Drug exposure impacts the antitumor activity for many chemotherapy agents. Maximum drug concentrations (Cmax), Area Under the Curve (AUC), and steady state concentrations (Css) may influence antitumor activity. We recently described development of an in vitro bioreactor tissue culture system that allows for changing drug exposure to mimic measured changes in in vivo drug concentrations. Previously, we infused gemcitabine over 0.5 hr, and then analyzed the MDA-231 cells. We observed a significant arrest of cells in S-phase compared to control cells, but not an increase in subG1 peak. Since cell death is a more desirable endpoint than S phase arrest for evaluating anticancer drug regimens, we describe in this abstract the determination of the most suitable cell harvest time from the bioreactor, and whether gemcitabine could induce cell death in this system. First, we performed cytotoxicity studies of MDA-231 cells treated with gemcitabine for 1 hr in 24-well plates (MTT). We measured absorbance 2 and 5 days after treatment. The mean ± SD IC50 two days after treatment was 0.14 ± 0.09 μM, and after five days, 0.22 ± 0.11 μM (p=0.5; n=3). The Emax data were 0.44 ± 0.13 after two days and 0.88 ± 0.02 after five days (p=0.02). DNA flow cytometry demonstrated a significant decrease in G1 phase and increase in S-phase two days after treatment, and complete degradation of DNA five days after treatment. We then seeded 5 X 106 MDA-231 cells into the extracapillary space of bioreactors. Two weeks later, we infused 5.3 mg gemcitabine over 0.5 hr (n=3). The AUC was 58.5 ± 3.9 μM*hr, the Cmax was 87.7 ± 2.3 μM, and the clearance was 4.7 ± 0.07 mL/min. Five days after treatment, 17.2 ± 1.5 X 106 cells were recovered from treated bioreactors and 32.7 ± 7.5 X 106 cells from control bioreactors. With flow cytometry, we observed 63.7 ± 2.9 % in G1 for the control and 30.9 ± 14.0% for treated cells (p=0.05); 11.9 ± 2.6% in S for the control and 24.9 ± 7.9% for treated cells (p=0.06); 9.1 ± 1% in G2/M for control and 6 ±1.3% for treated cells (p=0.14); and 16.3 ± 3.6% in subG1 for the control and 40 ± 9.5% for treated cells (p=0.05). In conclusion, apoptosis, as measured by subG1 peak, can be detected 5 days after drug exposure in this system. Thus, this system can be used to determine the effects of Cmax, AUC, and Css on the induction of cell death. These data can be used to plan phase II clinical trial designs. Supported by Cancer Center Translational Breast Cancer Award.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]