The efficacy of HI-6 DMS in a sustained infusion against percutaneous VX poisoning in the guinea-pig.

Post-exposure nerve agent treatment usually includes administration of an oxime, which acts to restore function of the enzyme acetylcholinesterase (AChE). For immediate treatment of military personnel, this is usually administered with an autoinjector device, or devices containing the oxime such as pralidoxime, atropine and diazepam. In addition to the autoinjector, it is likely that personnel exposed to nerve agents, particularly by the percutaneous route, will require further treatment at medical facilities. As such, there is a need to understand the relationship between dose rate, plasma concentration, reactivation of AChE activity and efficacy, to provide supporting evidence for oxime infusions in nerve agent poisoning. Here, it has been demonstrated that intravenous infusion of HI-6, in combination with atropine, is efficacious against a percutaneous VX challenge in the conscious male Dunkin-Hartley guinea-pig. Inclusion of HI-6, in addition to atropine in the treatment, improved survival when compared to atropine alone. Additionally, erythrocyte AChE activity following poisoning was found to be dose dependent, with an increased dose rate of HI-6 (0.48mg/kg/min) resulting in increased AChE activity. As far as we are aware, this is the first study to correlate the pharmacokinetic profile of HI-6 with both its pharmacodynamic action of reactivating nerve agent inhibited AChE and with its efficacy against a persistent nerve agent exposure challenge in the same conscious animal.

Efficacy of the antinicotinic compound MB327 against soman poisoning - Importance of experimental end point.

Medical countermeasures for acute poisoning by organophosphorus nerve agents are generally assessed over 24h following poisoning and a single administration of treatment. At 24h, the antinicotinic bispyridinium compound MB327 (1,10-(propane-1,3-diyl)bis(4-tert-butylpyridinium)) dimethanesulfonate is as effective as the oxime HI-6 against poisoning by soman, when used as part of a treatment containing atropine and avizafone. In this study, we hypothesised that an earlier endpoint, at 6h, would be more appropriate for the pharmacokinetics and mechanism of action of MB327 and would therefore result in improved protection. MB327 diiodide (33.8mg/kg) or the oxime HI-6 DMS (30mg/kg), in combination with atropine and avizafone (each at 3mg/kg) was administered intramuscularly to guinea pigs 1min following subcutaneous soman and the LD50 of the nerve agent was determined at 6h after poisoning for each treatment. The treatment containing HI-6 gave a similar level of protection at 6h as previously determined at 24h (protection ratios 3.9 and 2.9, respectively). In contrast, the protection achieved by treatment containing MB327 showed a striking increase at 6h (protection ratio >15.4) compared to the 24h end point (protection ratio 2.8). The treatment gave full protection for at least 5h against doses of soman up to 525µg/kg; in contrast, mortality began in animals treated with HI-6 after 1h. This study demonstrates the importance of using an appropriate end point and has shown that treatment including MB327 was far superior to oxime-based treatment for poisoning by soman, when assessed over a pharmacologically-relevant duration. The improved outcome was seen following a single dose of treatment: it is possible that additional doses to maintain therapeutic plasma concentrations would further increase survival time. Antinicotinic compounds therefore offer a promising addition to treatment, particularly for rapidly aging or oxime-insensitive nerve agents.

Bioscavenger is effective as a delayed therapeutic intervention following percutaneous VX poisoning in the guinea-pig.

The prolonged systemic exposure that follows skin contamination with low volatility nerve agents, such as VX, requires treatment to be given over a long time due to the relatively short half-lives of the therapeutic compounds used. Bioscavengers, such as butyrylcholinesterase (BChE), have been shown to provide effective post-exposure protection against percutaneous nerve agent when given immediately on signs of poisoning and to reduce reliance on additional treatments. In order to assess the benefits of administration of bioscavenger at later times, its effectiveness was assessed when administration was delayed for 2h after the appearance of signs of poisoning in guinea-pigs challenged with VX (4×LD50). VX-challenged animals received atropine, HI-6 and avizafone on signs of poisoning and 2h later the same combination with or without bioscavenger. Five out of 6 animals which received BChE 2h after the appearance of signs of poisoning survived to the end of the study at 48h, compared with 6 out of 6 which received BChE immediately on signs. All the animals (n=6+6) that received only MedCM, without the addition of BChE, died within 10h of poisoning. The toxicokinetics of a sub-lethal challenge of percutaneous VX were determined in untreated animals. Blood VX concentration peaked at approximately 4h after percutaneous dosing with 0.4×LD50; VX was still detectable at 36h and had declined to levels below the lower limit of quantification (10pg/mL) by 48h in 7 of 8 animals, with the remaining animal having a concentration of 12pg/mL. These studies confirm the persistent systemic exposure to nerve agent following percutaneous poisoning and demonstrate that bioscavenger can be an effective component of treatment even if its administration is delayed.

Affinities of bispyridinium non-oxime compounds to [(3)H]epibatidine binding sites of Torpedo californica nicotinic acetylcholine receptors depend on linker length.

The toxicity of organophosphorus nerve agents or pesticides arises from accumulation of acetylcholine and overstimulation of both muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs) due to inhibition of acetylcholinesterase (AChE). Standard treatment by administration of atropine and oximes, e.g., obidoxime or pralidoxime, focuses on antagonism of mAChRs and reactivation of AChE, whereas nicotinic malfunction is not directly treated. An alternative approach would be to use nAChR active substances to counteract the effects of accumulated acetylcholine. Promising in vitro and in vivo results were obtained with the bispyridinium compounds SAD-128 (1,1'-oxydimethylene bis(4-tert-butylpyridinium) dichloride) and MB327 (1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium) di(iodide)), which were partly attributed to their interaction with nAChRs. In this study, a homologous series of unsubstituted and 4-tert-butyl-substituted bispyridinium compounds with different alkane linker lengths was investigated in competition binding experiments using [(3)H]epibatidine as a reporter ligand. Additionally, the effect of the well-characterised MB327 on the [(3)H]epibatidine equilibrium dissociation (KD) constant in different buffers was determined. This study demonstrated that divalent cations increased the affinity of [(3)H]epibatidine. Since quaternary ammonium molecules are known to inhibit AChE, the obtained affinity constants of the tested bispyridinium compounds were compared with the inhibition of human AChE. In competition experiments, bispyridinium derivatives of longer linker length displaced [(3)H]epibatidine and inhibited AChE strongly. Bispyridinium compounds with short linkers, at most, have an allosteric interaction with the [(3)H]epibatidine binding sites and barely inhibited AChE. In dependence on alkane linker length, the bispyridinium compounds seemed to interact at different binding sites. However, the exact binding sites of the bispyridinium compounds responsible for the positive pharmacological effects have still not been identified, making predictive drug design difficult.

Restoration of soman-blocked neuromuscular transmission in human and rat muscle by the bispyridinium non-oxime MB327 in vitro.

The standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes is not sufficiently effective against all types of nerve agents. Alternative therapeutic strategies are required and bispyridinium non-oximes, acting as nicotinic antagonists, were identified as promising compounds. A previous study showed that the di(methanesulfonate) salt of the bispyridinium compound MB327 could restore soman-impaired neuromuscular function in vitro and improve survival of sarin, soman and tabun poisoned guinea pigs in vivo. Here, by using the indirect field stimulation technique, the ability of MB327 to counteract soman-impaired neuromuscular transmission was investigated in human intercostal muscle and rat diaphragm preparations. MB327 restored muscle force in a concentration-dependent manner in both species without reactivating soman-inhibited acetylcholinesterase. The therapeutic effect of MB327 could be washed out, indicating a direct effect at the nicotinic receptor level. Also the ability of MB327 to restore respiratory muscle function could be demonstrated for the first time in rat and human tissue. In combination with previous in vitro and in vivo findings MB327 may be considered a promising compound for the treatment of nerve agent poisoning and further studies are needed to identify optimized drug combinations, concentrations and dosing intervals to provide an effective therapy for OP poisoning.

Competition radioligand binding assays for the investigation of bispyridinium compound affinities to the human muscarinic acetylcholine receptor subtype 5 (hM(5) ).

Standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes lacks efficacy with some nerve agents. Promising in vitro and in vivo results were obtained with the bispyridinium compound SAD-128 which was partly attributed to its interaction with nicotinic acetylcholine receptors. Previous studies indicate that bispyridinium compounds interact with muscarinic acetylcholine receptors as well. The muscarinic M(5) receptor is not well investigated compared to other subtypes, but could be important in the search for new drugs for treating nerve agent poisoning. A set of bispyridinium compounds structurally related to SAD-128 were tested in competition binding experiments with recombinant human M(5) muscarinic acetylcholine receptors. Five of the six investigated bispyridinium compounds interacted with the orthosteric binding site, with affinities in the low micromolar range. These data indicate that interaction of bispyridinium compounds with muscarinic receptors may contribute to their therapeutic efficacy.

Protection against nerve agent poisoning by a noncompetitive nicotinic antagonist.

The acute toxicity of organophosphorus (OP) nerve agents arises from accumulation of acetylcholine (ACh) and overstimulation of ACh receptors. The mainstay of current pharmacotherapy is the competitive muscarinic antagonist, atropine. Nicotinic antagonists have not been used due to the difficulties of administering a dose of a competitive neuromuscular blocker sufficient to antagonise the effects of excessive ACh, but not so much that it paralyses the muscles. An alternative approach would be to use a noncompetitive antagonist whose effects would not be overcome by increasing ACh concentrations. This study demonstrates that the compound 1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium), which blocks open nicotinic ion channels noncompetitively, is able to reverse the neuromuscular paralysis after nerve agent poisoning in vitro and to protect guinea pigs against poisoning by nerve agents when used as part of a therapeutic drug combination including a muscarinic antagonist. In contrast to the oxime HI-6, this compound was equally effective in protecting against poisoning by sarin or tabun. Further studies should identify more effective compounds with this action and optimise doses for protection against nerve agent poisoning in vivo.

Interaction of bispyridinium compounds with the orthosteric binding site of human α7 and Torpedo californica nicotinic acetylcholine receptors (nAChRs).

Standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes lacks efficacy with different nerve agents. A direct pharmacologic intervention at the nicotinic acetylcholine receptor (nAChR) was proposed as an alternative therapeutic approach and promising in vitro and in vivo results were obtained with the bispyridinium compound SAD-128. In addition, a number of SAD-128 analogues improved neuromuscular transmission of soman-poisoned diaphragms in vitro. We investigated the interaction of six of these SAD-128 analogues with the orthosteric binding site of the human α7 nAChR and Torpedo californica nAChR with a high-throughput assay using radioactive ligands. The determined affinity constants indicate a weak interaction of three test compounds (K(i) in the micromolar range) with both receptors, but no interaction could be recorded with the other three test compounds. The six SAD-128 analogues showed a low intrinsic inhibitory potency with human acetylcholinesterase (IC50 > 400 µM). In conclusion, the results of the present study do not indicate a correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission and it is assumed that other mechanisms contribute to the therapeutic effect of the tested compounds.

An investigation of the effects of TETRA RF fields on intracellular calcium in neurones and cardiac myocytes.

PURPOSE: This study aimed to determine whether Terrestrial Trunked Radio (TETRA) fields can affect intracellular calcium signalling in excitable cells. MATERIALS AND METHODS: Intracellular calcium concentration ([Ca(2 +) ](i)) was measured in cultured rat cerebellar granule cells and cardiac myocytes during exposure to TETRA fields (380.8875 MHz pulse modulated at 17.6 Hz, 25% duty cycle). [Ca(2 +) ](i) was measured as fura-PE3, fluo-3 or fluo-4 fluorescence by digital image analysis. RESULTS: Granule cells exposed at specific absorption rates (SARs) of 5, 10, 20, 50 or 400 mW x kg(-1) showed no significant changes in resting [Ca(2 +) ](i). Increases in [Ca(2 +) ](i) in response to potassium-induced depolarization were significantly different from sham controls in TETRA-exposed cells, but the majority of the difference was attributable to initial biological variation between cell cultures. No difference was found between fura-PE3 (UV excitation) and fluo-3 (visible light excitation) measurements in these cells. Exposure to TETRA (50 or 400 mW x kg(-1)) had no significant effect on either the rate or amplitude of spontaneous Ca(2 +) transients in cardiac myocytes. The cells showed normal responses to salbutamol (50 microM) and acetylcholine (10 microM). CONCLUSIONS: Overall, these results showed no evidence of any consistent or biologically relevant effect of TETRA fields on [Ca(2 + )](i) in granule cells and cardiac myocytes at any of the SAR tested.