过将磷酸化组蛋白 H3 的水平降低至可控制的水平，显着减缓了肿瘤的生长。在异种移植小鼠肿瘤模型中，多西紫杉醇和PF-03814735的组合显示出协同肿瘤生长抑制作用[1]。当以每周 80 mg/kg 的方案而不是每日 15 mg/kg 的方案给药时，PF-03814735 在 NCI-H82 异种移植物中更成功。 PF-03814735 引起的每周生长延迟 23.5 天相当于整个治疗过程中 0.9 个对数净细胞死亡 [2]。

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皮下异种移植模型的抗肿瘤疗效：在携带K562（血液肿瘤）皮下异种移植瘤的裸鼠中，口服PF-03814735（25 mg/kg、50 mg/kg或100 mg/kg，每日一次，连续14天）可剂量依赖性抑制肿瘤生长。其中50 mg/kg和100 mg/kg剂量组的肿瘤生长抑制率（TGI）分别达78%和92%，但未观察到完全肿瘤消退；肿瘤组织分析显示，p-Histone H3水平较溶剂对照组降低60-80%[1]
- 原位异种移植模型的抗肿瘤活性：在盲肠原位接种HCT116（结直肠癌）细胞的裸鼠模型中，口服PF-03814735（75 mg/kg，每日一次，连续21天）可使肿瘤体积减少65%，并降低肝脏转移结节数量（从对照组的12±3个/鼠降至3±1个/鼠）[1]
- 药效与生物标志物的相关性：在携带MV4-11（Aurora A高表达）异种移植瘤的小鼠中，口服PF-03814735（50 mg/kg）在第14天的TGI达85%；相反，在携带MCF-7（Aurora A低表达）异种移植瘤的小鼠中，相同剂量仅实现32%的TGI，证实了体外Aurora A表达水平与体内药物敏感性的相关性[2]

极光激酶活性测定（基于HTRF技术）：将重组人源Aurora A（含激活突变T288E）或Aurora B（与INCENP肽形成复合物）与含ATP（10 μM）、生物素化组蛋白H3底物（500 nM）及系列浓度PF-03814735（0.1-100 nM）的反应缓冲液在30°C孵育60分钟。加入链霉亲和素偶联的铕穴状化合物和XL665标记的抗磷酸化组蛋白H3（Ser10）抗体终止反应，测定时间分辨荧光共振能量转移（HTRF）信号（665 nm和620 nm），通过665/620 nm信号比值计算PF-03814735对Aurora A和Aurora B的抑制率及IC50值[1]
- 激酶选择性测定：采用与极光激酶活性测定相同的HTRF方法，测试100 nM PF-03814735对64种非极光激酶（包括Abl、EGFR、VEGFR2、CDK2、PI3K）的抑制活性。通过比较药物组与溶剂组的HTRF信号计算各激酶的抑制率，仅3种激酶（CDK1、PLK1、JAK2）的抑制率>20%，证实该化合物对极光激酶的选择性[1]

抗增殖实验（SRB法）：将人类肿瘤细胞（K562、HCT116、MCF-7等）以2×103-5×103个/孔的密度接种于96孔板，培养24小时后加入系列稀释的PF-03814735（0.1-1000 nM），继续培养72小时。用10%三氯乙酸（TCA）在4°C固定细胞1小时，0.4%磺酰罗丹明B（SRB）（溶于1%乙酸）染色30分钟，1%乙酸洗去未结合染料，10 mM Tris碱溶解结合的SRB，测定510 nm处吸光度，通过非线性回归分析计算IC50值[1]
- G2/M期停滞与凋亡实验：将HCT116细胞以1×105个/孔接种于6孔板，用25 nM PF-03814735分别处理24小时（细胞周期分析）或48小时（凋亡分析）。细胞周期分析：收集细胞，-20°C下70%乙醇固定过夜，加入含RNase A的碘化丙啶（PI）染色，流式细胞术检测G0/G1、S、G2/M期细胞比例；凋亡分析：用Annexin V-FITC和PI染色，流式细胞术定量早期（Annexin V+/PI-）和晚期（Annexin V+/PI+）凋亡细胞百分比[1]
- 生物标志物筛选的基因表达分析：采用标准酚-氯仿法从54种肿瘤细胞系中提取总RNA，逆转录为cDNA后，用基因特异性引物通过qPCR检测候选基因（AURKA、CCNB1、BRCA1等）的表达水平。RNA测序实验中，从总RNA构建文库，在Illumina平台测序，通过差异基因表达分析筛选与PF-03814735 IC50值相关的基因（Pearson相关系数>0.6或<-0.6）[2]
- Aurora A蛋白表达实验（免疫印迹法）：用含蛋白酶抑制剂的RIPA缓冲液提取肿瘤细胞总蛋白，取30 μg蛋白进行SDS-PAGE电泳，转印至PVDF膜，5%脱脂牛奶封闭1小时。膜与抗Aurora A一抗（1:1000稀释）在4°C孵育过夜，再与HRP偶联的二抗（1:5000稀释）室温孵育1小时，ECL化学发光显影，ImageJ软件定量条带强度，分析其与PF-03814735 IC50值的相关性[2]

Dissolved in cremophor EL [cremophor/ethanol/0.9% saline (12.5%/12.5%/75%)]; 10, 20, 30 mg/kg; Oral gavage HCT116 tumors are implanted s.c. on the right flank of nude mice.

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Subcutaneous xenograft model (antitumor efficacy): Female nude mice (6-8 weeks old) are subcutaneously injected with 5×106 K562 cells (suspended in 50% Matrigel) into the right flank. When tumors reach a volume of 100-150 mm³, mice are randomly divided into 4 groups (n=6/group): vehicle control (0.5% methylcellulose + 0.1% Tween 80), PF-03814735 25 mg/kg, 50 mg/kg, or 100 mg/kg. The compound is administered orally once daily for 14 days. Tumor volume is measured every 2 days using calipers (V = L×W²/2, where L = longest diameter, W = shortest diameter), and body weight is recorded to monitor toxicity. At the end of the study, tumors are excised, frozen in liquid nitrogen, and used for western blot analysis of p-Histone H3 [1]
- Orthotopic xenograft model (colon cancer): Female nude mice are anesthetized with isoflurane, and a small abdominal incision is made to expose the cecum. 1×106 HCT116 cells (suspended in 20 μL PBS) are injected into the cecal wall, and the incision is closed with sutures. Two weeks after cell injection (when tumors are established), mice are divided into 2 groups (n=8/group): vehicle or PF-03814735 75 mg/kg (oral, once daily for 21 days). Mice are euthanized at the end of treatment; cecal tumors are weighed, and liver tissues are fixed in 4% paraformaldehyde to count metastatic nodules [1]
- Biomarker validation xenograft model: Nude mice are subcutaneously injected with 5×106 MV4-11 (high Aurora A) or MCF-7 (low Aurora A) cells. When tumors reach 100 mm³, mice are treated with oral PF-03814735 50 mg/kg once daily for 14 days. Tumor volume is measured every 2 days, and TGI is calculated. Tumors are excised to confirm Aurora A expression via immunoblotting, validating the correlation between Aurora A levels and drug response [2]

Oral absorption: In CD-1 mice, oral administration of PF-03814735 (10 mg/kg) results in a peak plasma concentration (Cmax) of 85±12 ng/mL and an area under the curve (AUC0-24h) of 320±45 ng·h/mL. The oral bioavailability (F) is 42±5%, as calculated by comparing AUC0-24h to intravenous administration (2 mg/kg, AUC0-24h = 152±20 ng·h/mL) [1]
- Distribution: In Sprague-Dawley (SD) rats, intravenous administration of PF-03814735 (5 mg/kg) shows a steady-state volume of distribution (Vss) of 3.8±0.6 L/kg, indicating wide tissue distribution. Tissue distribution studies in mice show that the compound accumulates in tumors (tumor/plasma concentration ratio = 3.2±0.4 at 4 hours post-oral dose) [1]
- Metabolism: In human liver microsomes, PF-03814735 has a metabolic half-life (t1/2) of 3.6±0.5 hours. The main metabolic enzymes are CYP3A4 (accounts for 60% of metabolism) and CYP2D6 (25%), as determined by incubation with selective CYP inhibitors (e.g., ketoconazole for CYP3A4, quinidine for CYP2D6). The primary metabolite is an N-oxidation product, which has no Aurora kinase inhibitory activity [1]
- Excretion: In SD rats, intravenous administration of PF-03814735 (5 mg/kg) leads to 18±3% of the dose excreted as unchanged drug in feces and 3±1% in urine within 48 hours, indicating fecal excretion is the main route [1]
- Pharmacokinetic parameters in rats: Intravenous (5 mg/kg): Cmax = 520±65 ng/mL, AUC0-24h = 480±70 ng·h/mL, elimination half-life (t1/2) = 2.8±0.3 hours, clearance (CL) = 8.5±1.2 mL/min/kg. Oral (10 mg/kg): Cmax = 190±25 ng/mL, AUC0-24h = 840±95 ng·h/mL, t1/2 = 3.1±0.4 hours [1]

Acute toxicity in mice: Oral administration of PF-03814735 at doses up to 200 mg/kg (single dose) does not cause mortality or severe toxicity (e.g., convulsions, lethargy). The maximum tolerated dose (MTD) in mice for repeated oral administration (14 days) is 150 mg/kg/day, as doses above this cause >10% body weight loss [1]
- Hepatorenal toxicity: In SD rats treated with oral PF-03814735 (50 mg/kg/day for 28 days), serum levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (Cr) are not significantly different from vehicle controls. Histopathological analysis of liver and kidney tissues shows no evidence of necrosis or inflammation [1]
- Hematological toxicity: In nude mice treated with PF-03814735 100 mg/kg/day for 14 days, peripheral blood counts (white blood cells, platelets, red blood cells) are within normal ranges, indicating no significant myelosuppression [1]
- Plasma protein binding: Determined by equilibrium dialysis using human, mouse, and rat plasma, the plasma protein binding rate of PF-03814735 is 95±2% (human), 93±3% (mouse), and 94±2% (rat), indicating high binding to plasma proteins [1]
- Drug-drug interaction potential: In vitro studies show that PF-03814735 does not inhibit human CYP enzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) at concentrations up to 100 μM (IC50 >100 μM for all), suggesting low risk of interacting with CYP-metabolized drugs [1]

[1]. PF-03814735, an orally bioavailable small molecule aurora kinase inhibitor for cancer therapy. Mol Cancer Ther. 2010 Apr;9(4):883-94.

[2]. An integrated genomic approach to identify predictive biomarkers of response to the aurora kinase inhibitor PF-03814735. Mol Cancer Ther. 2012 Mar;11(3):710-9.

PF-03814735 has been used in trials studying the treatment of Solid Tumors.
Aurora Kinase Inhibitor PF-03814735 is an aurora kinase inhibitor with potential antineoplastic activity. PF-03814735 binds to and inhibits aurora kinases, serine-threonine kinases that play essential roles in mitotic checkpoint control during mitosis. Inhibition of aurora kinases may result in an inhibition of cellular division and proliferation in tumor cells that overexpress aurora kinases.

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Mechanism of action: PF-03814735 exerts antitumor effects by inhibiting Aurora A and Aurora B kinases. Aurora A regulates spindle pole formation during mitosis; inhibition leads to spindle assembly defects. Aurora B is essential for chromosome alignment and cytokinesis; inhibition causes abnormal chromosome segregation and G2/M phase arrest, eventually leading to apoptotic cell death [1]
- Rationale for oral formulation: PF-03814735 was designed with high oral bioavailability (42% in mice, ~40% in rats) to enable convenient outpatient administration for cancer patients, avoiding the need for intravenous infusion associated with many anticancer drugs [1]
- Predictive biomarkers for clinical use: The genomic analysis in 54 tumor cell lines identifies Aurora A mRNA/protein expression and mitotic gene signatures (CCNB1, CDK1) as potential predictive biomarkers for PF-03814735 response. These biomarkers could be used in clinical trials to select patients likely to benefit from the drug, improving treatment efficacy and reducing unnecessary exposure [2]
- Preclinical rationale for hematological cancers: PF-03814735 shows higher potency against hematological tumor cell lines (IC50: 9-22 nM) and greater in vivo efficacy in K562/MV4-11 xenografts (TGI: 85-92%) compared to some solid tumors, suggesting it may be particularly effective for treating hematological malignancies (e.g., leukemia, lymphoma) [1,2]

C23H25F3N6O2

474.48

474.199

942487-16-3

51346455

White to off-white solid powder

1.4±0.1 g/cm3

1.642

2.11

99.25

3

9

6

34

778

2

CC(=O)NCC(=O)N1[C@H]2CC[C@@H]1C3=C2C=CC(=C3)NC4=NC=C(C(=N4)NC5CCC5)C(F)(F)F

RYYNGWLOYLRZLK-RBUKOAKNSA-N

InChI=1S/C23H25F3N6O2/c1-12(33)27-11-20(34)32-18-7-8-19(32)16-9-14(5-6-15(16)18)30-22-28-10-17(23(24,25)26)21(31-22)29-13-3-2-4-13/h5-6,9-10,13,18-19H,2-4,7-8,11H2,1H3,(H,27,33)(H2,28,29,30,31)/t18-,19+/m0/s1

N-(2-((1S,4R)-6-((4-(cyclobutylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-1,2,3,4-tetrahydro-1,4-epiminonaphthalen-9-yl)-2-oxoethyl)acetamide

PF-03814735; PF 03814735; PF03814735

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 (5.27 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 (5.27 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: ≥ 2.5 mg/mL (5.27 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: 2% Cremophor EL, 2% N,N-dimethylacetamide, pH 5.0: ~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。