Anti-angiogenic and anti-tumor effects of TAK-593, a potent and selective inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptor tyrosine kinase

Abstract

We recently reported that TAK-593, a novel imidazo[1,2-b]pyridazine derivative, is a highly potent and selective inhibitor of the vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) receptor tyrosine kinase families. Moreover, TAK-593 exhibits a uniquely long-acting inhibitory profile towards VEGF receptor 2 (VEGFR2) and PDGF receptor β (PDGFRβ). In this study, we demonstrated that TAK-593 potently inhibits VEGF- and PDGF-stimulated cellular phosphorylation and proliferation of human umbilical vein endothelial cells and human coronary artery smooth muscle cells. TAK-593 also potently inhibits VEGF-induced tube formation of endothelial cells co-cultured with fibroblasts. Oral administration of TAK-593 exhibited strong anti-tumor effects against various human cancer xenografts along with good tolerability despite a low level of plasma exposure. Even after the blood and tissue concentrations of TAK-593 decreased below the detectable limit, a pharmacodynamic marker (phospho VEGFR2) was almost completely suppressed, indicating that its long duration of enzyme inhibition might contribute to the potent activity of TAK-593. Immunohistochemical staining indicated that TAK-593 showed anti-proliferative and pro-apoptotic effects on tumors along with a decrease of vessel density and inhibition of pericyte recruitment to microvessels in vivo. Furthermore, dynamic contrast-enhanced magnetic resonance imaging revealed that TAK-593 reduced tumor vessel permeability prior to the onset of anti-tumor activity. In conclusion, TAK-593 is an extremely potent VEGFR/PDGFR kinase inhibitor whose potent anti-angiogenic activity suggests therapeutic potential for the treatment of solid tumors.

Figure 1

TAK‐593 inhibits cellular vascular endothelial growth factor/platelet‐derived growth factor (VEGF/PDGF) signaling and VEGF‐induced tube formation. (a) Human umbilical vein endothelial cells (HUVECs) or coronary artery smooth muscle cells (CASMCs) were treated with TAK‐593 and then stimulated with VEGF (100 ng/mL) or PDGF‐BB (20 ng/mL), respectively. (b) Human umbilical vein endothelial cells co‐cultured with normal human dermal fibroblasts (NHDF) were treated with TAK‐593 in the presence of VEGF (10 ng/mL) for 7 days. Endothelial cells were visualized and quantified with anti‐CD31 staining and fluorescent imaging; representative images are shown (×20). The experiments were performed in duplicate.

Figure 2

TAK‐593 causes tumor regression and prolongs survival in a mouse xenograft model. (a) Data represent the mean and standard deviation (SD) (n = 5). The T/C (%) was calculated relative to the vehicle control group on Day 42. *P ≤ 0.025 versus the vehicle control group by one‐tailed Williams' test. (b) U87 MG human glioblastoma cells were orthotopically implanted into the brains of nude mice. Twice daily oral administration of TAK‐593 (1 and 4 mg/kg) or vehicle was initiated at 4 days after inoculation, and the mice were monitored for survival (n = 10). #P ≤ 0.025 versus vehicle control by Tarone's test.

Figure 3

Pharmacodynamic and pharmacokinetic correlation of TAK‐593. (a,b) TAK‐593 was orally administered at (a) 0.125 mg/kg and (b) 1 mg/kg. Plasma and lung tissue concentration of TAK‐593 were determined by High performance liquid chromatography (HPLC)/MS/MS or HPLC with a fluorescence detector at the indicated times (n = 3). (c) Athymic nude mice (n = 3) were treated with a single oral dose of TAK‐593 (1 mg/kg) and the lung tissue was collected at the indicated times after vascular endothelial growth factor (VEGF)injection (20 μg/mouse). Tissues were lysed and subjected to western blot analysis for phospho‐VEGFR2 and VEGFR2.

Figure 4

TAK‐593 shows anti‐angiogenic, anti‐proliferative, and pro‐apoptotic effect in vivo. TAK‐593 was orally administered to A549 xenograft mice for up to 7 days and tumors were harvested on Days 3 and 7. (a) Endothelial cells were visualized and quantified by CD31 immunostaining. (b) Tumor cells were immunostained with anti‐Ki67 antibody and proliferative activity was quantified. (c) Apoptosis of tumor cells was quantified by the terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL) method. Data represent the mean and standard deviation (SD) (n = 4). *P ≤ 0.025 versus the vehicle control group by the one‐tailed Williams' test.

Figure 5

TAK‐593 decreases pericyte coverage of vessels. TAK‐593 or the vehicle was administered to A549 xenograft mice. (a) Tumors were resected and processed for immunohistochemical analysis at 4 h after the last dose. Sections were simultaneously stained for CD31 and α‐smooth muscle actin (α‐SMA) to detect endothelial cells and pericytes, respectively. The region positive for α‐SMA surrounding the CD31‐positive vessels was quantified in five randomly selected fields. Data represent the mean and standard deviation (SD) (n = 3). *P ≤ 0.025 versus the vehicle control group by the one‐tailed Williams' test. (b) Representative tumor sections from mice treated with the vehicle or TAK‐593 (1.5 mg/kg). White arrowhead in tumors from TAK‐593‐treated mice indicates the pericyte layer that was substantially thinner than that seen in vehicle‐treated tumors. Bar = 100 μm. Red = CD31‐positive endothelial cells; Green = α‐SMA‐positive pericytes

Figure 6

TAK‐593 affects tumor vascular permeability on dynamic constant enhanced magnetic resonance imaging (DCE‐MRI). TAK‐593 or the vehicle was administered for 3 days and once on Day 4 to HT‐29 xenograft mice. Before and after treatment, gadolinium tetraazocyclododecane‐tetraacetate (Gd‐DTPA was injected intravenously and MRI was conducted. Ktrans values were obtained by calculating the Gd‐DTPA concentrations in tumor tissue and plasma on the basis of a two‐compartment model (n = 6–8 per group). (a) Ktrans values of tumors from vehicle or TAK‐593‐treated mice. Data represent the mean and SD. #P ≤ 0.025 versus pretreatment by the one‐tailed Shirley‐Williams' test. (b) Representative Ktrans maps of tumors before and after treatment with TAK‐593 or the vehicle. The colored portion of the spectrum map represents higher vascular permeability. Bar = 2 mm.