Cell experiment:

Primary HUVECs or HLECs in exponential phase are suspended in 100 μL of RPMI-1640 media containing 0.5% FBS, and seeded at 5000 cell/well in 96-well plates pre-coated with 0.2% gelatin or fibronectin, and incubated overnight in a 5% CO2, 37℃ incubator. Fruquintinib and VEGF-A165 or VEGF-C (50 ng/mL) are added and incubated for 48 hours. Viability of the cells is determined using CCK-8 assay format[1].

Animal experiment:

Mice: The patient derived xenograft models are established after the primary tumor adopted serial passages in vivo. Once tumors have grown to 100-300 mm3, the animals are randomly assigned with 6-8 animals per group. The mice are treated orally with the vehicle (control group) or fruquintinib at a dose range of 0.5-20 mg/kg suspended in the vehicle (treated group) once daily for 3 weeks. In combination studies, docetaxel (Taxotere, 5 mg/kg) or oxaliplatin (10 mg/kg) is administered to nude mouse via intravenous injection, once a week. Tumor size and body weights are measured 3 times a week. Tumor volumes (TV) are calculated[1].

IC50: 33 nmol/L, 35 nmol/L and 0.5 nmol/L for VEGFR1, 2, 3

VEGF/VEGFR signal has been proven to be an important target for development of novel cancer therapies. One challenging aspect in VEGFR inhibitors is to achieve sustained target inhibition at tolerable doses previously seen only with the long-acting biologics. Fruquintinib is a potent and highly selective small molecule inhibitor of VEGFR 1, 2, 3 tyrosine kinases for cancer therapy.

In vitro: Fruquintinib was found to inhibit VEGFR2 with an IC50 of 25 nmol/L. The kinase selectivity of fruquintinib was evaluated against a panel of 253 kinases. The results showed that fruquintinib inhibited VEGFR family members with weak inhibition of RET, FGFR-1 and c-kit kinases [1].

In vivo: Anti-tumor activity of fruquintinib was evaluated in a variety of tumor xenografts. The results from gastric cancer BGC-823 model seemed to indicate that the drug concentration needs to be at least maintained above EC85 for around 8 hours in order to achieve >80% tumor growth inhibition. BGC-823 was found to be most sensitive to fruquintinib [1].

Clinical trial: Fruquintinib (HMPL-013) is currently in Phase II clinical studies. Analysis of Phase I pharmacokinetic data revealed that at the maximum tolerated dose of once daily oral administration fruquintinib achieved complete VEGFR2 suppression for 24 hours/day [1].

Reference:
[1] Sun Q, Zhou J, Zhang Z, Guo M, Liang J, Zhou F, Long J, Zhang W, Yin F, Cai H, Yang H, Zhang W, Gu Y, Ni L, Sai Y, Cui Y, Zhang M, Hong M, Sun J, Yang Z, Qing W, Su W, Ren Y.  Discovery of fruquintinib, a potent and highly selective small molecule inhibitor of VEGFR 1, 2, 3 tyrosine kinases for cancer therapy. Cancer Biol Ther. 2014;15(12):1635-45.