Preclinical anticancer activity of the potent, oral Src inhibitor AZD0530

Abstract

AZD0530, an orally available Src inhibitor, demonstrated potent antimigratory and anti-invasive effects in vitro, and inhibited metastasis in a murine model of bladder cancer. Antiproliferative activity of AZD0530 in vitro varied between cell lines (IC(50) 0.2 ->10μM). AZD0530 inhibited tumor growth in 4/10 xenograft models tested and dynamically inhibited in vivo phosphorylation of Src substrates paxillin and FAK in both growth-inhibition-resistant and -sensitive xenografts. The activity of AZD0530 in NBT-II bladder cancer cells in vitro was consistent with inhibition of cell migration and stabilization of cell-cell adhesion. These data suggest a dominant anti-invasive pharmacology for AZD0530 that may limit tumor progression in a range of cancers. AZD0530 is currently in Phase II clinical trials.

Figures

Figure 1

AZD0530 antimigratory and anti‐invasive activity. (A) An illustration of the microdroplet migration assay. The image shows human NSCLC A549 cells migrating out from an agarose microdroplet after 72h of culture, and indicates how cell migration is measured. (B) Representative time‐lapse photography images (magnification ×200) in which AZD0530 shows dose‐dependent antimigratory activity compared with untreated controls in human breast cancer cells (MDA‐MB‐231) after 18h treatment in a monolayer scratch assay. (C) AZD0530 exhibited dose dependent inhibition of NBT‐II cell motility. EGF (20ng/mL)‐ and collagen‐induced cell motility assessed by videomicroscopy in vitro (mean+SEM of at least three experiments). (D) In vitro invasion of HT1080 cells through a 3D fibrillar collagen matrix. Exposure of HT1080 cells to AZD0530 resulted in a dose‐dependent reduction in the proportion of cells invading beyond a depth of 60μm into a 3D collagen type I matrix. Bars represent mean±SEM of three experiments. Representative images (magnification ×200) of invaded, Hoechst‐labeled cells at a depth of 60μm into the 3D collagen matrix are shown.

Figure 2

AZD0530 effects on cell scattering and paxillin phosphorylation. (A) AZD0530 inhibited EGF (20ng/mL)‐mediated loss of desmoplakin immunoreactivity from the surface of NBT‐II cells in monolayer culture. (B) AZD0530 inhibited tyrosine phosphorylation of paxillin Y118 in NBT‐II cells in vitro. Relative phosphorylation levels were quantified by densitometry. (C) Effects of AZD0530 on the intracellular distribution of phosphorylated paxillin in NBT‐II cells in vitro.

Figure 3

AZD0530 enzyme kinetics. (A) Competitive kinetics when the concentrations of AZD0530 and ATP are varied in the presence of 1mM Src II peptide, with best‐fit values of Kis 1.5nM, maximum velocity (Vmax) 0.03μM/min, and ATP KM 190μM. (B) Pure noncompetitive kinetics when the concentrations of AZD0530 and Src II peptide are varied in the presence of 1.6mM ATP, with best‐fit values of apparent Ki′ 10nM; Vmax 0.04μM/min, and Src peptide II KM 330μM.

Figure 4

AZD0530 pharmacokinetics and effects on xenograft tumor growth. (A) AZD0530 plasma concentration (ng/mL) 6h post‐terminal dose in female nude mice bearing LoVo tumors (n=7 per group). (B–D) Effect of once‐daily AZD0530 on tumor growth in female nude mice (n=7 per group) bearing (B) Src3T3 allografts, (C) human NSCLC (Calu‐6) xenografts, and (D) human colon cancer (LoVo) xenografts. (E) Effects of AZD0530 at 10 (n=14), 25 (n=14) or 50mg/kg/day po (n=7) on the same day as cell inoculation or 1 week after inoculation on the growth of NBT‐II tumor cells grown as sc xenografts in nude mice. Mean±SEM.

Figure 5

AZD0530 effects on metastasis and phosphorylation of Src substrates FAK and paxillin. (A) Effects of increasing doses of AZD0530 on metastasis to lymph nodes after injection of NBT‐II cells into nude mice. (B–D) Immunohistochemical analysis after 28 (Calu‐6) or 14 (LoVo) days once‐daily oral treatment with vehicle alone or AZD0530 50mg/kg. AZD0530 inhibited phosphorylation of paxillin (pY31) in (B) human Calu‐6, and (C) LoVo xenografts grown subcutaneously in nude mice. (D) AZD0530 inhibited phosphorylation of FAK (pY861) in LoVo human xenografts grown subcutaneously in nude mice. Images are at magnifications of (B) ×40 and (C and D) ×20.