The enhanced in vivo activity of the combination of a MEK and a PI3K inhibitor correlates with [18F]-FLT PET in human colorectal cancer xenograft tumour-bearing mice

Abstract

Combined targeting of the MAPK and PI3K signalling pathways in cancer may be necessary for optimal therapeutic activity. To support clinical studies of combination therapy, 3'-deoxy-3'-[(18)F]-fluorothymidine ([(18)F]-FLT) uptake measured by Positron Emission Tomography (PET) was evaluated as a non-invasive surrogate response biomarker in pre-clinical models. The in vivo anti-tumour efficacy and PK-PD properties of the MEK inhibitor PD 0325901 and the PI3K inhibitor GDC-0941, alone and in combination, were evaluated in HCT116 and HT29 human colorectal cancer xenograft tumour-bearing mice, and [(18)F]-FLT PET investigated in mice bearing HCT116 xenografts. Dual targeting of PI3K and MEK induced marked tumour growth inhibition in vivo, and enhanced anti-tumour activity was predicted by [(18)F]-FLT PET scanning after 2 days of treatment. Pharmacodynamic analyses using the combination of the PI3K inhibitor GDC-0941 and the MEK inhibitor PD 0325901 revealed that increased efficacy is associated with an enhanced inhibition of the phosphorylation of ERK1/2, S6 and 4EBP1, compared to that observed with either single agent, and maintained inhibition of AKT phosphorylation. Pharmacokinetic studies indicated that there was no marked PK interaction between the two drugs. Together these results indicate that the combination of PI3K and MEK inhibitors can result in significant efficacy, and demonstrate for the first time that [(18)F]-FLT PET can be correlated to the improved efficacy of combined PI3K and MEK inhibitor treatment.

Figure 1. Plasma and tumour concentrations of GDC-0941 and PD 0325901 from mice bearing human tumour xenografts.

Plasma and tumour concentrations of the PI3K inhibitor GDC-0941 (GDC) (A) and the MEK inhibitor PD 0325901 (PD) (B) measured by HPLC in samples from HCT116 tumour xenograft-bearing mice at the indicated time points after a single p.o. dose of either 100 mg/kg GDC-0941 alone, 1 mg/kg PD 0325901 alone or the combination of 1 mg/kg PD 0325901 and 100 mg/kg GDC-0941. Data are presented as the mean concentration from 3 mice in each group ± standard error. The horizontal dashed line indicates the in vitro GI₅₀ concentration (previously determined in [38]).

Figure 2. Pharmacodynamic analysis of the in vivo effect of PD 0325901 and GDC-0941 on signal transduction.

Effects on the MAPK and PI3K/AKT signal transduction pathways after HCT116 tumour xenograft-bearing mice were treated with a single p.o. dose of either 1 mg/kg of the MEK inhibitor PD 0325901 alone (A), 100 mg/kg of the PI3K inhibitor GDC-0941 alone (B) or the combination of 1 mg/kg of the MEK inhibitor PD 0325901 and 100 mg/kg of the PI3K inhibitor GDC-0941 (C). After 0.25–24 hours, tumours were removed and lysates subjected to electrophoresis, followed by Western blotting using the indicated phospho-specific antibody. Blots were then stripped and re-probed with the corresponding total protein antibody to confirm equal protein loading. A–C represents samples from the three mice in each treatment group.

Figure 3. Efficacy and tolerability of GDC-0941 and PD 0325901 in mice bearing human colorectal tumour xenografts.

HCT116 (A, C, E) and HT29 (B, D, F) tumour xenografts were treated with either vehicle control, 1 mg/kg of the MEK inhibitor PD 0325901 and 100 mg/kg of the PI3K inhibitor GDC-0941 alone, or 1 mg/kg of the MEK inhibitor PD 0325901 and 100 mg/kg of the PI3K inhibitor GDC-0941 in combination, p.o. once daily for 14 days. [A–B] Tumour growth curves: Data are presented as the median relative tumour volume (RTV), where the growth is calculated for each tumour relative to its size on day 0. Points represent the median of the 10 mice in each group. The dashed line shows the point at which tumours reached four times the initial volume (RTV4). [C–D] Effects on body weight: Data are presented as a percentage of starting body weight. Points represent the mean of the 10 mice in each group ± standard error. [E–F] Time taken for xenografts to reach four times the initial volume (time to RTV4): Data are presented as the time taken by each individual tumour in each group to quadruple in size, and lines to represent the mean of the mice in each group ± standard error. P values are given where the combination is significantly different from either agent alone (p≤0.05).

Figure 4. [¹⁸F]-FLT uptake in human tumour xenografts before and after treatment with GDC-0941 and PD 0325901.

[A–D] HCT116 tumour xenograft [¹⁸F]-FLT uptake over the 1 hour dynamic PET scan at baseline and after treatment with either vehicle control (A) 100 mg/kg of the PI3K inhibitor GDC-0941 alone (B), 1 mg/kg of the MEK inhibitor PD 0325901 alone (C) or the combination of 1 mg/kg of the MEK inhibitor PD 0325901 and 100 mg/kg of the PI3K inhibitor GDC-0941 (D), p.o. once daily for 2 days. Data are presented as the mean standardised uptake value (SUV), where the tissue concentration is calculated relative to injected dose and body weight. Points represent the mean of the 5–7 mice in each group ± standard deviation. [E] Percentage change HCT116 tumour xenograft [¹⁸F]-FLT uptake at 1 hour before and after treatment with either vehicle control or the combination of 1 mg/kg PD 0325901 and 100 mg/kg GDC-0941, p.o. once daily for 2 days. Data are presented as the percentage change in the area under the curve (AUC) relative to the corresponding baseline AUC, derived from Figures A–D, and bars represent the mean of the 5–7 mice in each group ± standard error. * denotes that the [¹⁸F]-FLT SUV AUC in the combination treated group were significantly different after 2 days treatment compared to baseline (p<0.01).