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

In HEK293 cells, VEGFR-1/2/3 autophosphorylation is dose-dependently inhibited by EVT801 (10 nM-1 μM), with IC50s of 39 nM (VEGFR-3), 2130 nM (VEGFR-1), and 260 nM (VEGFR-2), respectively[1]. Proliferation of VEGFR-3-positive cells, such as human lymphatic microvascular endothelial cells (hLMVEC), is inhibited by EVT801 (1 nM-1 μM). EVT801 has dose-dependent inhibitory concentrations (IC50s) of 15 nM for VEGF-C, 8 nM for VEGF-D, and 155 nM for VEGF-A that prevent the induction of hLMVECs proliferation[1]. VEGFR-3-positive tumor cells' proliferation and tumor growth are inhibited by EVT801 (1 μM)[1].

EVT801 (30 mg/kg; po; twice daily for 7 d) inhibits tumors that are positive for VEGFR-3 in mouse models, including the RT-001-HAM Subcutaneous Patient-derived xenograft (PDx) Tumor Mouse Model, the 4T1 Mammary Carcinoma Mouse Model, the N-Diethylnitrosamine-Induced Hepatocarcinoma Mouse Model, the NCI-H1703 Subcutaneous Xenograft Tumor Mouse Model, the Rip1-Tag2/transgenic Mouse Models, and the CT26 Ectopic Tumor Mouse Model. In addition to tumor cells found in endothelial malignancies, EVT801 is expressed in blood arteries containing primary tumors and metastases of kidney cancer[1].

Cell Proliferation Assay[1]
Cell Types: VEGFR-3-positive cells, human lymphatic microvascular endothelial cells (hLMVEC)
Tested Concentrations: 1 nM-1 μM
Incubation Duration:
Experimental Results: demonstrated a maximum inhibition of 74%, 100%, and 65% against VEGF -C, VEGF-D, VEGF-A induction, respectively.

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

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)