EVT801

VEGFR3 11 nM (IC50); VEGFR1 396 nM (IC50); VEGFR2 130 nM (IC50); ERK 13 nM (IC50)

在 HEK293 细胞中，EVT801 (10 nM-1 μM) 剂量依赖性地抑制 VEGFR-1/2/3 自磷酸化，IC50 分别为 39 nM (VEGFR-3)、2130 nM (VEGFR-1) 和 260 nM（ VEGFR-2），分别[1]。 EVT801 (1 nM-1 μM) 抑制 VEGFR-3 阳性细胞（例如人淋巴微血管内皮细胞 (hLMVEC)）的增殖。 EVT801 的 VEGF-C 抑制浓度 (IC50) 为 15 nM，VEGF-D 抑制浓度为 8 nM，VEGF-A 抑制浓度为 155 nM，可防止 hLMVEC 增殖的诱导[1]。 EVT801 (1 μM) 可抑制 VEGFR-3 阳性肿瘤细胞的增殖和肿瘤生长[1]。

EVT801（30 mg/kg；口服；每天两次，持续 7 天）抑制小鼠模型中 VEGFR-3 阳性的肿瘤，包括 RT-001-HAM 皮下患者来源的异种移植 (PDx) 肿瘤小鼠模型、4T1 乳腺癌小鼠模型、N-二乙基亚硝胺诱导的肝癌小鼠模型、NCI-H1703皮下异种移植肿瘤小鼠模型、Rip1-Tag2/转基因小鼠模型和CT26异位肿瘤小鼠模型。除了在内皮恶性肿瘤中发现的肿瘤细胞外，EVT801还在含有原发性肿瘤和肾癌转移灶的动脉中表达[1]。

细胞增殖测定[1]
细胞类型： VEGFR-3 阳性细胞、人淋巴微血管内皮细胞 (hLMVEC)
测试浓度： 1 nM- 1 μM
孵育时间：
实验结果： 对 VEGF -C、VEGF-D 的最大抑制率为 74%、100% 和 65% 、 VEGF-A 诱导，分别。

RT-001-HAM Subcutaneous Patient-derived xenograft (PDx) Tumor Mouse Model[1]
Patient-derived tumors of the same passage were transplanted subcutaneously outbred athymic (nu/nu) female HSD mice. When tumor volume reached 726 to 1,437 mm3, 4 donor mice were sacrificed by cervical dislocation, and tumors were aseptically excised and dissected. After removing necrotic areas, tumor fragments of approximately 20 mm3 were transferred to culture medium before grafting. A total of 108 mice were anesthetized with 100 mg/kg ketamine hydrochloride (Virbac) and 10 mg/kg xylazine. Then, a tumor fragment was placed in the subcutaneous tissue of an incision at the level of the interscapular region as described previously. All mice from the same experiment were implanted on the same day. A total of 54 mice with RT-001-HAM established growing tumor (P6.0.0/0) between 62.5 and 196 mm3 were allocated according to tumor volume, ensuring homogenous median and mean tumor volume in each treatment arm. Treatments with 5 mL/kg vehicle, 30 mg/kg EVT801, or 30 mg/kg pazopanib started 15 days after tumor implantation and continued twice per day until termination 7 days later. Tumor volumes were measured via a caliper three times a week during the treatment period. All animals were weighed at the same time as tumor size measurement. Plasma and fresh tumor samples were collected at different timepoints after final dose at day 7 from all mice.

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4T1 Mammary Carcinoma Mouse Model[1]
The 4T1 mammary carcinoma mouse models were used as reported previously. Briefly, 1 × 105 4T1 cells were implanted into mammary fat pads of BALB/c mice. Twice daily treatments with 30 mg/kg EVT801 by oral gavage and either 10 mg/kg anti-PD-1 (BioXcell, BP0146) or anti-CTLA-4 weekly commenced when tumors reached 50 mm3 and lasted 3 weeks. Tumors were measured two to three times weekly with calipers. The tumor volume (V) was calculated using the formula V = 0.52 × a2 × b (a: smallest tumor diameter; b: largest tumor diameter). The tumors, lungs, and axillary lymph nodes were removed at day 21 for treatment with anti-PD-1, and day 28 for treatment with anti-CTLA-4. The tumors and the lungs were embedded in paraffin for histology studies. Intraperitoneal injection (10 mg/kg of antibody diluted at 1 mg/mL) was performed at day 6, day 11, and day 16 for treatment with anti-PD-1, and at day 6, day 11, day 16, and day 21 for treatment with anti-CTL-4 by using a 26G needle fitted to a plastic syringe in the right lower quarter of mice abdomen. The metastatic scoring was determined as follows: (0) no metastasis; (1) 1 to 20 metastases < 50 μm; (2) between 21 and 50 metastases < 50 μm; (3) 1 or more metastases > 50 μmol/L; (4) 1 or more metastases > 200 μmol/L.

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N-diethylnitrosamine–Induced Hepatocarcinoma Mouse Model[1]
After weighing, 38 male C3/H mice received a 10 mg/kg dose of N-diethylnitrosamine (DEN) intraperitoneally, whereas a group of 5 animals was kept without DEN injection as control group. After 12 months, mice that received DEN injection were randomized by weight in two groups of each 19 animals. The mice were dosed daily orally for 2 months either with EVT801 or vehicle. On the day of termination, mice treated with EVT801 received a last dose of 30 mg/kg to take blood and tumor samples for compound concentration determination. After incision of the peritoneal cavity, blood was taken from the vena cava. Following excision and weighing of the liver, the number of tumors per liver was counted. The tumor burden was calculated as the sum of individual tumor volumes for each mouse. No blood sampling was performed on control and vehicle groups. Plasma and tumor samples were stored at −20°C until analysis. Concentration of EVT801 was determined by LC/MS-MS.

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NCI-H1703 Subcutaneous Xenograft Tumor Mouse Model[1]
A total number of 40 homozygous NOD.CB17 Prkdcscid/NCrHsd mice were used for the xenograft tumor model. On the day of implantation, NCI-H1703 cells were harvested and suspended at a concentration of 1 × 108 cells per mL in an equal mix of Cultrex:RPMI without supplements. A volume of 100 μL was injected into the right hind flank of each animal. Tumor volumes were monitored until mean tumor volume reached 150 mm3. Then, mice were stratified into four treatment groups of each 10 mice and orally dosed twice per day with either EVT801 30 mg/kg and EVT801 vehicle [Soluplus (BASF)/water/hydroxypropylcellulose SL], or 30 mk/kg pazopanib and pazopanib vehicle (0.5% Hydroxypropyl methylcellulose trimellitate (HPMCT) + 0.1% Tween-80). A period of 8 hours was observed between the first and second daily dose.

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Rip1-Tag2/transgenic Mouse Models[1]
The Rip1-Tag2/transgenic mouse models were used as reported previously. Briefly, treatment of mice started at the age of 12 weeks. Mice were treated daily for 16 days, and volume of each tumor was measured and calculated as described for the 4T1 mammary carcinoma mouse model below. The tumor burden was calculated as the sum of individual tumor volumes for each mouse. For the survival study, daily treatment started at the age of 12 weeks, and mice were monitored daily to detect moribund mice for euthanasia. CD31-positive vessel density within the tumors were quantified by density index (1 vessel/mm2) measured using the Definiens software. Individual density indices were plotted as the mean ± SD for each group. Statistical significance was assessed by Kruskall–Wallis followed by Dunnet multiple comparison test.

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CT26 Ectopic Tumor Mouse Model[1]
BALB/c mice were anesthetized with ketamine (100 mg/kg) combined with xylazine (10 mg/kg) via intraperitoneal injection. A total of 5 × 104 CT26 tumor cells were suspended in 200 μL of Matrigel matrix, and then inoculated in the flank of legs. After implantation, mice were randomly separated into two groups. One group received EVT801 by oral gavage and anti-PD-1 by intraperitoneal injection in a volume of 10 mL/kg, whereas the mice in the second group served as control, and were injected with IgG isotype. PD-1 was injected at day 11, day 14, and day 18 after CT26 inoculation. EVT801 was administrated daily by oral route at 30 mg/kg from day 11 until day 21. Tumor volume was measured on days 11, 13, 15, 18, 19 and 21 following tumor cell injection. The selected groups received vehicle or EVT801 orally in a volume of 10 mL/kg. At day 21, 60 mg/kg hypoxyprobe was injected 30 minutes before sacrifice of mice. The tumors were embedded in paraffin for histology studies.

[1]. Targeting Tumor Angiogenesis with the Selective VEGFR-3 Inhibitor EVT801 in Combination with Cancer Immunotherapy. Cancer Research Communications, 2022, 2(11): 1504-1519.

The receptor tyrosine kinase VEGFR-3 plays a crucial role in cancer-induced angiogenesis and lymphangiogenesis, promoting tumor development and metastasis. Here, we report the novel VEGFR-3 inhibitor EVT801 that presents a more selective and less toxic profile than two major inhibitors of VEGFRs (i.e., sorafenib and pazopanib). As monotherapy, EVT801 showed a potent antitumor effect in VEGFR-3-positive tumors, and in tumors with VEGFR-3-positive microenvironments. EVT801 suppressed VEGF-C-induced human endothelial cell proliferation in vitro and tumor (lymph)angiogenesis in different tumor mouse models. In addition to reduced tumor growth, EVT801 decreased tumor hypoxia, favored sustained tumor blood vessel homogenization (i.e., leaving fewer and overall larger vessels), and reduced important immunosuppressive cytokines (CCL4, CCL5) and myeloid-derived suppressor cells (MDSC) in circulation. Furthermore, in carcinoma mouse models, the combination of EVT801 with immune checkpoint therapy (ICT) yielded superior outcomes to either single treatment. Moreover, tumor growth inhibition was inversely correlated with levels of CCL4, CCL5, and MDSCs after treatment with EVT801, either alone or combined with ICT. Taken together, EVT801 represents a promising anti(lymph)angiogenic drug for improving ICT response rates in patients with VEGFR-3 positive tumors.
Significance: The VEGFR-3 inhibitor EVT801 demonstrates superior selectivity and toxicity profile than other VEGFR-3 tyrosine kinase inhibitors. EVT801 showed potent antitumor effects in VEGFR-3-positive tumors, and tumors with VEGFR-3-positive microenvironments through blood vessel homogenization, and reduction of tumor hypoxia and limited immunosuppression. EVT801 increases immune checkpoint inhibitors' antitumor effects.

C19H21N5O3

367.40

367.16

C, 62.11; H, 5.76; N, 19.06; O, 13.06

1412453-70-3

71001861

Typically exists as solid at room temperature

0.3

117Ų

3

7

6

27

681

1

CCN1C(=C(C(=O)C2=C1N=C(C=C2)C#C[C@](C)(COC)O)C3=NC=CN3)N

FQPLKTQWEHDNAB-LJQANCHMSA-N

InChI=1S/C19H21N5O3/c1-4-24-16(20)14(17-21-9-10-22-17)15(25)13-6-5-12(23-18(13)24)7-8-19(2,26)11-27-3/h5-6,9-10,26H,4,11,20H2,1-3H3,(H,21,22)/t19-/m1/s1

(R)-2-amino-1-ethyl-7-(3-hydroxy-4-methoxy-3-methylbut-1-yn-1-yl)-3-(1H-imidazol-2-yl)-1,8-naphthyridin-4(1H)-one

338JH2SY4A; 1412453-70-3; 1,8-Naphthyridin-4(1H)-one 2-amino-1-ethyl-7-((3R)-3-hydroxy-4-methoxy-3-methyl-1-butyn-1-yl)-3-(1H-imidazol-2-yl)-; 1,8-Naphthyridin-4(1H)-one, 2-amino-1-ethyl-7-((3R)-3-hydroxy-4-methoxy-3-methyl-1-butyn-1-yl)-3-(1H-imidazol-2-yl)-; 2-Amino-1-ethyl-7-((3R)-3-hydroxy-4-methoxy-3-methyl-1-butyn-1-yl)-3-(1H-imidazol-2-yl)-1,8-naphthyridin-4(1H)-one; 2-Amino-1-ethyl-7-((3R)-3-hydroxy-4-methoxy-3-methylbut-1-yn-1-yl)-3-(1H-imidazol-2-yl)-1,4-dihydro-1,8-naphthyridin-4-one; 1,8-Naphthyridin-4(1H)-one, 2-amino-1-ethyl-7-[(3R)-3-hydroxy-4-methoxy-3-methyl-1-butyn-1-yl]-3-(1H-imidazol-2-yl)-; UNII-338JH2SY4A;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。