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.

Optimization of the convection volume in online post-dilution haemodiafiltration: practical and technical issues.

In post-dilution online haemodiafiltration (ol-HDF), a relationship has been demonstrated between the magnitude of the convection volume and survival. However, to achieve high convection volumes (>22 L per session) detailed notion of its determining factors is highly desirable. This manuscript summarizes practical problems and pitfalls that were encountered during the quest for high convection volumes. Specifically, it addresses issues such as type of vascular access, needles, blood flow rate, recirculation, filtration fraction, anticoagulation and dialysers. Finally, five of the main HDF systems in Europe are briefly described as far as HDF prescription and optimization of the convection volume is concerned.

Resolution of low-grade proteinuria is associated with improved outcomes after renal transplantation-a retrospective longitudinal study.

Low-grade proteinuria and systolic hypertension (SHT) are risk factors for allograft failure. Both are dynamic variables and their relationship is not independent. We have simultaneously analyzed the effects of proteinuria and SHT on graft outcomes in 805 adult Kidney Transplant Recipients and impact of their changes over time. Proteinuria and systolic blood pressure (SBP) were recorded for years 1 and 3 posttransplantation. Subjects with proteinuria >1 g/day were excluded. Patients were divided into groups based on proteinuria (Absent(A) <150 mg/day or low-grade(P)150 mg-1 g/day) and blood pressure (Normotensive-SBP <140 mmHg or hypertensive-SBP ≥ 140 mmHg). Graft survival was assessed in all four groups over 10 years by multivariate analysis. At the three annual time points (Year 1, 2 and 3) hypertensive patients with proteinuria had the worst graft survival. Patients with persistent proteinuria between years 1-2 and 2-3 had the poorest graft survival with an improvement if proteinuria regressed (P-A), especially in the Hypertensive group. The impact of proteinuria was highest in persistently hypertensive patients between years 1-3. Thus both proteinuria and SHT were associated with poor graft survival and the combination of the two led to the worst outcomes. Importantly, SHT was associated with significantly worse outcomes in patients with proteinuria. Patient cohort with SHT and low-grade proteinuria represent a selective group that might benefit from intervention.

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.

Effect of surgical technique on radiographic fusion of the anconeus in the treatment of ununited anconeal process.

OBJECTIVES: The aim of this study was to determine if internal fixation of the anconeus combined with a proximal ulnar osteotomy was more likely to result in fusion of the anconeus to the ulna compared with a proximal ulnar osteotomy alone. METHODS: A total of 12 orthopaedic referral clinics reviewed their clinical databases for cases of ununited anconeal process. Demographic and clinical parameters were collected along with radiographic follow-up at a minimum of four weeks. Cases treated with proximal ulnar osteotomy alone were compared with those treated with proximal ulnar osteotomy + internal fixation. Both groups were compared for background and disease variables. We tested for an association between treatment method and whether radiographic anconeal union had occurred. RESULTS: A total of 47 elbows (44 dogs) were identified. Of these, 28 cases (average age 7.6 months) were treated with proximal ulnar osteotomy (of which eight were stabilised with an intramedullary pin) alone. Nineteen cases (average age 7.1 months) were treated with proximal ulnar osteotomy + internal fixation. The two groups were not significantly different in age (P=0.638, Mann-Whitney U test). Fourteen of 28 cases with proximal ulnar osteotomy alone displayed anconeal union at follow-up compared with 16 of 19 cases of proximal ulnar osteotomy + internal fixation, and this difference was statistically significant (P=0.029, Fisher's exact test). CLINICAL SIGNIFICANCE: These data suggest that use of a lag screw to stabilise and compress the ununited anconeal process in addition to proximal ulnar osteotomy produces a better radiographic outcome. It is argued that radiographic union of the anconeus is likely to be associated with better long-term clinical outcome but further studies are required to confirm this.

Retrospective analysis of the relationship between time of thoracostomy drain removal and discharge time.

OBJECTIVES: The objective of this study was to evaluate the relationship between the volume of fluid being produced at the time of thoracostomy drain removal and the time to hospital discharge in dogs and cats. METHODS: Records of 101 dogs and 26 cats with thoracostomy drains were reviewed. Three subgroups were created according to the reason for thoracostomy drain placement: P (postsurgical), A (air) and F (fluid). A generalised linear model with Poisson Errors was performed to test the relationship between the volume of fluid produced at the time of thoracostomy drain removal and the time to discharge. The volume of fluid produced and the time to discharge were compared between species and subgroups. RESULTS: No significant relationship was found between the volume of fluid produced at the time of thoracostomy drain removal and the time to discharge in either species or between the time to discharge and the reason for thoracostomy drain placement. Animals with a volume of fluid higher than 2 ml/kg/day at the time of thoracostomy drain removal did not have increased hospitalisation times. CLINICAL SIGNIFICANCE: Thoracostomy drain can be removed, without clinical compromise, when the volume of fluid produced exceeds 2 ml/kg/day. However, other clinical parameters must be taken into consideration.

Factors influencing the short-term outcome following thoracic surgery in 98 dogs.

OBJECTIVES: To evaluate the factors influencing the short-term (<14 days) outcome of thoracic surgery in dogs. METHODS: A retrospective review of 98 dogs undergoing thoracotomy over a five-year period was undertaken. RESULTS: A pre-operative diagnosis was achieved in 69 per cent of cases. Intrathoraic neoplasia had the lowest pre-operative diagnosis rate (5.5 per cent). Mortality rates of 21 per cent were recorded and were significantly higher for intrathoracic neoplasia (50 per cent) and significantly lower for persistent ductus arteriosus (7.4 per cent) and vascular ring anomaly (0 per cent). Median sternotomy was the preferred approach for pyothorax (85 per cent) and penetrating thoracic injuries (66 per cent). Intercostal thoracotomy was the preferred approach for all other diseases. Postoperative complications occurred in 39 per cent of cases. Wound complications were more common for pyothorax (45 per cent) and following median sternotomy (71 per cent). Thoracic drains were placed in 77 per cent of cases and complications were recorded in 23 per cent. Pyothorax and chylothorax had thoracic drains maintained for significantly longer periods of time. Longer thoracic drain duration was correlated significantly with increased complication rates. CLINICAL SIGNIFICANCE: The short-term outcome following thoracic surgery is influenced by diagnosis. The thoracic approach is determined by intrathoracic disease, but may influence outcome by affecting the incidence of postoperative wound complications. The risk of thoracic drain complications increases with drain duration, which is influenced by the underlying disease. Drains should be maintained for the minimal amount of time possible.

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.

Artificial intelligence: a new approach for prescription and monitoring of hemodialysis therapy.

The effect of dialysis on patients is conventionally predicted using a formal mathematical model. This approach requires many assumptions of the processes involved, and validation of these may be difficult. The validity of dialysis urea modeling using a formal mathematical model has been challenged. Artificial intelligence using neural networks (NNs) has been used to solve complex problems without needing a mathematical model or an understanding of the mechanisms involved. In this study, we applied an NN model to study and predict concentrations of urea during a hemodialysis session. We measured blood concentrations of urea, patient weight, and total urea removal by direct dialysate quantification (DDQ) at 30-minute intervals during the session (in 15 chronic hemodialysis patients). The NN model was trained to recognize the evolution of measured urea concentrations and was subsequently able to predict hemodialysis session time needed to reach a target solute removal index (SRI) in patients not previously studied by the NN model (in another 15 chronic hemodialysis patients). Comparing results of the NN model with the DDQ model, the prediction error was 10.9%, with a not significant difference between predicted total urea nitrogen (UN) removal and measured UN removal by DDQ. NN model predictions of time showed a not significant difference with actual intervals needed to reach the same SRI level at the same patient conditions, except for the prediction of SRI at the first 30-minute interval, which showed a significant difference (P = 0.001). This indicates the sensitivity of the NN model to what is called patient clearance time; the prediction error was 8.3%. From our results, we conclude that artificial intelligence applications in urea kinetics can give an idea of intradialysis profiling according to individual clinical needs. In theory, this approach can be extended easily to other solutes, making the NN model a step forward to achieving artificial-intelligent dialysis control.

Interaction of channel-blocking bispyridinium compounds with supported phospholipid layers.

The interaction of phospholipid monolayers of dioleoyl phosphatidylcholine (DOPC) on mercury electrodes with bispyridinium compounds is reported in this paper. The interaction is related to the modification of the capacitance-potential plot and in particular of two well defined phase transitions of the phospholipid monolayer. The order of the extent of interaction of the test compounds with the monolayer can be related to their structure and is: P65>Toxogonin>BPE>HS6>TMB4>HI6>BPT>P2S. The penetration of the compound into the monolayer depends on potential. At potentials more negative than the occurrence of the two phase transitions, the test compounds penetrate further and disrupt the monolayers. At more positive potentials this effect is reversed.

Effects of low intensity radiofrequency electromagnetic fields on electrical activity in rat hippocampal slices.

Slices of rat hippocampus were exposed to 700 MHz continuous wave radiofrequency (RF) fields (25.2-71.0 V m(-1), 5-15 min exposure) in a stripline waveguide. At low field intensities, the predominant effect on the electrically evoked field potential in CA1 was a potentiation of the amplitude of the population spike by up to 20%, but higher intensity fields could produce either increases or decreases of up to 120 and 80%, respectively, in the amplitude of the population spike. To eliminate the possibility of RF-induced artefacts due to the metal stimulating electrode, the effect of RF exposure on spontaneous epileptiform activity induced in CA3 by 4-aminopyridine (50-100 microM) was investigated. Exposure to RF fields (50.0 V m(-1)) reduced or abolished epileptiform bursting in 36% of slices tested. The maximum field intensity used in these experiments, 71.0 V m(-1), was calculated to produce a specific absorption rate (SAR) of between 0.0016 and 0.0044 W kg(-1) in the slices. Measurements with a Luxtron fibreoptic probe confirmed that there was no detectable temperature change (+/- 0.1 degrees C) during a 15 min exposure to this field intensity. Furthermore, imposed temperature changes of up to 1 degrees C failed to mimic the effects of RF exposure. These results suggest that low-intensity RF fields can modulate the excitability of hippocampal tissue in vitro in the absence of gross thermal effects. The changes in excitability may be consistent with reported behavioural effects of RF fields.

An improved brain slice model of nerve agent-induced seizure activity.

A brain slice model was developed to investigate the mechanisms of seizure activity induced by soman and the effectiveness of potential anticonvulsant drugs. Unlike previously reported slice studies with nerve agents, this model contains the entorhinal cortex as well as the hippocampus. This allows the study of the spread of seizure discharges within the limbic system and the development of prolonged, sustained discharges that are rarely seen in the simple hippocampal slice preparation. Soman (1 microM) induced a second population spike in the evoked field potential in the CA1 or CA3 region within 15-20 min. In almost all the slices tested, this developed into spontaneous seizure activity within 30-40 min. As well as interictal bursts, many slices also showed longer periods of high-frequency bursting analogous to ictal seizure activity that originated in the entorhinal cortex. This activity appeared similar to that induced by the muscarinic agonist pilocarpine. Both the second population spike and the spontaneous discharges could be blocked by diazepam and by AMPA/kainate antagonists, but not by the NMDA antagonists AP5 and MK-801. This study confirms that the combined hippocampal-entorhinal cortex slice preparation is a suitable model for investigating the origin and propagation of nerve-agent-induced seizures within the limbic system.