Pharmacodynamic characterization of gemcitabine cytotoxicity in an in vitro cell culture bioreactor system
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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.
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.
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.
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.
KeywordsGemcitabine Bioreactor SubG1 MDA-MB-231 cells Pharmacokinetics
This work was supported in part by the Cancer Center Translational Breast Cancer Award to M.N.K.
- 1.Spratlin J, Sangha R, Glubrecht D, Dabbagh L, Young JD, Dumontet C, Cass C, Lai R, Mackey JR (2004) The absence of human equilibrative nucleoside transporter 1 is associated with reduced survival in patients with gemcitabine-treated pancreas adenocarcinoma. Clin Cancer Res 10(20):6956–6961PubMedCrossRefGoogle Scholar
- 13.Bengala C, Guarneri V, Giovannetti E, Lencioni M, Fontana E, Mey V, Fontana A, Boggi U, Del Chiaro M, Danesi R, Ricci S, Mosca F, Del Tacca M, Conte PF (2005) Prolonged fixed dose rate infusion of gemcitabine with autologous haemopoietic support in advanced pancreatic adenocarcinoma. Br J Cancer 93(1):35–40PubMedCrossRefGoogle Scholar
- 14.Tempero M, Plunkett W, Ruiz Van Haperen V, Hainsworth J, Hochster H, Lenzi R, Abbruzzese J (2003) Randomized phase II comparison of dose-intense gemcitabine: thirty-minute infusion and fixed dose rate infusion in patients with pancreatic adenocarcinoma. J Clin Oncol 21(18):3402–3408PubMedCrossRefGoogle Scholar
- 20.D’Argenio DZaS A (1997) ADAPT II User’s Guide: Pharmacokinetic/Pharmacodynamic Systems Analysis Software. In: Biomedical Simulations Resource, Los AngelesGoogle Scholar
- 22.Dumez H, Louwerens M, Pawinsky A, Planting AS, de Jonge MJ, Van Oosterom AT, Highley M, Guetens G, Mantel M, de Boeck G, de Bruijn E, Verweij J (2002) The impact of drug administration sequence and pharmacokinetic interaction in a phase I study of the combination of docetaxel and gemcitabine in patients with advanced solid tumors. Anticancer Drugs 13(6):583–593PubMedCrossRefGoogle Scholar
- 23.Kroep JR, Giaccone G, Voorn DA, Smit EF, Beijnen JH, Rosing H, van Moorsel CJ, van Groeningen CJ, Postmus PE, Pinedo HM, Peters GJ (1999) Gemcitabine and paclitaxel: pharmacokinetic and pharmacodynamic interactions in patients with non-small-cell lung cancer. J Clin Oncol 17(7):2190–2197PubMedGoogle Scholar
- 34.Hernandez-Vargas H, Rodriguez-Pinilla SM, Julian-Tendero M, Sanchez-Rovira P, Cuevas C, Anton A, Rios MJ, Palacios J, Moreno-Bueno G (2006) Gene expression profiling of breast cancer cells in response to gemcitabine: NF-kappaB pathway activation as a potential mechanism of resistance. Breast Cancer Res Treat 102(2):157–172PubMedCrossRefGoogle Scholar