YN968D1 is a novel and selective inhibitor of vascular endothelial growth factor receptor‐2 tyrosine kinase with potent activity in vitro and in vivo

Abstract

Angiogenesis is an important process in cell development, especially in cancer. Vascular endothelial growth factor (VEGF) signaling is an important regulator of angiogenesis. Several therapies that act against VEGF signal transduction have been developed, including YN968D1, which is a potent inhibitor of the VEGF signaling pathway. This study investigated the antitumor activity of YN968D1 (apatinib mesylate) in vitro and in vivo. YN968D1 potently suppressed the kinase activities of VEGFR‐2, c‐kit and c‐src, and inhibited cellular phosphorylation of VEGFR‐2, c‐kit and PDGFRβ. YN968D1 effectively inhibited proliferation, migration and tube formation of human umbilical vein endothelial cells induced by FBS, and blocked the budding of rat aortic ring. In vivo, YN968D1 alone and in combination with chemotherapeutic agents effectively inhibited the growth of several established human tumor xenograft models with little toxicity. A phase I study of YN968D1 has shown encouraging antitumor activity and a manageable toxicity profile. These findings suggest that YN968D1 has promise as an antitumor drug and might have clinical benefits. (Cancer Sci 2011; 102: 1374–1380)

Angiogenesis is an important process that is involved in many physiological processes, including cell development and wound healing, and in pathological processes, especially cancer.^{( 1 )} As a tumor grows, it needs nutrients to support its proliferation, growth and metastasis, and the tumor will develop new capillaries from pre‐existing blood vessels to meet its metabolic demands. Activation of tumor angiogenesis is due to an imbalance between pro‐ and anti‐angiogenesis factors.^{( 2} , ^{3 )} Among the positive regulators, vascular endothelial growth factor (VEGF) signaling is one of the most prominent and best characterized mediators. Vascular endothelial growth factor regulates endothelial cell function through binding to three membrane receptor tyrosine kinases (RTK): VEGFR‐1 (Flt1); VEGFR‐2 (KDR); and VEGFR‐3 (Flt4).^{( 4 )} Among these receptors, VEGFR‐2, mainly expressed on endothelial cells, is principally responsible for mediating the mitogenic, angiogenic and permeability‐enhancing effects of VEGF.^{( 4} , ^{5 )} As tumor angiogenesis is one of the hallmarks of cancer, the inhibition of VEGF signaling has become an attractive antitumor approach.^{( 6 )}

Several strategies targeting the VEGF signaling pathway have been developed. These include neutralizing antibodies to VEGF or VEGF receptors (VEGFR), soluble VEGFR/VEGFR hybrids and small molecule VEGFR inhibitors. Several of these agents have shown promising clinical efficacy, either as a single agent or in combination with existing therapies against diverse tumor types. Bevacizumab is a monoclonal neutralizing antibody targeting VEGF, and is the first drug that inhibits VEGF signaling to be approved by USA FDA for cancer treatment, including in combination with chemotherapy for metastatic colorectal cancer, metastatic breast cancer and nonsquamous non‐small‐cell lung cancer (NSCLC),^{( 7} , ⁸ , ^{9 )} and in combination with interferon‐α for renal cell carcinoma (RCC).^{( 10 )} Both CDP791 and IMC‐1121B are humanized monoclonal antibodies that bind directly to the extracellular domain of VEGFR‐2, and are being evaluated in several ongoing clinical trials.^{( 11} , ^{12 )} Aflibercept (VEGF Trap) is a recombinant fusion protein of the human VEGFR‐1 and VEGFR‐2 extracellular domains and the Fc portion of human immunoglobulin G1.^{( 13 )} Aflibercept, which binds to all isoforms of VEGF and to placental growth factor with high affinity, might be a promising anti‐VEGFR agent. Phase III trials of aflibercept in several diseases are ongoing, including ovarian, prostate, pancreatic, colorectal and NSCLC.^{( 14 )}

A number of small molecule inhibitors of the VEGFR have been developed, which target the intracellular ATP‐binding site of RTK, preventing phosphorylation and subsequent downstream signaling.^{( 15 )} Sorafenib is a multitargeted tyrosine kinase inhibitor that has been approved for the treatment of patients with advanced RCC and hepatocellular carcinoma.^{( 16} , ^{17 )} Sunitinib is an oral multikinase inhibitor with antiangiogenic and antitumor properties that targets VEGFR, c‐kit, platelet derived growth factor receptors (PDGFR), FLT‐3, CSF‐1R and Ret, and is approved for clinical use in RCC and gastrointestinal stromal tumor.^{( 18} , ^{19 )} Several other tyrosine kinase inhibitors (TKI) with varying VEGFR selectivity are in phase II/III development, including vandetanib, vatalanib, cediranib, axitinib, motesanib, pazopanib and exelixis.^{( 14 )} Here we report another small molecule inhibitor of VEGFR‐2 tyrosine kinase, YN968D1 (apatinib mesylate, N‐[4‐ (1‐cyano‐cyclopentyl) phenyl]‐2‐(4‐pyridylmethyl) amino‐3‐pyridine carboxamide mesylate) (Fig. 1), provided by Shanghai Hengrui Pharmaceutical Co., Ltd (Shanghai, China). YN968D1 impairs the function of human umbilical vein endothelial cells (HUVEC), including proliferation, migration and tube formation, and blocks rat aortic ring budding in vitro. Furthermore, YN968D1 shows antitumor efficacy against a variety of experimental tumor models. YN968D1 is currently in phase II/III evaluation for the treatment of metastatic gastric adenocarcinoma.

Figure 1.

 Chemical structure of YN968D1.

Materials and Methods

Substances.  The agents used in the present study were YN986D1, sunitinib, oxaliplatin, 5‐fluorouracil (5‐FU), doxorubicin and docetaxel. For the in vitro assays, the stock solution containing YN986D1 and sunitinib were prepared in dimethyl sulfoxide. For the in vivo studies, YN986D1 and oxaliplatin were diluted in 0.5% (w/v) carboxymethyl cellulose and 5% (w/v) glucose solution. 5‐Fluorouracil and doxorubicin were dissolved in physiological saline. Docetaxel was suspended in a mixture of Cremophor EL and ethanol.

Cells and culture conditions.  The following human tumor cell lines were used: Ls174t, HCT 116, SGC‐7901, HT‐29, A549, NCI‐H460, Mo7e, A431, BT474 and NIH‐3T3 (Cell Bank, Chinese Academy of Science, Shanghai, China). The cells were cultured in RPMI‐1640 or DMEM supplemented with 10% FBS at 37°C with 5% carbon dioxide. Primary HUVEC were isolated from segments of normal‐term cords by digestion with type I collagenase, and were pooled and cultured in Medium 199 (M199) supplemented with 20% FBS and endothelial cell growth factor (Sigma, St Louis, MO, USA). Cells in the exponential growth phase were used in the experiments.

Enzyme‐linked immunosorbent assay.  The inhibitory activity of YN968D1 against tyrosine kinases was determined using ELISA methodology described previously.^{( 20 )} VEGFR‐2 and PDGFR were purchased from Upstate Biotechnology (Lake Placid, NY, USA) and EGFR1 and Ret from Sigma; Her‐2, c‐kit and c‐src were activated intracellular protein tyrosine kinases expressed by Bab‐to‐Bac Baculovirus Expression Vector System and purified by Ni‐NTA spin columns (Amresco, Solon, OH, USA). The optical density was measured at 490 nm using VERSAmax (Molecular Devices, Sunnyvale, CA, USA). The inhibitory activity was expressed as IC₅₀, which was calculated from three independent experiments by the Logit method.

Cell proliferation assays.  The HUVEC were seeded into 96‐well plates. After 24 h of incubation, cells were exposed to the test agents (vehicle as control) together with 20 ng/mL VEGF or 20% FBS for another 72 h. After fixation with 10% trichloroacetic acid, the cells were stained with 0.4% sulforhodamine B for 30 min at 37°C and then washed with 1% acetic acid. Tris was added to dissolve the complex, and the optical density was measured at 520 nm.

Migration assay.  The bottoms of the Transwell inserts (8 μm; Corning Costar Corp., Cambridge, MA, USA) were coated with 1% glutin and balanced with serum‐free M199 for 1 h. Then, 1.5 × 10⁴ HUVEC were trypsinized, resuspended in 2% FBS M199 with various concentrations of test agents, and seeded into the top chamber of each insert. Six hundred microlitres M199 supplemented with or without 20% FBS was injected into the lower chambers. After incubation for 6 h at 37°C, cells that had migrated to the bottom of the membrane were fixed with 90% ethanol and stained in a dyeing solution containing 0.1% crystal violet. The migrated cells were then imaged via an inverse microscope (Olympus IX70; Olympus Optical Co., Ltd, Osaka, Japan).

Tube formation assay.  Thawed matrigel (Becton Dickinson Labware, Bedford, MA, USA), 60 μL per well, was added to a prechilled 96‐well sterile plate and incubated at 37°C for 1 h. Then, 1.5 × 10⁴ HUVEC per well suspended in M199 culture medium containing 20% FBS were added into each well, together with various concentrations of test agents. After incubation for 8 h at 37°C, cells were imaged using a high magnification field (Olympus Optical Co., Ltd.).

Rat aortic ring assay.  The aorta of Sprague Dawley rats (6 weeks) was isolated under ether anesthesia, rinsed with serum‐free M199, and cut into 1‐mm ring sections. The sections were then placed in a 96‐well plate, embedded with 70 μL Matrigel for each well, and incubated at 37°C for 1 h. The serum‐free M199 medium was subsequently added into each well, with or without 20% FBS, and various concentrations of test agents. On the sixth day, images were taken through an inverse microscope.

Western blot analysis.  Serum‐starved HUVEC, NIH‐3T3, Mo7e and A431 cells were treated with different concentrations of test agents for 1.5 h and then stimulated with 50 ng/mL VEGF165, 10 ng/mL PDGF‐BB, 10 ng/mL stem cell factor (SCF) and 10 ng/mL EGF (R&D systems Inc., Minneapolis, MN, USA) for 5, 10, 10 and 5 min, respectively. BT474 cells naturally expressing Her‐2 were treated with test agents for 1.5 h. Cell lysates containing equal amounts of protein were separated by SDS‐PAGE and transferred to PVDF membranes (Amersham Life Science, Arlington Heights, IL, USA). Blots were probed with the appropriate primary antibodies, obtained from Cell Signaling Technology (Beverly, MA, USA) except for tubulin (Sigma), and then probed with secondary antibodies (Calbiochem, Darmstadt, Germany). Immunoreactive proteins were visualized using enhanced chemiluminescence reagents.

Nude mouse human tumor xenograft model.  The effects of YN968D1 on tumor growth were tested against various human tumors grown subcutaneously in BALB/cA nude mice. Tumor growth was initiated by subcutaneous inoculation of cells into mice. Tumors were allowed to establish and grow to 100–300 mm³, at which time the mice were randomized into experimental groups. YN968D1 was administered once daily by oral gavage for the indicated periods (Table 1). In combination treatment experiments, mice were administered YN968D1 alone by oral gavage; 5‐FU, oxaliplatin, docetaxel and doxorubicin alone by intravenous injection; or YN968D1 in combination with each cytotoxic drug at the indicated dose and schedule (Table 2). Tumor volume and bodyweight were monitored every other day or every 3 days, with the means indicated for groups of six (treated) or 12 (vehicle control) animals. Tumor volumes were determined by measuring the largest diameter (a) and its perpendicular (b) according to the formula (a × b²)/2. The evaluation index for inhibition was the relative tumor growth ratio according to the equation: T/C (%) = mean increase of tumor volumes of treated groups/mean increase of tumor volumes of control groups × 100%.

Table 1.

  In vivo efficacy of YN968D1 alone against a variety of established tumor xenografts

Tumor model	Tumor origin	YN968D1 dose (mg/kg)	Route and schedule	T/C (%)	P‐value
NCI‐H460	Lung	100	p.o. qd × 14	57	0.017
		200		17	<0.001
A549	Lung	50	p.o. qd × 14	73	0.198
		100		41	0.012
		200		17	0.001
HCT 116	Colon	50	p.o. qd × 21	63	0.009
		100		40	<0.001
		200		14	<0.001
HT‐29	Colon	50	p.o. qd × 21	59	0.005
		100		42	<0.001
		200		18	<0.001
Ls174t	Colon	50	p.o. qd × 14	72	0.014
		100		43	<0.001
		200		8	<0.001
SGC‐7901	Stomach	50	p.o. qd × 18	58	0.067
		100		33	0.006
		200		12	0.001

T/C (%) = mean increase of tumor volumes of treated groups/mean increase of tumor volumes of control groups × 100%. n = 6 per treated group or 12 for the vehicle control group. p.o., per os (by mouth); qd, quaque die (every day).

Table 2.

  In vivo efficacy of YN968D1 in combination with cytotoxic agents against established tumor xenografts

Tumor model	Tumor origin	Agent	Dose (mg/kg)	Route and schedule	T/C (%)
NCI‐H460	Lung	YN968D1	150	p.o. qd × 14	46
		Docetaxel	12	i.v. q4d × 3	37
		YN968D1 +docetaxel			7*,**
		Doxorubicin	10	i.v. single dose	35
		YN968D1 +doxorubicin			14*,**
Ls174t	Colon	YN968D1	150	p.o qd × 14	28
		Oxaliplatin	6	i.v. q4d × 3	54
		YN968D1 +oxaliplatin			5*,**
		YN968D1	75	p.o. qd × 14	60
		5‐Fu	50	i.v. q4d × 3	60
		YN968D1 +5‐Fu			40*,**

*P < 0.05 versus YN968D1 alone. **P < 0.05 versus cytotoxic drugs alone. n = 6 per treated group or 12 for the vehicle control group. Results from all treatment groups were significant compared with vehicle control (P < 0.05). p.o., per os (by mouth); qd, quaque die (every day); q4d, once a day for four days.

Immunohistochemistry assay.  Immunohistochemistry was used to determine vessel density by analyzing the expression of CD31, an endothelial marker. Briefly, nude mice xenografted with NCI‐H460 tumor were treated with 200 mg/kg YN968D1 by oral garage for 14 days and tumor sections were prepared from formalin‐fixed and paraffin‐embedded tumor tissues. Slides were treated with 3% H₂O₂ for 10 min and then incubated in 2% goat serum for 20 min to block the nonspecific antibody binding. Slides were stained with anti‐CD31 antibody (Sigma) at room temperature for 2 h, followed by treatment with biotinylated goat anti‐mouse IgG and SABC complex at 37°C for 30 min. Finally, diaminobenzidine tetrachloride was used for color development and the slides were counterstained with hematoxylin. Positive cells (brown color, indicated by arrows) in images were measured with Image‐Pro Plus software (Media Cybernetics, Silver Spring, MD, USA).

Phase I clinical trial.  A phase I study was performed to determine the maximum tolerated dose (MTD), pharmacokinetic (PK) parameters, and to observe the safety and efficacy of YN968D1 for patients with advanced solid tumors (ClinicalTrials.gov number: NCT00633490).^{( 21 )} Study approval was provided by the Medical Ethical Committees of the Fudan University Cancer Hospital, Shanghai, China. Written informed consent was obtained from all patients.

Statistical analysis.  Data are represented as mean ± standard error. Differences between groups were calculated by Student’s t‐test. Statistical significance was defined as a P‐value of <0.05 for a two‐tailed test.

Results

Inhibition of tyrosine kinase activities.  In vitro enzyme experiments showed that YN968D1 was an even more selective inhibitor of VEGFR‐2 than sunitinib, with an IC₅₀ of 0.001 μM and 0.005 μM, respectively. YN968D1 could also potently suppress the activities of Ret, c‐kit and c‐src with an IC₅₀ of 0.013 μM, 0.429 μM and 0.53 μM, respectively (Table 3). YN968D1 had no significant effects on EGFR, Her‐2 or FGFR1 in concentrations up to 10 μM.

Table 3.

  In vitro kinase activity of YN968D1

Kinase	IC50 (μM)
	YN968D1	Sunitinib
VEGFR‐2	0.001	0.005
c‐kit	0.429	0.001
PDGFRα	>1	0.013
Ret	0.013	0.072
c‐src	0.53	2.2
EGFR	>10	>10
Her‐2	>10	>10
FGFR1	>10	0.51

Inhibition of growth factor‐stimulated receptor phosphorylation at the cellular level.  In HUVEC, VEGF‐stimulated phosphorylation of VEGFR‐2/KDR was decreased by YN968D1 in a concentration‐dependent manner. YN968D1 completely blocked VEGFR‐2 activation at a concentration of 0.1 μM, which was comparable to sunitinib. The phosphorylation of ERK1/2, a downstream of VEGF signaling, was inhibited concomitantly (Fig. 2A). In addition, YN968D1 suppressed the phosphorylation of c‐kit and PDGFRβ in Mo7e and NIH‐3T3 cells stimulated with the relevant ligand, respectively, in a concentration‐dependent manner. Both concentrations of YN968D1 required to completely block c‐kit and PDGFRβ activation were a bit higher than those of sunitinib (Fig. 2B,C). YN968D1 had no effect on the phosphorylation of EGFR and Her‐2 at a concentration of 10 μM (Fig. 2D,E).

Figure 2.

 Effects of YN968D1 on various growth factor‐stimulated receptor phosphorylation at the cellular level detected by western blot analysis. VEGFR, ERK1/2, c‐kit and PDGFR phosphorylation was suppressed by YN968D1 in a dose‐dependent manner, which were comparable with sunitinib (A–C). YN968D1 had no effect on the phosphorylation of EGFR and Her‐2, and lapatinib served as a positive control (D,E). Tubulin was used as a loading control. VEGF, vascular endothelial growth factor. VEGFR, vascular endothelial growth factor receptors; PDGF, platelet derived growth factor; PDGFR, PDGF receptors; SCF, stem cell factor.

Inhibition of proliferation, migration and tube formation of HUVEC in vitro and blocking of rat aortic ring budding.  YN968D1 slightly inhibited proliferation of HUVEC stimulated by 20% FBS (IC₅₀ = 23.4 μM), whereas YN968D1 significantly inhibited proliferation stimulated by 20 ng/mL VEGF (IC₅₀ = 0.17 μM). The IC₅₀ values of sunitinib were lower under the same conditions (7.4 μM and 0.034 μM, respectively) (Fig. 3A).

Figure 3.

 Inhibition of vascular endothelial growth factor (VEGF)‐stimulated HUVEC proliferation, HUVEC tubule formation, HUVEC migration and microvessel outgrowth from rat aortic ring by YN968D1. (A) HUVEC were incubated with test agents together with 20% FBS or 20 ng/mL VEGF. The inhibitory activity was expressed as IC₅₀. (B) HUVEC suspended in M199 medium with 20% FBS were added into solidified matrigel, with different test agents for 8 h, and the cells were imaged using a high magnification field. The tube formation ability of HUVEC was impaired by 1 μM YN968D1 and sunitinib. (C) Transwell migration assays using the Corning chamber showed that the migration of HUVEC induced by 20% FBS was inhibited by 1 μM YN968D1 and sunitinib. (D) The aortic rings were embedded in matrigel and incubated in supplemented media at 37°C, 5% CO₂ for 6 days. Images taken through an inverse microscope showed YN968D1 and sunitinib suppressed vessels sprouting from the rat aorta rings. Columns, mean; bars, SE. *P < 0.05 versus vehicle control.

It has been reported that VEGF cannot induce the tube formation of HUVEC when cultured on matrigel,^{( 22 )} so 20% FBS was selected as a stimulating factor to evaluate the effect of YN968D1 on the tube formation of HUVEC. YN968D1 impaired the ability of HUVEC to form tubes in a dose‐dependent manner. When treated with 1 μM of YN968D1, there was no enclosed tube. Similarly, at a concentration of 1 μM, sunitinib also inhibited tube formation completely (Fig. 3B).

Figure 3(C) shows that 1 μM YN968D1 significantly inhibited the migration of HUVEC induced by FBS, but did not affect proliferation of HUVEC, indicating that the inhibitory effect of YN968D1 on FBS‐induced migration was not due to the suppression of proliferation. At a concentration of 1 μM, sunitinib also inhibited the migration of HUVEC.

As shown in Figure 3(D), there were few microvessels surrounding the rat aortic ring when cultured in serum‐free medium. When stimulated with FBS, the microvessels were greater than the control. YN968D1 significantly suppressed budding of the aortic ring in a concentration‐dependent manner, and the inhibition was comparable with that of sunitinib.

Potency of antitumor effect in vivo.  The antitumor potential of YN968D1 was evaluated in six human tumor xenografts in immunodeficient mice. Once‐daily oral administration of YN968D1 produced a dose‐dependent inhibition of tumor growth in all tumor models examined (Table 1). Statistically significant growth inhibition was obtained with 50 mg/kg/day YN968D1 in three of five tumor xenografts tested. Each tumor xenograft model was significantly growth inhibited by YN968D1 at the dose of 100/kg/day. Similar tumor growth inhibition was observed (T/C%, 8% to 18%) in mice following treatment with YN968D1 at the dose of 200/kg/day. Full growth inhibition profiles are shown for three of the xenografts (Fig. 4A). Compared with the control animals, no effect of YN968D1 treatment on bodyweight was observed at any dose level, which suggested that YN968D1 was well tolerated.

Figure 4.

 Antitumor activity of YN968D1 against human tumor xenografts in nude mice. (A) YN968D1 inhibited growth of established NCI‐H460 human lung tumors, HCT 116 human colon tumors, or SGC‐7901 human gastric tumors in nude mice. Tumor volume was measured on the indicated days, with the mean tumor volume indicated for groups of 6 (treated) or 12 (vehicle control) animals. *P < 0.05 versus vehicle control. (B) YN968D1 in combination with docetaxel or oxaliplatin showed synergistic tumor growth inhibition effects against NCI‐H460 and Ls174t xenografts, respectively. *P < 0.05 versus YN968D1 alone. **P < 0.05 versus cytotoxic drugs alone. Data are represented as mean ± standard error. (C) Angiogenesis was inhibited markedly by YN968D1 within NCI‐H460 xenograft tumor tissues as evidenced by CD31 staining (positive cells, brown color, indicated by arrows). Columns, mean; bars, SE. *P < 0.05 versus vehicle control.

Inhibition of tumor growth in combination with chemotherapy.  The antitumor efficacy of YN968D1 was further assessed in combination with docetaxel or doxorubicin (in lung cancer models), oxaliplatin or 5‐FU (in colon cancer models). Each agent alone significantly inhibited tumor growth compared with the vehicle‐treated groups (P < 0.05). In mice bearing NCI‐H460 tumor xenografts, the antitumor activity of either YN968D1 or docetaxel alone was enhanced by the combination of the two agents, resulting in a 93% inhibition after 14 days of dosing (P < 0.05 for combination therapy versus either YN968D1 or docetaxel alone). The combination of YN968D1 and doxorubicin against the NCI‐H460 xenograft also exerted stronger tumor growth inhibition than that attained by either drug alone. Additionally, the combination of YN968D1 with oxaliplatin or with 5‐FU displayed synergistic inhibition effects on the growth of Ls174t xenografts compared with each treatment alone (Table 2, Fig. 4B). Moreover, the results from immunohistochemistry indicated that the expression of CD31, an endothelial marker, significantly decreased after YN968D1 treatment, suggesting that YN968D1 inhibits tumor growth in vivo mainly through inhibiting angiogenesis (Fig. 4C).

Preliminary antitumor activity of YN968D1 in clinical practice.  A phase I study was performed to determine the MTD, safety profile, pharmacokinetic (PK) parameters and preliminary antitumor activity of YN968D1. YN968D1 was well tolerated and showed encouraging antitumor activity across a broad range of malignancies.^{( 21 )} A 46‐year‐old woman with metastatic rectal cancer involving the liver and lung treated at the 750 mg daily dose level from November 2007 had a partial response (PR). Compared with the baseline computed tomography (CT) scan, the same lesions at day 53 showed cavity formation and density decrease. Further CT scan confirmed the PR (Fig. 5).^{( 21 )} Due to hand‐foot syndrome, diarrhea and oral ulcer, she received a reduced dose of 500 mg daily until progression (on day 255). This activity of YN968D1 in patients with a broad range of advanced solid tumors, especially gastric and colorectal cancers, deserves further investigation.

Figure 5.

 Pulmonary computed tomography (CT) images of a patient with metastatic rectal cancer involving the liver and lung who was treated with YN968D1 and obtained a partial response.^{( 21 )} (A) Baseline CT image. (B) CT image 53 days after treatment.

Discussion

Vascular endothelial growth factor signaling plays a pivotal role in the angiogenic process of solid malignancies.^{( 1 )} The most pro‐angiogenic effects of VEGF are thought to be mediated through binding to VEGFR‐2. VEGFA, VEGFC, VEGFD and VEGFE can bind to VEGFR‐2 and induce activation of the downstream molecules of VEGFR‐2. When stimulated by VEGF, VEGFR‐2 is auto‐phosphorylated at the carboxy terminal tail and kinase‐insert region.^{( 15 )} The phosphorylation of specific sites creates binding sites for the SH2 domains of various signaling molecules and has subsequent effects on the vascular endothelium, including cell proliferation, migration, permeability and survival. VEGFR‐2 is mainly expressed on vascular endothelial cells and upregulated in a wide range of tumors.^{( 23 )} Overexpression of VEGF and VEGFR correlates with increased tumor growth rate, microvessel density, proliferation, tumor metastatic potential and poor patient prognosis in a variety of malignancies.^{( 23 )} Therefore, inhibition of VEGFR signaling is an attractive therapeutic target in clinical practice.

In this report, the characterization of YN968D1, a novel, orally bioavailable, selective inhibitor of VEGFR‐2 is described. In addition, YN968D1 has been shown to inhibit a subclass of RTK, including, c‐kit, Ret and c‐src. YN968D1 is a potent and selective inhibitor against VEGFR‐2 with an IC₅₀ concentration of 0.001 μM, which is even lower than sunitinib as reported previously.^{( 24 )} It has been shown to more selectively inhibit HUVEC proliferation in response to VEGF, which is comparable with sunitinib. Further study indicated that YN968D1 inhibited the migration and tube formation of HUVEC with a concentration equal to sunitinib. Moreover, YN968D1 could block the formation of rat aortic ring, which is a better verification of its anti‐angiogenic activity, as rat aortic ring budding mimics the multiple steps of angiogenesis in vitro, including proliferation, migration and tube formation of vascular endothelial cells. This inhibitory effect occurred at a YN968D1 dose (1 μM) that was significantly lower than that required to affect the proliferation of endothelial cells. YN968D1 blocked VEGFR‐2, c‐kit and PDGFRβ activation in a concentration‐dependent manner in several cell lines, which was consistent with the in vitro enzyme experiments. VEGFR‐2 stimulates ERK phosphorylation and proliferation via a PKC‐dependent pathway involving activation of PLC‐γ.^{( 25 )} YN968D1 reduced the phosphorylation of ERK through binding to VEGFR‐2, thus preventing endothelial cell proliferation.

By virtue of its inhibitory activity against VEGFR‐2 tyrosine kinase, the potential antitumor effect of YN968D1 in vivo was further investigated. In the present study, YN968D1 inhibited the growth of a broad range of human tumor xenografts in a significant dose‐dependent manner. These models represent a diversity of tumor histology and genotype. YN968D1 did not inhibit the growth of these cancer cell lines in vitro (data not shown), suggesting that the antitumor activity is not due to a direct inhibition of cell proliferation, but via an anti‐angiogenic mechanism, which is different from chemotherapy agents. The in vivo antitumor effect of YN968D1 is generally attributable to its potent activity against VEGFR‐2. PDGFR, mainly expressed on the pericytes, is also a contribution to the process of tumor neovascularization.^{( 26 )} Modulation of these key mediators should provide more inhibition of angiogenesis. The experimental outcomes also suggested that YN968D1 was well tolerated when achieving effective antitumor activity, without weight loss.

Targeting angiogenesis agents could benefit patients in a synergistic way when combined with chemotherapy. It has been hypothesized that anti‐angiogenesis therapy does not only reduce the formation of new blood vessels, but also induces normalization of tumor blood vessels and facilitates the delivery of cytotoxic drugs.^{( 27 )} Combination treatments of anti‐angiogenic therapy plus conventional chemotherapeutic agents are currently being investigated and have proven clinically successful in several cancers.^{( 7} , ⁸ , ^{9 )} Specifically, our data demonstrated a synergistic inhibition effect of YN968D1 when combined with several chemotherapeutic drugs in tumor xenografts. The detailed interaction mechanisms between YN968D1 and these cytotoxic drugs are still unclear, and should be explored in further analyses. The possible therapeutic benefit of YN968D1 in combination with chemotherapy should be further evaluated in clinical practice.

In summary, YN968D1 is a highly potent and selective VEGF signaling inhibitor. Due to the inhibitory activity against VEGF signaling, YN968D1 effectively inhibited the proliferation, migration and tube formation of HUVEC. Additionally, YN968D1 blocked the microvessel budding of rat aortic ring. YN968D1 showed antitumor efficacy in vivo when administrated alone or in combination with chemotherapy against a variety of established tumor xenografts with good tolerance.

Preliminary investigation of YN968D1 in patients with advanced solid malignancies in a phase I study has shown encouraging antitumor activity (80% of overall control rate) and a manageable toxicity profile.^{( 21 )} Several patients achieved complete or partial response. Compared with sunitinib, YN968D1 showed specific inhibition of VEGFR‐2 tyrosine kinase activity but more tolerability (MTD 850 mg/day), which makes it more clinically acceptable. Based on the promising phase I clinical result, a phase II/III study with single agent YN968D1 is currently being evaluated in patients with metastatic gastric adenocarcinoma who failed two lines of chemotherapy (NCT00970138). More studies should be developed to facilitate better understanding of YN968D1 in the future.

Disclosure Statement

The authors have no conflict of interest.