Article Figures & Data
Figures
- Figure 1.
Chemical structure of AZD4547.
- Figure 2.
Inhibition of in vitro cell proliferation by AZD4547 correlates with deregulated FGFR expression in tumor cell lines. Western blot analyses confirming high expression of FGFR proteins from cell lines with known deregulations. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
- Figure 3.
Inhibition of FGFR signaling by AZD4547. All cell lines were incubated for 3 hours with the stated AZD4547 concentrations and then stimulated with acidic FGF (aFGF)/basic FGF (bFGF; 10 ng/mL) and heparin (10 μg/mL) for 20 minutes. Cells were then lysed and immunoblotted for the proteins indicated. A–C, KMS11, Sum52-PE, and KG1a were analyzed for total FGFR1–3, phospho- and total FGFR, MAPK, and AKT. D, phospho- and total PLCγ, and Erk. E, phospho- and total FRS2.
- Figure 4.
The mechanism of in vitro growth inhibition by AZD4547 is cell line–dependent. A–C, cells were exposed to AZD4547 for 72 hours and then analyzed for their cell-cycle distribution using propidium iodide and a FACSCalibur flow cytometric system. D, quantified Annexin V flow cytometric data from cells treated in vitro with 100 nmol/L AZD4547 for 72 hours. P values were calculated using the Student t test and refer to the comparison between dimethyl sulfoxide (DMSO) and AZD4547 for each cell line. Data are representative of 3 independent experiments. NS, not significant. E, cells were exposed to 100 nmol/L AZD4547 for 72 hours and then lysed and immunoblotted for the proteins indicated.
- Figure 5.
AZD4547 exposure correlates with in vivo antitumor activity, pharmacodynamic modulation of phospho-FGFR, and reduced tumor cell proliferation. A, AZD4547 was administered by oral gavage once (qd) or twice (bid) daily to SCID mice bearing established s.c. KMS11 human tumor xenografts at the doses indicated. Tumor volumes are plotted against time. B, pharmacokinetic (PK)/pharmacodynamic relationship in KMS11 xenograft. AZD4547 was dosed orally at a range of concentrations, and tumors and blood collected at various time points for ELISA analysis of phospho-FGFR3 and AZD4547 drug levels, respectively. Total plasma drug levels and the percentage inhibition of phospho-FGFR3 (PD inhibition) compared with control are plotted. C, AZD4547 was dosed orally at 6.25 mg/kg (single dose) and plasma and tumors collected over a 16-hour time course. D, Ki-67, cleaved caspase-3 (CC3), and CD31 immunohistochemical staining of KMS11 tumor sections removed after 7 days twice daily dosing of 6.25 mg/kg AZD4547 (4 hours after final dose). E, image analysis data for Ki-67, CC3, and CD31 immunostained tumors showing mean values for each group (n = 4 animals per group). Ki-67 shows statistical significance with one-tailed t test. *, P = 0.012. NS, not significant.
- Figure 6.
AZD4547 antitumor efficacy is not attributable to inhibition of KDR. A, mean diastolic blood pressure measurements of a group of unrestrained conscious telemetered rats (n = 3) given 2 oral doses at 180 and 540 minutes (arrows): on day 1, vehicle control (o) and the following day (day 2) AZD4547 at 10 mg/kg (•). bid, twice daily; po, orally. B, nude mice bearing established Calu-6 human tumor xenografts were treated orally, twice daily with AZD4547 (6.25 mg/kg) or cediranib (3 mg/kg) once daily.
Tables
- Table 1.
AZD4547 kinase activity
Kinase Enzyme IC50,a nmol/L Cellular IC50, nmol/L FGFR1 0.2 ± 0.06 12 ± 3 FGFR3 1.8 ± 0.33 40 ± 1 FGFR2 2.5 ± 0.23 2 ± 0.2 KDR 24 ± 0.001 258 ± 17 FGFR4 165 ± 30.3 142 ± 29 IGFR 581 ± 0.02 828 ± 41 AXL >2,000 CDK2 >10,000 FAK >10,000 ROCK2 >50,000 AKT1 >100,000 ALK1 >100,000 p38 >100,000 PI3Ka >100,000 -
↵aThe ability of AZD4547 to inhibit a range of human recombinant kinase activities was tested using ATP concentrations at, or just below, the respective Km. Data represent the mean ± SEM of at least 3 separate determinations. IC50 values denoted as “greater than” denote the inability to reach 50% inhibition of maximal activity at the highest tested concentration.
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- Table 2.
Summary of AZD4547 in vitro antiproliferative IC50 values obtained by MTS proliferation assay
Tumor cell line Deregulated FGFR member Proliferation IC50, μmol/L SEM KG1a FGFR1 0.018 (n = 3) 0.0017 Sum52-PE FGFR2 0.041 (n = 4) 0.0185 KMS11 FGFR3 0.281 (n = 5) 0.0294 MCF7 None >30 (n = 6) NA Abbreviation: NA, not applicable.
- Table 3.
Tumor growth inhibition in nude mice bearing established Calu-6 human tumor xenografts
Inhibition of tumor growth, % Tumor model AZD4547 Cediraniba LoVo 0 63 HCT-15 4.7 82b NOTE: Mice were treated orally, twice daily with AZD4547 (6.25 mg/kg) for 14 to 17 days or cediranib (LoVo, 1.5 mg/kg; Calu-6, 6 mg/kg; HCT-15, 6 mg/kg) once daily for up to 28 days. Percentage of tumor growth inhibition was calculated as the difference between the mean change in control versus treated tumor volumes over the period of treatment.
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure 1 - PDF file - 68K
- Supplementary Figure 2 - PDF file - 61K
- Supplementary Figure Legends 1-2 - PDF file - 59K
- Supplementary Table 1 - PDF file - 52K
Volume 72, Issue 8
