Sequential Exposure of Bortezomib and Vorinostat is Synergistic in Multiple Myeloma Cells

Abstract

Purpose: To examine the combination of bortezomib and vorinostat in multiple myeloma cells (U266) and xenografts, and to assess the nature of their potential interactions with semi-mechanistic pharmacodynamic models and biomarkers.

Methods: U266 proliferation was examined for a range of bortezomib and vorinostat exposure times and concentrations (alone and in combination). A non-competitive interaction model was used with interaction parameters that reflect the nature of drug interactions after simultaneous and sequential exposures. p21 and cleaved PARP were measured using immunoblotting to assess critical biomarker dynamics. For xenografts, data were extracted from literature and modeled with a PK/PD model with an interaction parameter.

Results: Estimated model parameters for simultaneous in vitro and xenograft treatments suggested additive drug effects. The sequence of bortezomib preincubation for 24 hours, followed by vorinostat for 24 hours, resulted in an estimated interaction term significantly less than 1, suggesting synergistic effects. p21 and cleaved PARP were also up-regulated the greatest in this sequence.

Conclusions: Semi-mechanistic pharmacodynamic modeling suggests synergistic pharmacodynamic interactions for the sequential administration of bortezomib followed by vorinostat. Increased p21 and cleaved PARP expression can potentially explain mechanisms of their enhanced effects, which require further PK/PD systems analysis to suggest an optimal dosing regimen.

Keywords: Bortezomib; pharmacodynamic modeling; synergistic combination; vorinostat.

Figure 1

U266 cell growth inhibition by bortezomib (a) and vorinostat (b). Symbols represent experimental observed values, and lines are model-fitted profiles (Equation 1).

Figure 2

Fitted 3D response surface (Equation 2) for combination effects in U266 cells treated with bortezomib and vorinostat simultaneously for 48 hours (a) and sequentially with bortezomib alone for the first 24 hours followed by vorinostat for another 24 hours (b). The mesh surface represents model fitting with the final estimates of ψ (0.976) and μ (0.708). Green symbols are experimental data above the surface, and red symbols are experimental data below the surface.

Figure 3

Model-fitted profiles of U266 cellular proliferation time-course with (a) vehicle control, and (b) bortezomib (3 nM) and vorinostat (2 μM) as single agents, on simultaneous exposure (bortezomib 3 nM and vorinostat 2 μM), and a 24-hour sequential regimen (bortezomib 3nM exposure alone for the first 24 hours followed by vorinostat 2 μM for next 24 hours). Symbols represent experimental observed values, and lines represent model-fitted profiles.

Figure 4

Model-fitted profiles of effects on tumor progression in LAGκ-1B tumor xenografts treated with vehicle (control), borteozmib (0.5 mg/kg twice weekly), vorinostat (100 mg/kg twice weekly), and their simultaneous combination (bortezomib 0.5 mg/kg twice weekly and vorinostat 100 mg/kg twice weekly) for 35 days. Symbols represent digitized data from the original publication (7), and lines represent fitted profiles or model simulations with ψ =1 and μ = 8.96 (black dashed and pink lines).

Figure 5

Cellular protein dynamics across different treatment arms in U266 multiple myeloma cells. Time-course of relative expression and representative western blots for p21 (a) and cleaved PARP (b) are shown for vehicle control, continuous exposure of bortezomib (3 nM), vorinostat (2 μM), simultaneous exposure (bortezomib 3 nM and vorinostat 2 μM) for 48 hours, and a 24-hour sequential regimen (bortezomib 3 nM exposure alone for the first 24 hours followed by vorinostat 2 μM for next 24 hours). Symbols represent the mean of three replicates, and error bars represent standard deviation. Groups not statistically different; Kruskal-Wallis One-Way ANOVA on Ranks with Tukey (data do not follow a normal distribution).