Radioisotopic assays involve expense, multistep protocols, equipment, and radioactivity safety requirements which are problematic in high-throughput drug testing. This study reports an alternative, simple, robust, inexpensive, one-step fluorescence assay for use in antimalarial drug screening. Parasite growth is determined by using SYBR Green I, a dye with marked fluorescence enhancement upon contact with Plasmodium DNA. A side-by-side comparison of this fluorescence assay and a standard radioisotopic method was performed by testing known antimalarial agents against Plasmodium falciparum strain D6. Both assay methods were used to determine the effective concentration of drug that resulted in a 50% reduction in the observed counts (EC 50 ) after 48 h of parasite growth in the presence of each drug. The EC 50 s of chloroquine, quinine, mefloquine, artemisinin, and 3,6-bis--(N,N-diethylamino)-amyloxyxanthone were similar or identical by both techniques. The results obtained with this new fluorescence assay suggest that it may be an ideal method for highthroughput antimalarial drug screening.The global scope of malaria and the spread of drug-resistant Plasmodium falciparum make the need for improved therapy undeniable (4). Assessment of both existing drugs and new antimalarials, alone or in combination, requires reliable methods for high-throughput testing. For decades, antimalarial drug effects have been measured in vitro by quantifying parasite uptake of radioactive substrates as a measure of growth and viability in the presence of the test drug (2, 3). [3 H]hypoxanthine is the most widely used radiolabel, but because it requires purine starvation prior to the assay, the [ 3 H]ethanolamine assay is favored by our laboratory. While these methods are accurate and reliable, they rely on relatively expensive radioisotopes and multistep procedures that become increasingly problematic and impractical as the volume of testing increases. We report on the development of an alternative, fluorescencebased in vitro assay for use for the high-throughput screening of the activities of drugs against P. falciparum. The goals for the assay included simplicity, cost savings, robust performance, applicability to automated analysis, and speed.The principle behind the assay is the contrast between host erythrocytes, which lack DNA and RNA, and the malaria parasites, which do not and which are thus readily stained with dyes that show enhanced fluorescence in the presence of nucleic acids (8). Fluorescence-based in vitro antimalarial assays have been developed previously (1, 12) but have required complex, multistep protocols or additional equipment not amenable to rapid, high-throughput use. This report establishes and validates a facile, one-step method that uses the same principle. MATERIALS AND METHODSChloroquine diphosphate, quinine sulfate, artemisinin, and saponin were purchased from Sigma Aldrich Chemical Company (St. Louis, Mo.); mefloquine was obtained from Walter Reed Army Institute for Research (Silver Spring, Md.); and 3,6-b...

During the investigational use of oral N-acetylcysteine as an antidote for poisoning with acetaminophen, 11,195 cases of suspected acetaminophen overdose were reported. We describe the outcomes of 2540 patients with acetaminophen ingestions treated with a loading dose of 140 mg of oral N-acetylcysteine per kilogram of body weight, followed four hours later by 70 mg per kilogram given every four hours for an additional 17 doses. Patients were categorized for analysis on the basis of initial plasma acetaminophen concentrations and the interval between ingestion and treatment. Hepatotoxicity developed in 6.1 percent of patients at probable risk when N-acetylcysteine was started within 10 hours of acetaminophen ingestion and in 26.4 percent of such patients when therapy was begun 10 to 24 hours after ingestion. Among patients at high risk who were treated 16 to 24 hours after an acetaminophen overdose, hepatotoxicity developed in 41 percent--a rate lower than that among historical controls. When given within eight hours of acetaminophen ingestion, N-acetylcysteine was protective regardless of the initial plasma acetaminophen concentration. There was no difference in outcome whether N-acetylcysteine was started zero to four or four to eight hours after ingestion, but efficacy decreased with further delay. There were 11 deaths among the 2540 patients (0.43 percent); in the nine fatal cases in which aminotransferase was measured before treatment, values were elevated before N-acetylcysteine was started. No deaths were clearly caused by acetaminophen among patients in whom N-acetylcysteine therapy was begun within 16 hours. We conclude that N-acetylcysteine treatment should be started within eight hours of an acetaminophen overdose, but that treatment is still indicated at least as late as 24 hours after ingestion. On the basis of available data, the 72-hour regimen of oral N-acetylcysteine is as effective as the 20-hour intravenous regimen described previously, and it may be superior when treatment is delayed.

Preventing and delaying the emergence of drug resistance is an essential goal of antimalarial drug development. Monotherapy and highly mutable drug targets have each facilitated resistance, and both are undesirable in effective long-term strategies against multi-drug-resistant malaria. Haem remains an immutable and vulnerable target, because it is not parasite-encoded and its detoxification during haemoglobin degradation, critical to parasite survival, can be subverted by drug-haem interaction as in the case of quinolines and many other drugs. Here we describe a new antimalarial chemotype that combines the haem-targeting character of acridones, together with a chemosensitizing component that counteracts resistance to quinoline antimalarial drugs. Beyond the essential intrinsic characteristics common to deserving candidate antimalarials (high potency in vitro against pan-sensitive and multi-drug-resistant Plasmodium falciparum, efficacy and safety in vivo after oral administration, inexpensive synthesis and favourable physicochemical properties), our initial lead, T3.5 (3-chloro-6-(2-diethylamino-ethoxy)-10-(2-diethylamino-ethyl)-acridone), demonstrates unique synergistic properties. In addition to 'verapamil-like' chemosensitization to chloroquine and amodiaquine against quinoline-resistant parasites, T3.5 also results in an apparently mechanistically distinct synergism with quinine and with piperaquine. This synergy, evident in both quinoline-sensitive and quinoline-resistant parasites, has been demonstrated both in vitro and in vivo. In summary, this innovative acridone design merges intrinsic potency and resistance-counteracting functions in one molecule, and represents a new strategy to expand, enhance and sustain effective antimalarial drug combinations.