pralidoxime and Neuroblastoma

Unforeseen toxic effects contribute to compound attrition during preclinical evaluation and clinical trials. Consequently, there is a need to correlate in vitro toxicity to in vivo and clinical outcomes quickly and effectively. We propose an expedited evaluation of physiological parameters in vitro that will improve the ability to predict in vivo toxicity of potential therapeutics. By monitoring metabolism, mitochondrial physiology and cell viability, our approach provides insight to the extent of drug toxicity in vitro. To implement our approach, we used human hepatocellular carcinoma cells (HepG2) and neuroblastoma cells (SH-SY5Y) to monitor hepato- and neurotoxicity of the experimental oxime K027. We utilized a trivalent approach to measure metabolism, mitochondrial stress and induction of apoptosis in 96-well formats. Any change in these three areas may suggest drug-induced toxicity in vivo. K027 and pralidoxime, an oxime currently in clinical use, had no effect on glycolysis or oxygen consumption in HepG2 and SH-SY5Y cells. Similarly, these oximes did not induce oxidant generation nor alter mitochondrial membrane potential. Further, K027 and pralidoxime failed to activate effector caspases, and these oximes did not alter viability. The chemotherapeutic agent, docetaxel, negatively affected metabolism, mitochondrial physiology and viability. Our studies present a streamlined high-throughput trivalent approach for predicting toxicity in vitro, and this approach reveals that K027 has no measurable hepatotoxicity or neurotoxicity in vitro, which correlates with their in vivo data. This approach could eliminate toxic drugs from consideration for in vivo preclinical evaluation faster than existing toxicity prediction panels and ultimately prevent unnecessary experimentation.

Topics: Apoptosis; Brain; Caspases; Cell Survival; Docetaxel; Glycolysis; Hep G2 Cells; Humans; Liver; Membrane Potential, Mitochondrial; Neuroblastoma; Oximes; Pralidoxime Compounds; Pyridinium Compounds; Taxoids; Toxicity Tests; Tumor Cells, Cultured

Our intent was to evaluate the C1300 neuroblastoma cell as an in vitro system for studying the mode of action and efficacy of drugs used to treat or prevent organophosphate intoxication. The anticholinergic drugs hexamethonium, trimethaphan, and hemocholinium and the triethylcholine and cholinesterase/reactivator 2-pyridine aldoxime methochloride (2-PAM) have been shown to be effective in preventing intoxication by diisopropyl phosphorofluoridate (also known as diisopropyl fluorophosphate, DFP) in vivo. We determined their efficacy in preventing cell death (as measured by trypan blue exclusion) of neuroblastoma cells alone or in combination. We also determined their efficacy in reversing the cytotoxic effects of DFP on cell DNA synthesis (as measured by [3H]-thymidine incorporation), cell RNA synthesis (as measured by [3H]uridine incorporation), and on cell protein synthesis (as measured by [3H]leucine incorporation). The maximal nontoxic doses of the drugs in vitro were determined. All anticholinergic agents studied reduced the cytotoxicity of DFP using one or more parameters. 2-PAM, the cholinesterase reactivator, enhanced the cytotoxicity of DFP on cultured cells at a high concentration (1 mg/mL) and reduced it at a lower concentration (0.3 mg/mL). All four anticholinergic agents were capable of enhancing the uptake of [3H]thymidine. Only hexamethonium and hemicholinium reversed DFP inhibition of DNA synthesis. RNA synthesis was not affected by any anticholinergic agent and no agent reversed DFP inhibition of RNA synthesis. Protein synthesis was enhanced by every anticholinergic agent except hemicholinium; the inhibition of protein synthesis by DFP was reversed by trimethaphan and triethylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cell Survival; Choline; Cholinergic Antagonists; Cholinesterase Reactivators; DNA, Neoplasm; Hemicholinium 3; Hexamethonium; Isoflurophate; Mice; Neoplasm Proteins; Neuroblastoma; Pralidoxime Compounds; RNA, Neoplasm; Trimethaphan; Tumor Cells, Cultured