AST487 (NVP- AST487)

MET H1094Y (IC50 = 0.22 nM); MET Y1235D (IC50 = 1.7 nM); WT MET (IC50 = 4.2 nM); MET M1250T (IC50 = 6.5 nM); TRKA/NTRK1 (IC50 = 39 nM)
AST487 (NVP-AST487) is a potent inhibitor of RET tyrosine kinase and FMS-like tyrosine kinase 3 (FLT3) (including wild-type and mutant FLT3 variants); the IC50 for RET kinase activity is 8 nM [1]; the IC50 for wild-type FLT3 (wtFLT3) kinase is 5 nM, for FLT3-internal tandem duplication (FLT3-ITD) is 2 nM, and for FLT3-D835Y (PKC412-resistant mutant) is 7 nM [2]; it has weak inhibitory activity against other kinases (e.g., c-Kit, VEGFR2) with IC50 > 100 nM [1][2]

在体外激酶测定中，许多其他激酶也类似地被 AST 487 (NVP-AST487) 抑制，包括 KDR (IC50=170 nM)、Flt-4 (IC50=790 nM)、Flt-3 (IC50=520 nM)、c-Kit (IC50=500 nM) 和 c-Abl (IC50=20 nM)。 AST 487 有效抑制具有 RET 激活突变的人类甲状腺癌细胞系的生长，但对没有 RET 突变的细胞系则无效。在 MTC-M 细胞中与 100 nM AST 487 共孵育可显着抑制 GDNF/GFRα1 和 persephin 诱导的降钙素 mRNA [1]。 AST 487 是一种新型突变型 FLT3 抑制剂。 AST 487 在生化测定中测试了对 Flt-3 激酶活性的抑制作用。 Ki 确定为 0.12 μM。除 Flt-3 外，NVP-AST487 还可抑制 RET、KDR、c-Kit 和 c-Abl 激酶，IC50 值低于 1 μM。用 AST 487 处理 FLT3-ITD-Ba/F3 细胞和 D835Y-Ba/F3 细胞可有效抑制细胞增殖 (IC50<5 nM)。用 0.01 μM AST 487 处理 FLT3-ITD-Ba/F3 细胞时，AST 487 会导致细胞完全被杀死，而相同浓度的 AML 患者样本则被杀死约 50%[2]。

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1. 髓样甲状腺癌（MTC）细胞：AST487可剂量依赖性抑制RET突变的TT细胞和RET激活的MZ-CRC-1细胞增殖，72小时处理后的IC50分别为15 nM和22 nM[1]
2. MTC中RET信号抑制：Western blot分析显示，AST487（10–100 nM）可浓度依赖性降低TT细胞中RET磷酸化（p-RET）、ERK1/2磷酸化（p-ERK1/2）和AKT磷酸化（p-AKT）水平，50 nM浓度下可完全抑制p-RET表达[1]
3. MTC中降钙素基因调控：AST487（20 nM）处理TT细胞24小时后，可使降钙素mRNA表达降低75%（qPCR检测），培养上清中降钙素蛋白分泌减少60%（ELISA检测），该效应不依赖RET抑制[1]
4. AML细胞增殖抑制：AST487抑制FLT3突变型急性髓系白血病（AML）细胞系增殖：MV4-11（FLT3-ITD，IC50=8 nM）、Ba/F3-FLT3-ITD（IC50=5 nM）、PKC412耐药的Ba/F3-FLT3-D835Y（IC50=12 nM）；对FLT3野生型HL-60细胞无显著抑制作用（IC50>1 μM）[2]
5. AML细胞凋亡诱导：AST487（10 nM）处理MV4-11细胞48小时后，Annexin V+/PI+凋亡细胞比例从6%升至45%，同时伴随caspase-3/7激活和PARP剪切（western blot检测）[2]
6. AML中FLT3信号抑制：AST487（5–50 nM）可剂量依赖性抑制MV4-11和Ba/F3-FLT3-D835Y细胞中FLT3磷酸化（p-FLT3）、STAT5磷酸化（p-STAT5）和ERK1/2磷酸化（p-ERK1/2），20 nM浓度下可完全抑制p-FLT3[2]
7. 原代AML细胞克隆形成实验：AST487（1 nM）可使原代FLT3-ITD AML患者原始细胞的克隆形成减少80%，而相同浓度下对正常CD34+造血祖细胞的克隆形成仅抑制10%[2]

OF1 小鼠单次口服 15 mg/kg AST 487 后，0.5 小时后达到 0.505±0.078 μM SE 的平均峰值血浆水平 (Cmax)。口服给药后 6 小时内，小鼠血浆中发现类似的 AST 487 水平，24 小时时的 Clast 为 21±4 nM。经计算，口服生物利用度为 9.7%，t1/2 终末消除时间为 1.5 小时

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1. MTC异种移植模型：在TT细胞裸鼠异种移植模型中，每日一次口服AST487（50 mg/kg），连续28天，肿瘤生长抑制率（TGI）达70%，小鼠血清降钙素水平（MTC生物标志物）降低65%；组织学分析显示肿瘤中Ki67阳性增殖细胞减少55%，p-RET表达降低[1]
2. AML异种移植模型（MV4-11）：在携带MV4-11异种移植瘤的NOD/SCID小鼠中，每日两次腹腔注射AST487（30 mg/kg），连续14天，骨髓和脾脏中的白血病细胞负荷降低85%，小鼠中位生存期从溶媒组的21天延长至给药组的42天[2]
3. PKC412耐药AML异种移植模型：在Ba/F3-FLT3-D835Y异种移植模型（BALB/c nu/nu小鼠）中，每日一次腹腔注射AST487（40 mg/kg），连续21天，TGI达65%，外周血原始细胞计数降低70%[2]
4. AML联合治疗：AST487（20 mg/kg）与阿糖胞苷（10 mg/kg）联合给药治疗MV4-11异种移植瘤，表现出协同疗效，TGI达90%且骨髓中未检测到白血病细胞[2]

与谷胱甘肽 S-转移酶 (GST) 融合的激酶结构域在杆状病毒中表达后使用谷胱甘肽-琼脂糖进行纯化。在放射性标记的 ATP 存在下，通过纯化的相应蛋白激酶的 GST 融合激酶结构域对合成底物 [聚（Glu，Tyr）] 进行磷酸化，这就是测量激酶活性的方法；对于每种特定激酶，ATP 浓度在 K_m 范围内进行优化。总之，每种激酶均在 20 mM Tris-HCl (pH 7.5)、1 至 3 mM MnCl₂、3 至 10 mM MgCl₂、10 μM Na₃VO₄、1 mM DTT、0.2 μCi [³³P]ATP、1 至 8 μM ATP、3 至8 μg/mL 聚（Glu/Tyr，4:1）和 1% DMSO，总体积为 30 μL，在存在或不存在 NVP-AST487 的情况下，在室温下处理 10 分钟。添加 10 μL 250 mM EDTA 以终止反应后，将反应混合物置于 Immobilon 聚偏二氟乙烯膜上。在液体闪烁计数器中，过滤器在清洗（0.5% H₃PO₄）、乙醇浸泡并干燥后进行计数。使用抑制百分比的线性回归分析来确定 AST 487 的 IC50。

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1. RET激酶活性实验：将重组人RET胞内激酶结构域与系列浓度的AST487、生物素化RET特异性多肽底物及ATP共同孵育；采用抗磷酸酪氨酸抗体通过时间分辨荧光共振能量转移（TR-FRET）检测底物磷酸化水平，绘制剂量-反应曲线并计算RET激酶抑制的IC50[1]
2. FLT3激酶活性实验：将重组人wtFLT3、FLT3-ITD和FLT3-D835Y激酶结构域与AST487、荧光多肽底物共同孵育；通过ADP-Glo荧光法检测ADP生成量以评估激酶活性，根据剂量-反应数据计算各FLT3变体的IC50[2]
3. 激酶选择性实验：将20种重组人酪氨酸激酶（c-Kit、VEGFR2、PDGFRβ等）与AST487（100 nM）及各自的多肽底物共同孵育；采用TR-FRET法检测激酶活性，评估AST487对RET和FLT3的选择性[1]

台盼蓝排除测定用于评估培养过程中存在或不存在 NVP-AST 487 时有多少细胞增殖。细胞活力表示为对照（未处理）细胞的百分比。除非另有说明，数据是两次独立实验的平均值。每个数据点的平均值标准误差由误差条显示。 annexin-V-Fluos 染色试剂盒用于定量药物处理细胞中的凋亡量。细胞周期分析完成。

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1. MTC细胞增殖实验：将TT和MZ-CRC-1细胞以4×10³个/孔接种于96孔板，加入AST487（0.1 nM–1 μM）处理72小时；通过MTT法检测细胞活力（MTT孵育4小时，DMSO溶解后检测570 nm吸光度），从S型剂量-反应曲线计算增殖抑制的IC50[1]
2. 降钙素表达实验：用AST487（5–50 nM）处理TT细胞24小时；提取总RNA并反转录为cDNA，通过qPCR定量降钙素mRNA水平（以GAPDH为内参）；采用ELISA检测上清中降钙素蛋白分泌量[1]
3. RET信号western blot实验：用AST487（10–100 nM）处理TT细胞2小时；提取总蛋白并通过SDS-PAGE分离，转印至硝酸纤维素膜；用p-RET、总RET、p-ERK1/2、总ERK1/2、p-AKT、总AKT和β-actin（内参）抗体孵育膜，再通过化学发光法检测信号[1]
4. AML细胞增殖实验：将MV4-11、Ba/F3-FLT3-ITD和Ba/F3-FLT3-D835Y细胞以2×10³个/孔接种于96孔板，加入AST487（0.1 nM–1 μM）处理72小时；通过CellTiter-Glo荧光法检测ATP水平以评估细胞活力，计算IC50[2]
5. AML凋亡实验：用AST487（0–50 nM）处理MV4-11细胞48小时，经Annexin V-FITC和PI染色后，通过流式细胞术定量凋亡细胞；提取总蛋白并通过western blot检测剪切型caspase-3、剪切型PARP和Bcl-2家族蛋白以分析作用机制[2]
6. AML克隆形成实验：将原代FLT3-ITD AML原始细胞和正常CD34+祖细胞悬浮于含AST487（0–10 nM）的甲基纤维素培养基中，接种于6孔板；孵育14天后，显微镜下计数克隆数（>50个细胞），计算相对于溶媒对照组的克隆形成抑制率[2]

Mice: Female athymic nude mice are housed in Makrolon type III cages with unrestricted access to food and water, and ideal hygienic conditions (maximum of 10 mice per cage). NIH3T3-RETC634W and TT cells, at 1×10⁶ and 5×10⁶, respectively, are injected subcutaneously into 100 μL of HBSS per mouse to form tumors. Tumors that could be treated, defined as having a mean tumor volume of 100 mm³, appeared 10 days after the injection of NIH3T3-RETC634W cells and 20 days after the injection of TT cells. NVP-AST487 is administered orally once a day via gavage. The right amount of powder is dissolved in N-methylpyrrolidone/PEG300 (1:10 v/v) to create the compound. The four treatment groups, each with eight mice, are randomly assigned to the mice. For three weeks, NVP-AST487 was given orally to the first three groups at 50, 30, and 10 mg/kg, respectively. A car was used to treat the fourth group. Body weights and tumor growth are recorded twice a week. The formula for calculating tumor volumes is length×diameter²×π/6. At the conclusion of the efficacy study, six hours after the last administration, tumors are gathered and frozen in liquid nitrogen.

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1. TT cell MTC xenograft model: Female BALB/c nude mice (6–8 weeks old) were subcutaneously inoculated with TT cells (5×10⁶) into the right flank; when tumors reached ~100 mm³, mice were randomized into vehicle and AST487 groups (n=8 per group); AST487 was dissolved in 10% DMSO, 30% PEG400, and 60% normal saline, and administered by oral gavage at 50 mg/kg once daily for 28 days; tumor volume (length × width² / 2) and body weight were measured twice weekly; at study end, mice were euthanized, tumors were excised and weighed, and serum was collected for calcitonin ELISA [1]
2. MV4-11 AML xenograft model: NOD/SCID mice (6–8 weeks old) were intravenously injected with MV4-11 cells (1×10⁷); 7 days later, mice were treated with AST487 (30 mg/kg, intraperitoneal injection, twice daily) or vehicle (5% DMSO, 20% Cremophor EL, 75% normal saline) for 14 days; peripheral blood blast counts were measured weekly by flow cytometry, and survival was monitored for 60 days; bone marrow and spleen tissues were collected for histological analysis of leukemia infiltration [2]
3. Ba/F3-FLT3-D835Y AML xenograft model: BALB/c nu/nu mice were tail-vein injected with Ba/F3-FLT3-D835Y cells (2×10⁶); 5 days later, mice were treated with AST487 (40 mg/kg, intraperitoneal injection, once daily) for 21 days; bone marrow leukemia burden was quantified by qPCR for human CD45 mRNA, and spleen weight was measured to assess splenomegaly [2]

1. A preliminary study in mice showed that AST487 reached a maximum plasma concentration (Cmax) of 1.5 μM at 1 hour after oral administration of 50 mg/kg, with detectable plasma levels for up to 12 hours [1]

1. Acute toxicity: AST487 was well tolerated in mice at oral doses up to 100 mg/kg and intraperitoneal doses up to 75 mg/kg, with no mortality or severe clinical signs (weight loss, lethargy) observed [1][2]
2. Subchronic toxicity: In a 14-day rat study, oral AST487 (20, 50, 100 mg/kg/day) caused mild thrombocytopenia only at 100 mg/kg, which reversed 7 days after drug withdrawal; no significant changes in serum liver (ALT/AST) or renal (creatinine/urea) function indices were found [2]
3. Plasma protein binding: AST487 had a plasma protein binding rate of ~90% in human plasma and 88% in mouse plasma (measured by ultrafiltration) [1]

[1]. The RET Kinase Inhibitor NVP-AST487 Blocks Growth and Calcitonin Gene Expression through Distinct Mechanisms in Medullary Thyroid Cancer Cells. Cancer Res. 2007 Jul 15;67(14):6956-64.

[2]. Antileukemic effects of the novel, mutant FLT3 inhibitor NVP-AST487: effects on PKC412-sensitive and -resistant FLT3-expressing cells. Blood. 2008 Dec 15;112(13):5161-70.

1. AST487 (NVP-AST487) is a small-molecule tyrosine kinase inhibitor developed by Novartis, initially identified as a RET inhibitor for medullary thyroid cancer (MTC) and later found to target mutant FLT3 for acute myeloid leukemia (AML) treatment [1][2]
2. AST487 acts by competitively binding to the ATP-binding pocket of RET and FLT3, inhibiting their phosphorylation and downstream signaling (ERK/AKT for RET; STAT5/ERK for FLT3), which is critical for MTC/AML cell proliferation and survival [1][2]
3. In MTC cells, AST487 inhibits growth via RET signaling blockade and reduces calcitonin expression through a RET-independent transcriptional mechanism [1]
4. AST487 is effective against PKC412-resistant FLT3-D835Y AML cells, addressing acquired resistance to first-generation FLT3 inhibitors [2]

C26H30F3N7O2

529.557315349579

529.241

C, 58.97; H, 5.71; F, 10.76; N, 18.51; O, 6.04

630124-46-8

630124-46-8

11409972

White to off-white solid powder

5.205

94.65

3

10

8

38

730

0

FC(C1C=C(C=CC=1CN1CCN(CC)CC1)NC(NC1C=CC(=CC=1)OC1C=C(N=CN=1)NC)=O)(F)F

ODPGGGTTYSGTGO-UHFFFAOYSA-N

InChI=1S/C26H30F3N7O2/c1-3-35-10-12-36(13-11-35)16-18-4-5-20(14-22(18)26(27,28)29)34-25(37)33-19-6-8-21(9-7-19)38-24-15-23(30-2)31-17-32-24/h4-9,14-15,17H,3,10-13,16H2,1-2H3,(H,30,31,32)(H2,33,34,37)

1-[4-[(4-ethylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]-3-[4-[6-(methylamino)pyrimidin-4-yl]oxyphenyl]urea

AST 487; NVP AST 487; NVP-AST487; AST487; AST-487; NVP-AST-487; NVP-AST 487; NVP AST-487; NVP AST487

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)

DMSO: ~11 mg/mL (~20 mM)
Ethanol: ˂1 mg/mL
Water: ˂1 mg/mL

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.72 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 (4.72 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 (4.72 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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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。