Investigation of cardiac glycosides from oleander in a human induced pluripotent stem cells derived cardiomyocyte model.

The ingestion of Nerium oleander and Thevetia peruviana are common causes for poisoning in Southeast Asia. All parts of the oleander shrub contain cardiac glycosides of the cardenolide type. These glycosides act via inhibition of a Na+/K+-ATPase which might cause severe arrhythmia and subsequent death in oleander-poisoned patients. The current study uses human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) in a microelectrode array (MEA) system to assess the cardiac effects of neriifolin, oleandrin, digitoxigenin, peruvoside and thevetin A from the oleander plant. Digoxin was used as established reference compound. All tested compounds showed a corrected field potential duration (FPDc) shortening and was the lowest for 600 nM digitoxigenin with -36.9 ± 1.2 %. Next to the dose-dependent pro-arrhythmic potential, a complete beat arrest of the spontaneously beating hiPSC-CM was observed at a concentration of 300 nM for neriifolin, 600 nM for oleandrin and 1000 nM for digitoxigenin and peruvoside. Thevetin A did not cause arrhythmia up to a final concentration of 1000 nM. Thus, it was possible to establish a cardiac effect rank order of the tested substances: neriifolin > oleandrin > digitoxigenin = peruvoside > digoxin > thevetin A.

In vitro kinetic interactions of pyridostigmine, physostigmine and soman with erythrocyte and muscle acetylcholinesterase from different species.

The low effectiveness of atropine and oxime treatment in soman poisoning may be enhanced by carbamates pre-treatment. For ethical reasons medical countermeasures can only be tested in animal models despite the fact of substantial species differences. With this kinetic in vitro study the interactions between pyridostigmine, physostigmine and soman with human, Rhesus monkey, swine and guinea pig erythrocyte AChE were investigated. In addition, the effect of the carbamates on the residual activity and enzyme recovery after soman inhibition was examined with erythrocyte and intercostal muscle AChE from these species with a dynamic in vitro model with real-time determination of AChE activity. Only small to moderate species differences of the inhibition and decarbamylation kinetics were recorded. It was possible to show that with erythrocyte and muscle AChE a similar level of protection by carbamates and reactivation after discontinuation of the carbamates and soman could be observed. Thus, these data indicate that carbamate pre-treatment is expected to protect a critical level of muscle AChE and confirm the presumption that erythrocyte AChE may serve as a surrogate for synaptic AChE. Hence, these and previous data fortify the notion that erythrocyte AChE is a proper tool for in vitro kinetic studies as well as for therapeutic monitoring in experimental and clinical studies.

Development of a high-throughput screening for nerve agent detoxifying materials using a fully-automated robot-assisted biological assay.

Developing improved medical countermeasures against chemical warfare agents (nerve agents) is urgently needed but time-consuming and costly. Here we introduce a robot-assisted liquid handling system with warming, cooling and incubating facilities to screen the detoxifying properties of biological and chemical materials against nerve agents. Two biological tests were established and plasma from various species, DFPase and three cyclodextrins were used as test materials. In test 1, plasma was mixed with sarin or VX and the inhibitory potency of the incubate was determined with human acetylcholinesterase (AChE) at 0, 30 and 60 min. In test 2, test materials and nerve agents were mixed and incubated. Between 0 and 40 min samples were taken and incubated for 3 min with AChE and the residual AChE inhibition was determined to enable the semi-quantitative evaluation of the detoxification kinetics. The automated assays proved to be highly reproducible. It was possible to pre-select detoxifying reagents with test 1 and to determine more detailed detoxifying kinetics with test 2. In conclusion, the automated assay may be considered as a versatile tool for the high-throughput screening of potential detoxifying materials against different nerve agents. With this two-step assay it is possible to screen effectively for detoxifying materials in a high-throughput system.

Reappraisal of indications and limitations of oxime therapy in organophosphate poisoning.

1 In vitro studies with human erythrocyte acetylcholinesterase (AChE) and the mouse diaphragm model were performed to unravel the various microscopic reaction parameters that contribute to the dynamic equilibrium of AChE inhibition, ageing and reactivation. These data may help to define more precisely the indications and limitations of oxime therapy in organophosphate (OP) poisoning. 2 Diethylphosphoryl-AChE resulting from intoxications with parathion, chlorpyrifos, chlorfenvinphos, diazinon and other OPs is characterized by slow spontaneous reactivation and low propensity for ageing. This kind of phosphorylated enzyme is particularly susceptible to reactivation by oximes. 3 None of the oximes tested (pralidoxime, obidoxime, HI 6 and HLö 7) can be regarded as a universally suitable reactivator. Obidoxime turned out to be the most potent and most efficacious oxime in reactivating AChE inhibited by various classes of OP insecticides and tabun. Obidoxime, however, was inferior to HI 6 against soman, sarin, cyclosarin and VX. Pralidoxime was generally less potent. 4 The kinetic data of reactivation established for diethylphosphoryl-AChE of human red cells indicate that the usually recommended dosage to attain a plasma concentration of 4 micrograms/ml does not permit exploitation of the full therapeutic potential of the oximes, in particular of pralidoxime. However, in suicidal mega-dose poisoning, oximes, even at optimal plasma concentrations, may be unable to cope with the fast re-inhibition of reactivated AChE in the first days following intoxication. 5 It is suggested that oximes be administered by continuous infusion following an initial bolus dose as long as reactivation can be expected and until permanent clinical improvement is achieved.

Cholinesterase status, pharmacokinetics and laboratory findings during obidoxime therapy in organophosphate poisoned patients.

1 The effectiveness of oxime therapy in organophosphate poisoning is still a matter of debate. It appears, however, that the often cited ineffectiveness of oximes may be due to inappropriate dosing. By virtue of in vitro findings and theoretical considerations we concluded in the preceding paper that oximes should preferably be administered by continuous infusion following an initial bolus dose for as long as reactivation of inhibited acetylcholinesterase (AChE) can be expected. This conclusion has called for a clinical trial to evaluate such oxime therapy on the basis of objective parameters. 2 Before transfer to the intensive care unit (ICU), 5 patients received primary care by an emergency physician. In the ICU, atropine sulphate was administered i.v. upon demand according to the endpoints: no bronchorrhoea, dry mucous membranes, no axillary sweating, heart rate of about 100/min. Obidoxime (Toxogonin) was given as an i.v. bolus (250 mg) followed by continuous infusion of 750 mg/24 h. 3 Intoxication and therapy were monitored by determining erythrocyte AChE (eryAChE) activity, reactivatability of the patient's eryAChE ex vivo, plasma cholinesterase activity, the presence of AChE inhibiting compounds, as well as the concentrations of obidoxime and atropine in plasma. 4 Obidoxime was effective in life-threatening parathion poisoning, in particular when the dose absorbed was comparably low. In mega-dose poisoning, net reactivation was not achieved until several days after ingestion, when the concentration of active poison in plasma had declined. Reactivatability in vivo lasted for a longer period than expected from in vitro experiments. 5 Obidoxime was quite ineffective in oxydemetonmethyl poisoning, when the time elapsed between ingestion and oxime therapy was longer than 1 day. When obidoxime was administered shortly after ingestion (1 h) reactivation was nearly complete. 6 Obidoxime levels of 10-20 microM were achieved by our regimen, and atropine could rapidly be reduced to approx. 20 microM, as attained by continuous infusion of 1 mg atropine sulphate/h. Maintenance of the desired plasma levels was not critical even when renal function deteriorated. 7 Signs of transiently impaired liver function were observed in patients who showed transient multiorgan failure. In the present stage of knowledge, we feel it advisable to keep the plasma concentration of obidoxime at 10-20 microM, although the full reactivating potential of obidoxime will not then be exploited. Still, the reactivation rate, with an apparent half-time of some 3 min, is twice that estimated for a tenfold higher pralidoxime concentration.