A cell-based screening platform for hypoxia-activated prodrugs

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The tumor microenvironment offers a unique target for developing selective anticancer drugs and a key characteristic of it is microregional hypoxia. We developed a sequence of cell-based assays to discover and characterize prodrugs specifically activated in hypoxic cells. We will illustrate this approach with a model compound based on an oxygen sensitive trigger linked to a cytotoxic effector molecule. TH-63 is a 2-nitroimidazole linked to daunorubicin, a potent topoisomerase II inhibitor. The primary screen uses 24 well plates and characterizes cell viability as a function of drug concentrations at selected oxygen concentrations in order to identify compounds that selectively kill hypoxic cells. Results are confirmed using the more stringent clonogenic cell survival assay. Compounds which exhibit the desired potency and selectivity are then advanced to multicellular assays designed to determine both drug penetrability and the ability of the hypoxia-activated drug to diffuse and kill nearby cells. Multilayer cell cultures (MCC’s) are an established model of tumor tissue grown on a solid support to a thickness of 150 to 200μm where diffusion-limited hypoxia develops and drug penetration can be assessed. Compounds are also then tested in multicellular spheroids (at 500μm in diameter) where oxygen gradients develop, creating a model with aerobic and hypoxic compartments. Prodrug activation is measured by clonogenic survival either alone or in combination with a chemotherapeutic drug that only targets the outer aerobic cells of the spheroid. The viability of H460 cells exposed to TH-63 for 2hrs was determined at anoxia (100% N₂), intermediate hypoxia (0.6% and 1% O₂), and aerobic conditions (10% and 20% O₂). There was an 80-fold difference in the IC₅₀’s comparing anoxia to air (0.5μM vs 40μM) indicating hypoxic selectivity. Clonogenic survival confirmed these findings. In addition, TH-63 under anoxia was similar in potency to daunorubicin (IC₉₀ 0.7μM vs 0.4μM) suggesting efficient drug activation. In MCC models where one side is closed off to establish temporarily hypoxic regions, TH-63 was able to penetrate significantly further than daunorubicin. H460 spheroids exposed to TH-63 exhibited increased penetration compared to daunorubicin. Therefore, the combination of daunorubicin (targeting the aerobic cells) with TH-63 (targeting the hypoxic cells) should result in complete cell kill and can be readily tested in the spheroid model. This linked series of in vitro assays identifies candidates for testing in in vivo models of cancer, and clinical assessment for addressing the problem of tumor resistance in patients.