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Pharmacodynamic characterization of gemcitabine cytotoxicity in an in vitro cell culture bioreactor system

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Abstract

Purpose

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 μM h and the maximum concentration was 87.5 ± 2.65 μM. 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.

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Acknowledgments

This work was supported in part by the Cancer Center Translational Breast Cancer Award to M.N.K.

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Authors and Affiliations

  1. Department of Experimental and Clinical Pharmacology, College of Pharmacy and Comprehensive Cancer Center, University of Minnesota, 308 Harvard St SE, Minneapolis, MN, 55455, USA

    Mark N. Kirstein

  2. Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, 308 Harvard St SE, Minneapolis, MN, 55455, USA

    Richard C. Brundage, Megan M. Moore, Brent W. Williams, Lisa A. Hillman, Jason W. Dagit & James E. Fisher

  3. Clinical Pharmacology Analytical Services, University of Minnesota, Minneapolis, USA

    Jason W. Dagit & James E. Fisher

  4. Department of Medicine and Stem Cell Institute, University of Minnesota, Minneapolis, USA

    Paul H. Marker

  5. Department of Medicine and Cancer Center, University of Minnesota, Minneapolis, USA

    Robert A. Kratzke & Douglas Yee

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  1. Mark N. Kirstein
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Correspondence to Mark N. Kirstein.

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Kirstein, M.N., Brundage, R.C., Moore, M.M. et al. Pharmacodynamic characterization of gemcitabine cytotoxicity in an in vitro cell culture bioreactor system. Cancer Chemother Pharmacol 61, 291–299 (2008). https://doi.org/10.1007/s00280-007-0474-z

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  • DOI: https://doi.org/10.1007/s00280-007-0474-z

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