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.

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.

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.

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.

Cutaneous receptive fields of somatic and viscerosomatic neurones in the thoracic spinal cord of the cat.

Extracellular single-unit recordings were made from 121 neurones in the thoracic spinal cord of the cat. All neurones could be driven by electrical stimulation of dorsal root afferent fibres. The neurones were classified, according to the absence or presence of inputs from the ipsilateral splanchnic nerve, as "somatic" or "viscerosomatic", respectively. Cutaneous receptive fields were identified for 75 of the neurones: 31 were somatic and 44 viscerosomatic. Only two of the somatic cells received cutaneous nociceptive inputs, compared with 33 of the viscerosomatic cells. Sixty-four percent of the whole sample of neurones had receptive fields which included three or more dermatomes. Viscerosomatic cells tended to have larger receptive fields than the somatic neurones, and six of them had fields which did not include the corresponding (T11) dermatome. Neurones with receptive fields in the dorsal one-third of the dermatome tended to be located in the lateral one-third of the dorsal horn, but those with receptive fields in the ventral two-thirds of the dermatome showed no differential distribution within the gray matter. This is discussed with respect to the results of anatomical studies on the dorsal horn projections of cutaneous afferent fibres from different regions of the dermatome. Preliminary results from intracellular staining with horseradish peroxidase reveal extensive branching of primary afferents in the dorsal horn, and large dendritic fields of dorsal horn neurones. Our physiological and morphological results indicate that the somatotopic organisation of the thoracic spinal cord is less well defined than that of the lumbosacral region.

Ion channel blockade by oximes and recovery of diaphragm muscle from soman poisoning in vitro.

1. The actions of oximes and related compounds on the nicotinic acetylcholine receptor ion channel at the adult mouse muscle endplate were investigated by use of single-channel recording techniques. The aim of the study was to determine whether the channel-blocking properties of the compounds could contribute to their therapeutic effectiveness against soman poisoning in vitro. 2. Therapeutic effectiveness was assessed in guinea-pig phrenic nerve-hemidiaphragm preparations by measuring the degree of recovery of neuromuscular function produced by the compounds following poisoning by soman. A number of the compounds, including some which lacked the oxime group, produced a significant recovery of neuromuscular function which was unrelated to acetylcholinesterase (AChE) reactivation; this was reversed by washing off the compound, and was therefore attributed to a direct pharmacological action on the muscle. 3. Single channel recordings showed that some of the compounds blocked open nicotinic receptor ion channels in preparations of mouse muscle fibres. The compounds which showed the greatest direct pharmacological actions in diaphragms produce a very fast, flickering blockade of the channels. Several quantitative measures of channel-blocking activity correlated very well with the direct pharmacological action. Furthermore, for two compounds studied in greater detail, the direct action and channel-blocking showed similar concentration-response relationships. 4. The results of this study indicate that the direct pharmacological action of oximes and their analogues against neuromuscular blockade by soman in vitro is due to their channel-blocking activity. The direct action does not correlate well with protection against soman poisoning in vivo, however, which suggests that additional non-reactivating properties of these compounds, at sites other than the neuromuscular junction, may also be important for their therapeutic effectiveness.

Effects of organophosphorus anticholinesterases on nicotinic receptor ion channels at adult mouse muscle endplates.

1. The effects of a range of organophosphorus anticholinesterases on the nicotinic acetylcholine receptor ion channel at the adult mouse muscle endplate were investigated by use of single-channel recording techniques. Diisopropylfluorophosphate (DFP), sarin and soman had no effect on open times at concentrations of up to 100 microM, but ecothiopate (Eco) and O-ethyl S-[2-(diisopropylamino)ethyl]methyl phosphonothiolate (VX) were found to have voltage- and concentration-dependent open channel-blocking actions at concentrations of 1-50 microM. In addition to its channel-blocking action, Eco (50 microM) had a weak agonist effect: it is suggested that this may be attributable to thiocholine produced by hydrolysis of Eco. 2. Rate constants for blockade by Eco and VX were determined according to a sequential model. The greater voltage-dependence of the block by Eco was due to a greater voltage sensitivity of the blocking rate constant compared to VX: the voltage-dependence of the unblocking rate constant was similar for both compounds. 3. In control recordings, the frequency of channel opening declined exponentially with time after formation of the gigaseal. Sarin and soman both increased the rate of this decline, indicating that they accelerated the rate of desensitization of the receptors. Eco and VX reduced the initial frequency of opening, which may have been due to enhancement of a fast phase of desensitization during gigaseal formation, or to blockade of closed channels. 4. It is concluded that the direct actions of organophosphates on nicotinic receptor ion channels are of little importance for their toxicity under normal conditions, since they occur only at much higher concentrations than those which cause inhibition of acetylcholinesterase. Such actions may become apparent, however, when therapies against the anticholinesterase effects of organophosphates increase their lethal dose sufficiently. These direct actions should also be taken into account when the effects of organophosphates on cholinergic transmission are studied.

Distribution of sural nerve afferent fibres within the dorsal horn of adult rats treated at birth with capsaicin.

The distribution of sural nerve afferent fibres within the spinal cord of normal adult rats and of adult rats treated at birth with capsaicin was examined using transganglionic transport of horseradish peroxidase (HRP). Labelled fibres were seen, in normal and in capsaicin-treated rats, in Laminae I-VI of the central third of the dorsal horn, extending rostrocaudally between the L3 and L5 segments. It is concluded that the changes in dorsal horn somatosensory systems induced by neonatal capsaicin are not due to anatomical redistribution of the areas of termination of peripheral nerves within the spinal cord.

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.

Supraspinal loops that mediate visceral inputs to thoracic spinal cord neurones in the cat: involvement of descending pathways from raphe and reticular formation.

Single unit electrical activity has been recorded from 29 viscero-somatic neurones in the T11 spinal cord segment of chloralose anaesthetized cats. Twenty-six of these neurones showed changes in their responses to electrical stimulation of the splanchnic nerve after reversible spinalization of the animals by cold block: 14 showed increased responses whereas 12 presented reduced or abolished responses during the spinal block. The majority of neurones in the first group were located in laminae IV, V and VII and were inhibited by electrical stimulation of the nucleus raphe magnus (NRM) and the reticular formation (Ret.F). Most neurones of the second type were located in the ventral horn, and the majority were excited by electrical stimulation of the NRM and the Ret.F. This second type of neurone may play a role in the maintenance of the excitation in the central nervous system which follows visceral noxious stimulation.

Compared toxicity of the potassium channel blockers, apamin and dendrotoxin.

The central toxicities of two potassium ion channel blockers, apamin and alpha-dendrotoxin (DTx), have been compared. Both apamin and dendrotoxin injected intracerebroventricularly produced signs of poisoning, including tremor and ataxia; however, only DTx produced changes in brain electrical activity, with high voltage spikes and epileptiform activity and subsequent brain damage. DTx, but not apamin, increased the amplitude of evoked field potentials and caused repetitive firing of neurones in hippocampal slices. Signs of poisoning following peripheral (intraperitoneal) administration of apamin were similar to those following central administration, including dramatic haemorrhagic effects on the lungs of decedent animals. These results are consistent with dendrotoxin being a centrally-active neurotoxin producing epileptiform activity and brain damage, whilst apamin produces its most significant pathology in the lung, possibly involving a neurogenic mechanism.

Modeling the postdialysis rebound: the reconciliation of current formulas.

Three approaches are currently used in kinetic models (UKMs) to account for the postdialysis rebound in urea concentration, and thereby accurately measure the hemodialysis dose, KT/V (where K, T, V denote dialyzer clearance, dialysis duration, and urea distribution volume, respectively). The approach developed by Smye uses an intradialytic sample to predict the postdialysis equilibrium concentration, Ce, which is then used in a single pool UKM to give KT/V. A second approach developed by Tattersall introduces a patient clearance time, tp. The true dialysis dose is then given by T/(T + tp) x apparent dose, and tp is estimated to be 36 minutes. The Daugirdas analysis uses an empiric regression equation to give the true dose; KT/V)true from the single pool value, KT/V)sp; KT/V)true = KT/V)sp - (36/T)(KT/V)sp + 0.03. The analysis confirms the equivalence of all three formulas, which arises from the observation that during the later stages of dialysis, the urea concentration decreases as a single exponential. The formulas are independent of whether a flow or diffusion model is used to describe the kinetics of urea removal. The original analysis assumed constant volumes, but the effect of ultrafiltration volume u on C(e) may be accounted for by multiplying by (1 + u/V). The Smye equation is more vulnerable to error in practice, because small errors in the intradialytic sample give larger errors in the equilibrium concentration estimate, whereas dose estimates based on the Tattersall and Daugirdas equations are less affected by sampling errors. However, unlike the Smye approach, these two formulas would need adaptation for use with other solutes. The advent of continuous urea monitoring should permit more accurate, prospective estimates of equilibrium concentrations and dialysis dose.

Fluid balance modelling in patients with kidney failure.

In patients with kidney failure, adequate control of fluid status remains one of the most difficult routine issues to be addressed in the modern style of dialysis. This is primarily due to the lack of quantitative methods for the assessment of fluid status and the reliance on subjective criteria. Fluid is removed from the blood during dialysis treatments using a process called ultrafiltration. The last decade has seen considerable developments in blood volume monitoring (BVM) technology which has enabled responses to ultrafiltration to be continually monitored on an individual basis. This has enabled feedback control of patients' blood volume to be applied with partial success, reducing the number of symptoms. The feedback control algorithms employed have been relatively unsophisticated, using simple proportional control with no attempt to include models of the patient fluid dynamics. This paper describes the development of some prototype fluid kinetic models which may be used in a more advanced control system. Initial results demonstrate the importance of active control processes in the patients' physiological compensatory mechanisms.

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.

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.

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.

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.