TAK-593

VEGFR1 (IC50 = 3.2 nM); VEGFR2 (IC50 = 0.95 nM); VEGFR3 (IC50 = 1.1 nM); PDGFRα (IC50 = 4.3 nM); PDGFRβ (IC50 = 13 nM); PDGFRα^V561D (IC50 = 1 nM)
TAK-593 suppresses HUVEC growth with an IC50 of 0.30 nM. It exhibits strong inhibitory action against the PDGFR (PDGFRα, β: IC50=4.3, 13 nM) and VEGFR (VEGFR1-3: IC50=3.2, 0.95, 1.1 nM) families. TAK-593 exhibits IC50 values greater than 100 nM against all kinases, with the exception of Fms (IC50 = 10 nM) and Ret (IC50 = 18 nM) kinases [1]. Human umbilical vein endothelial cells and human coronary artery smooth muscle cells' cellular phosphorylation and proliferation are both potently inhibited by TAK-593 in response to VEGF and PDGF stimulation. Additionally, TAK-593 strongly suppresses the formation of tubes in co-cultured endothelial cells and fibroblasts caused by VEGF[2].

TAK-593 抑制 HUVEC 生长，IC50 为 0.30 nM。它对 PDGFR (PDGFRα, β: IC50=4.3, 13 nM) 和 VEGFR (VEGFR1-3: IC50=3.2, 0.95, 1.1 nM) 家族表现出强烈的抑制作用。 TAK-593 对所有激酶的 IC50 值均大于 100 nM，但 Fms (IC50 = 10 nM) 和 Ret (IC50 = 18 nM) 激酶除外 [1]。 TAK-593 响应 VEGF 和 PDGF 刺激，有效抑制人脐静脉内皮细胞和人冠状动脉平滑肌细胞的细胞磷酸化和增殖。此外，TAK-593 强烈抑制共培养的内皮细胞和成纤维细胞中由 VEGF 引起的管的形成[2]。

---

TAK-593 能有效抑制 VEGF 诱导的人脐静脉内皮细胞 (HUVEC) 中 VEGFR2 的磷酸化，IC₅₀ 为 0.34 nM。
它还能抑制 PDGF-BB 诱导的冠状动脉平滑肌细胞 (CASMC) 中 PDGFRβ 的磷酸化，IC₅₀ 为 2.1 nM。
与此一致，TAK-593 抑制了 VEGF 刺激的 HUVEC 增殖 (IC₅₀ = 0.30 nM) 和 PDGF-BB 刺激的 CASMC 增殖 (IC₅₀ = 3.5 nM)。
相比之下，它对多种人类癌细胞系 (A549、CFPAC-1、DU-145、HT-29、MDA-MB-231) 和 MRC-5 人成纤维细胞的增殖抑制较弱，IC₅₀ 值在 8.6 至 30 µM 之间。
在模拟血管生成的管形成实验中，TAK-593 强烈抑制了 VEGF 诱导的内皮细胞管形成，IC₅₀ 为 0.32 nM。[2]

TAK-593 抑制 HUVEC 生长，IC50 为 0.30 nM。它对 PDGFR (PDGFRα, β: IC50=4.3, 13 nM) 和 VEGFR (VEGFR1-3: IC50=3.2, 0.95, 1.1 nM) 家族表现出强烈的抑制作用。 TAK-593 对所有激酶的 IC50 值均大于 100 nM，但 Fms (IC50 = 10 nM) 和 Ret (IC50 = 18 nM) 激酶除外 [1]。 TAK-593 响应 VEGF 和 PDGF 刺激，有效抑制人脐静脉内皮细胞和人冠状动脉平滑肌细胞的细胞磷酸化和增殖。此外，TAK-593 强烈抑制共培养的内皮细胞和成纤维细胞中由 VEGF 引起的管的形成[2]。

---

口服 TAK-593（每日两次）在多种皮下异种移植瘤模型中表现出广谱抗肿瘤活性，包括肺癌 (A549)、结肠癌 (HT-29)、乳腺癌 (MDA-MB-231)、前列腺癌 (DU145)、胰腺癌 (CFPAC-1)、肾癌 (RCC-02-JCK)、甲状腺癌 (TT)、胶质母细胞瘤 (U87MG)、卵巢癌 (SK-OV-3) 和胃癌 (MKN45)。低至 0.25 mg/kg 的剂量在大多数模型中显示出统计学上显著的肿瘤生长抑制。
在已建立的 A549 肺癌模型（较大肿瘤）中，0.125 和 0.25 mg/kg（每日两次）的 TAK-593 强烈抑制肿瘤生长，而 1.5 和 3 mg/kg（每日两次）的剂量则诱导了肿瘤消退。停止治疗后肿瘤会再生长，但重新开始治疗可再次发挥疗效。
在 U87MG 胶质母细胞瘤原位模型中，TAK-593 (1 和 4 mg/kg，每日两次) 显著延长了中位生存时间。
对 A549 肿瘤的免疫组织化学分析表明，TAK-593 治疗显著降低了肿瘤微血管密度 (CD31染色) 和周细胞覆盖 (αSMA染色)，同时增加了肿瘤细胞凋亡 (TUNEL检测) 并减少了增殖 (Ki-67染色)。
在 HT-29 荷瘤小鼠中进行的动态对比增强磁共振成像 (DCE-MRI) 显示，TAK-593 治疗 4 天后显著降低了肿瘤血管通透性 (Ktrans 值)。[2]

溶于 50 mM TrisHCl pH 7.5、5 mM MnCl₂、5 mM MgCl₂、0.01% Tween-20 和 2 mM DTT，含 10 μM ATP，0.1 μg/ mL 生物素化聚 GluTyr (4:1) 和 0.1 nM VEGFR2 进行酶反应。预孵育包括将化合物 (TAK-593) 和酶在室温下孵育五分钟，然后用 ATP 进行催化启动。通过添加 25 μL 100 mM EDTA、10 μg/mL 供体和受体链霉亲和素珠（溶于 62.5 mM HEPES pH 7.4、250 mM NaCl 和 0.1% BSA）来终止反应。在黑暗中孵育整夜后，酶标仪可读取酶标板的读数[1]。

HUVEC 以 3000 个细胞/孔的密度接种到 96 孔板中，使用人内皮细胞 SFM 生长培养基（ Invitrogen）补充有 3% 胎牛血清 (FBS)。添加不同浓度的测试化合物（TAK-593）并添加60 ng/mL VEGF后，将细胞再培养五天。 WST-8 甲臜测定使用 Cell Counting Kit-8，用于测量细胞增殖[1]。

---

对于受体磷酸化实验，HUVEC 或 CASMC 先用 TAK-593 处理 2 小时，然后用 VEGF 或 PDGF-BB 刺激 5 分钟。随后通过蛋白质印迹法，使用抗磷酸化 VEGFR2、总 VEGFR2 或 PDGFRβ 的抗体分析细胞裂解液。磷酸化 PDGFRβ 则是先用抗 PDGFRβ 抗体进行免疫沉淀，再用抗磷酸酪氨酸抗体进行检测。
对于细胞增殖实验，HUVEC 与 TAK-593 和 VEGF 共处理 5 天，CASMC 经饥饿处理后与 TAK-593 和 PDGF-BB 共处理 6 天，癌细胞系/成纤维细胞则与 TAK-593 共处理 3 天。使用比色法细胞计数试剂盒测定细胞活力。
对于管形成实验，HUVEC 与正常人真皮成纤维细胞在 VEGF 和 TAK-593 存在下共培养 7 天。随后用抗 CD31 抗体对内皮细胞进行染色，并通过荧光成像观察。使用图像分析软件对管状结构面积进行定量。[2]

Rats: Diethyl ether is used to induce anesthesia during the intravenous administration of medication to rats. Blood is drawn from the femoral vein in monkeys, and the tail vein in rats, at 5, 10, (only for IV dosing), 15, 30 min, and 1, 2, 3, 4, 6, 8, 12, 24, 32, and 48 hours (only for monkeys) following dosing. After that, the plasma fraction is extracted from the blood by centrifugation. Until analysis, the plasma is maintained frozen at 20°C. Utilizing a fluorescence detector and high-performance liquid chromatography, the concentration of TAK-593 in plasma is ascertained. The wavelengths of the excitation and emission are 346 and 420 nm, respectively.
Mice: Test compounds are given to female BALB/cAJcl mice that have not been fasted at a dose of 10 mg/kg via cassette dosing. Blood samples are taken following oral administration. To extract the plasma fraction, the blood samples are centrifuged. Acetonitrile with an internal standard is used to deproteinize the plasma samples. Following centrifugation, the supernatant is diluted and then centrifuged once more using a mixture of acetonitrile (9:1, v/v) and 0.01 M ammonium formate solution. By using LC/MS/MS, the compound concentrations in the supernatant are determined[1].

---

TAK-593 was prepared as a 0.5% (w/v) methylcellulose suspension for oral administration.
For subcutaneous xenograft efficacy studies, tumor cells or tissue fragments were implanted into the flank of athymic nude or SCID mice. When tumors reached a predetermined size (e.g., ~120-200 mm³), mice were randomized and orally administered TAK-593 or vehicle twice daily (BID) for 10-21 days. Tumor volumes and body weights were monitored regularly.
For pharmacodynamic (PD) and pharmacokinetic (PK) analysis, A549 tumor-bearing nude mice were orally administered a single dose of TAK-593. Blood and tissue samples were collected at various time points for drug concentration measurement by HPLC-MS/MS or HPLC-fluorescence. For PD analysis, VEGF was injected intravenously 5 minutes before euthanasia, and lung tissues were collected for phospho-VEGFR2 analysis by western blot.
For immunohistochemistry studies, A549 tumor-bearing mice were treated with TAK-593 BID for specified durations. Tumors were harvested, embedded, sectioned, and stained for CD31 (vessels), Ki-67 (proliferation), TUNEL (apoptosis), and αSMA (pericytes).
For the orthotopic glioblastoma model, U87MG cells were injected intracranially. Mice were treated with TAK-593 BID starting 4 days post-inoculation and monitored for survival.
For DCE-MRI studies, HT-29 tumor-bearing mice were treated with TAK-593 BID for 3 days. MRI scans were performed before and after treatment to assess changes in vascular permeability using a gadolinium-based contrast agent.[2]

Following a single oral dose of 0.125 mg/kg in mice, TAK-593 achieved a maximum plasma concentration (Cmax) of 0.069 µg/mL at 15 minutes and an area under the plasma concentration-time curve from 0 to 24 hours (AUC₀₋₂₄) of 0.078 µg·h/mL.
At a dose of 1 mg/kg, plasma Cmax was 0.451 µg/mL (at 1 hour) and AUC₀₋₂₄ was 0.883 µg·h/mL. In lung tissue, Cmax was 0.242 µg/mL and AUC₀₋₂₄ was 0.556 µg·h/mL.
TAK-593 exhibited rapid absorption and clearance; it was undetectable in plasma or lung tissue of most mice by 8 hours after a 1 mg/kg dose.
Despite rapid clearance from plasma, a single oral dose of 1 mg/kg led to almost complete suppression of VEGF-induced VEGFR2 phosphorylation in lung tissue for up to 8 hours, with recovery by 16 hours, indicating a long duration of target inhibition.[2]

In multiple xenograft studies, oral administration of TAK-593 at efficacious doses (up to 4 mg/kg twice daily) did not cause significant body weight loss or obvious adverse effects in mice, indicating good tolerability.[2]

[1]. Discovery of N-[5-({2-[(cyclopropylcarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide (TAK-593), a highly potent VEGFR2 kinase inhibitor. Bioorg Med Chem. 2013 Apr 15;21(8):2333-2345.

[2]. Anti-angiogenic and anti-tumor effects of TAK-593, a potent and selective inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptor tyrosine kinase. Cancer Sci. 2013 Apr;104(4):486-94.

TAK-593 has been used in trials studying the treatment of Solid Tumors.
VEGFR/PDGFR Tyrosine Kinase Inhibitor TAK-593 is an oral formulation containing a small-molecule receptor tyrosine kinase inhibitor of both vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) with potential antineoplastic activity. TAK-593 selectively binds to and inhibits VEGFR and PDGFR, which may result in the inhibition of angiogenesis and tumor cell proliferation.

---

TAK-593 is a novel imidazo[1,2-b]pyridazine derivative with a "pseudo-irreversible" or long-residence time mechanism of binding to VEGFR2 and PDGFRβ, contributing to its prolonged pharmacodynamic effect despite short systemic exposure.
Its anti-tumor efficacy is primarily mediated through an anti-angiogenic mechanism, involving inhibition of endothelial cell signaling (VEGFR2), pericyte recruitment (PDGFRβ), leading to vessel regression, reduced vascular permeability, and subsequent tumor cell death.
The study suggests that the dual inhibition of VEGF and PDGF signaling by TAK-593 may be more effective than VEGF inhibition alone in disrupting tumor vasculature.[2]

C23H23N7O3

445.4738

445.186

C, 62.01; H, 5.20; N, 22.01; O, 10.77

1005780-62-0

1005780-62-0

24767976

White solid powder

3.618

115.44

2

6

6

33

735

0

O=C(C1([H])C([H])([H])C1([H])[H])N([H])C1=C([H])N2C(C([H])=C([H])C(=N2)OC2C([H])=C([H])C(C([H])([H])[H])=C(C=2[H])N([H])C(C2=C([H])C(C([H])([H])[H])=NN2C([H])([H])[H])=O)=N1

DZFZXPPHBWCXPQ-UHFFFAOYSA-N

InChI=1S/C23H23N7O3/c1-13-4-7-16(11-17(13)24-23(32)18-10-14(2)27-29(18)3)33-21-9-8-20-25-19(12-30(20)28-21)26-22(31)15-5-6-15/h4,7-12,15H,5-6H2,1-3H3,(H,24,32)(H,26,31)

N-[5-[2-(cyclopropanecarbonylamino)imidazo[1,2-b]pyridazin-6-yl]oxy-2-methylphenyl]-2,5-dimethylpyrazole-3-carboxamide

TAK-593; TAK593; TAK 593

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: ≥ 48.5 mg/mL (~108.9 mM)

配方 1 中的溶解度: ≥ 2.67 mg/mL (5.99 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 26.7 mg/mL的澄清DMSO储备液加入到400 μL PEG300中并混合均匀；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。

---

配方 2 中的溶解度: ≥ 2.67 mg/mL (5.99 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 26.7 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。

---

View More

配方 3 中的溶解度: ≥ 2.67 mg/mL (5.99 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 26.7 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

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。