Discovery of fruquintinib, a potent and highly selective small molecule inhibitor of VEGFR 1, 2, 3 tyrosine kinases for cancer therapy

Figures & data

Table 1. VEGFR kinase activity and selectivity profile and bioactivities

Kinase assay	IC50 (nmol/L) or Inhibition rate (%)
Biochemical Activities
VEGFR2 （KDR）	35* (25)
VEGFR3 （Flt4）	0.5*
VEGFR1（Flt1）	33*
Ret	128*
FGFR1	181*
c-kit	458*
Flt3	>10000
PDGFRβ	>10000
EGFR	>30000
Tie2	>10000
c-Met	>10000
EphB4	>3000
Akt	>3000
CHK1	>10000
CDK1	>10000
CDK2	>10000
CDK5	>10000
Cell-based activity
bFGF stimulated p-FGFR1 in HUVEC	>1000
VEGF-A stimulated p-KDR in HEK293-KDR	0.6 ± 0.2, n = 3
VEGF-C stimulated p-VEGFR3 in HLEC	1.5
VEGF-A dependent HUVEC proliferation  VEGF-C dependent HLEC proliferation	1.7 4.2
HUVEC tube formation	94% at 300 nmol/L
Anti-angiogenesis activity
Chorioallantoic Membrane (CAM)	strong inhibition at 0.1 and 1 nmol/egg

* IC₅₀ provided by Millipore using ³²p-ATP incorporation, and other biochemical IC₅₀s were from HMP based on Z-lyte assay, except c-Met using fluorescence polarization method.

Figure 1. Fruquintinib is a highly selective and potent VEGFR1, 2, 3 kinase inhibitor. (A) Chemical structure of fruquintinib. (B) Kinome selectivity of fruquintinib at 1 μmol/L against 253 kinases using ³²p-ATP incorporation method generated at Millipore. The Kinome tree was downloaded from http://www.cellsignal.com. Percentage (%) denoted the inhibition of fruquintinib at 1 μmol/L to the recombinant kinases. Over 90% inhibition was observed for 3 VEGFR family members (1, 2, 3) and 70～90% inhibition on 4 other kinases, including Fms(Y969C), Ret, and FGFR1 and little effect on remaining kinases tested. IC₅₀s were generated for the kinases of interest and shown in Table 1.

Figure 2. Inhibition on VEGF stimulated activation of KDR and VEGFR3. (A) Fruquintinib inhibited VEGF-A-stimulated KDR phosphorylation and downstream signaling in HUVEC. (b) Fruquintinib abrogated VEGF-A-stimulated KDR phosphorylation and downstream signaling in HEK-293-KDR cell line. (C) Fruquintinib suppressed VEGF-C stimulated VEGFR3 phosphorylation in VEGF-C-stimulated HLEC.

Figure 3. Fruquintinib inhibited HUVEC tubule growth and CAM angiogenesis. Tube formation was suppressed significantly after treatment with fruquintinib at 0.3 μmol/L for 18 hours (A and B) No cytotoxicity was seen at the same concentration of fruquinitib in HUVECs. The plates were incubated for 3 hours at 37°C and fluorescence value was read at Ex 530 nm and Em 590 nm on Tecan (C) Fruquintinib displayed strong inhibition on the development of new blood vessels in the chick embryo (D) Left and right panels, as arrows indicated, were treated with saline and compound, respectively. Pseudolarix acid B was used as a positive control.

Figure 4. Fruquintinib inhibited p-KDR in lung tissues of mice. (A) Fruquintinib inhibited VEGF-A induced p-KDR in lung tissues. Each group was composed of 3 mice (m1, m2, m3). Animals were treated as described in Method section. (B) Fruquintinib concentration in plasma (purple line) in relation to its inhibition on p-KDR phosphorylation (green bar).

Table 2. Anti-tumor activity of fruquintinib in multiple xenograft tumor models

Tumor origin	Xenograft model	Dose (mg/kg)	%TGI (% regression)	Significance (t- test, two-tailed vs control)	Body weight change (%)*
Gastric cancer	BGC823 (exp. 1)	0.5	62.3	<0.05	+1.9
		1	74.3	<0.05	+4.9
		2	95.4	<0.01	+3.2
Gastric cancer	BGC-823 (exp. 2)	2	98.6	<0.01	+5.8
		5	106.5(24.1)	<0.01	+6.9
		20	113.3 (48.6)	<0.01	+4.6
Colon cancer	HT-29	0.77	46.4	<0.05	+4.2
		1.92	60.8	<0.01	+3.5
		4.8	80.6	<0.01	+7.4
		12	89.2	<0.01	+7.2
Renal clear cell	Caki-1	0.8	34.0	<0.05	+9.5
carcinoma		2	51.5	<0.01	+6.7
		5	63.2	<0.01	+4.6
Lung cancer	NCI-H460	0.77	40.9	<0.05	+8.1
		1.92	56.9	<0.01	+8.5
		4.8	71.8	<0.01	+6.6
		12	79.3	<0.01	+7.3
Gastric cancer	GAS1T0113	1	44.0	<0.05	−3.6
		Taxotere	46.3	<0.05	−20.9
		1+Taxotere	73.4	<0.01	−21.8
Colon cancer	COL1T0117	1	36.3	<0.05	−0.2
		Oxaliplatin	23.9	>0.05	−12.2
		1+Oxaliplatin	68.1	<0.01	−12.6

* , body weight change was calculated as (body weight_final / body weight_initial −1) × 100%.

Figure 5. Fruquintinib inhibited BGC-823 and Caki-1 tumor growth and anti-angiogenesis in tumor tissues. (A) Dose-dependent tumor growth inhibition in BGC-823 tumor following once daily oral treatment of fruquintinib at 0.5, 1 and 2 mg/kg. (B) Anti-tumor effect of fruquintinib at 2, 5 and 20 mg/kg in BGC-823. BGC-823 tumor was regressed by fruquintinib at 5 and 20 mg/kg. (C) Plasma exposures of fruquintinib at 2, 5 and 20 mg/kg in BGC-823 tumor bearing mice following consecutive oral daily dosing. The red line indicated the plasma EC85 for p-KDR inhibition in nude mouse. (D) Dose-dependent tumor growth inhibition in Caki-1 tumor model. (E) CD31 immunohistochemistry staining in Caki-1 xenografts. The pictures were taken at 200× magnification under microscope. Vascular vessels were stained as brown, representing the tumor angiogenesis. (E) Semi-quantification of IHC image. The percentage of CD31 positive area to tumor area was analyzed with NIKON BR-3.0 software. *P < 0.05, **P < 0.01 compared with vehicle group.

Figure 6. Combination of fruquintinib with chemo drugs shows enhanced anti-tumor effect in PDX models. (A) Fruquintinib in combination with taxotere (docetaxel) in gastric cancer PDX model. (B) Fruquintinib in combination with oxaliplatin in colon cancer PDX model. Percentage (%) represented tumor growth inhibition (TGI) rate. Two-tailed t-test for fruquintinib treatment versus the control group: *, P < 0.05; ^**, P < 0.01. The combination vs. single agent: ^#, P < 0.05; ^##, P < 0.01.

Huang YW, Xu LQ, Luo RZ, Huang X, Hou T, Zhang YN. VEGF-c expression in an in vivo model of orthotopic endometrial cancer and retroperitoneal lymph node metastasis. Reprod Biol Endocrinol 2013; 11:49; PMID:23693075; http://dx.doi.org/10.1186/1477-7827-11-49

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