Pharmacodynamic characterization of gemcitabine cytotoxicity in an in vitro cell culture bioreactor system.
OBJECTIVE
Gemcitabine, a pyrimidine nucleoside, is approved for the treatment of non-small cell lung cancer, pancreatic carcinoma, and breast cancer. Chemotherapy regimens are determined experimentally with static tissue culture systems, animal models, and in Phase I clinical trials. The aim of this study was to assess for gemcitabine-induced cell death following infusion of drug under clinically-relevant conditions of infusion rate and drug exposure in an in vitro bioreactor system.


METHODS
To estimate an appropriate harvest time for cells from the bioreactor after drug treatment, we estimated the temporal relationship between gemcitabine treatment for 1 h and cell death at a later time point with monolayer growth assays (i.e., static culture). Afterward, 5.3 mg gemcitabine was infused over 0.5 h in the bioreactor, followed by mono-exponential decay, simulating patient concentration-time profiles (n = 4). Controls were run with drug-free media (n = 4). Cells were harvested from the bioreactor at a later time point and assessed for cell death by flow cytometry.


RESULTS
According to monolayer growth assay results, cytotoxicity became more apparent with increasing time. The E Max for cells 48 h after treatment was 50% and after 144 h, 93% (P = 0.022; t test), while flow cytometry showed complete DNA degradation by 120 h. Gemcitabine was infused in the bioreactor. The gemcitabine area under the concentration-time curve (AUC) was 56.4 microM h and the maximum concentration was 87.5 +/- 2.65 microM. Flow cytometry results were as follows: the G1 fraction decreased from 65.1 +/- 4.91 to 28.6 +/- 12% (P = 0.005) and subG1 increased from 14.1 +/- 5.28 to 42.6 +/- 9.78% (P = 0.004) relative to control. An increase in apoptotic cells was observed by TUNEL assay.


CONCLUSIONS
The in vitro bioreactor system will be expanded to test additional cell lines, and will serve as a useful model system for assessing the role of drug pharmacokinetics in delivery of optimized anticancer treatment.
Kirstein
Mark N
MN
Department of Experimental and Clinical Pharmacology, College of Pharmacy and Comprehensive Cancer Center, University of Minnesota, 308 Harvard St SE, Minneapolis, MN 55455, USA. kirst002@umn.edu
Brundage
Richard C
RC
Moore
Megan M
MM
Williams
Brent W
BW
Hillman
Lisa A
LA
Dagit
Jason W
JW
Fisher
James E
JE
Marker
Paul H
PH
Kratzke
Robert A
RA
Yee
Douglas
D
D000964:Antimetabolites, Antineoplastic / Q000493:pharmacokinetics* / Q000633:toxicity*; D019540:Area Under Curve; D019149:Bioreactors; D002453:Cell Cycle / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000633:toxicity; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling; D013778:Tetrazolium Salts; D013844:Thiazoles; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D004273:DNA, Neoplasm; D013778:Tetrazolium Salts; D013844:Thiazoles; D003841:Deoxycytidine; C022616:thiazolyl blue; D000093542:Gemcitabine