A novel high-performance liquid chromatography with diode array detector method for the simultaneous quantification of the enzyme-reactivating oximes obidoxime, pralidoxime, and HI-6 in human plasma.

Oximes such as pralidoxime (2-PAM), obidoxime (Obi), and HI-6 are the only currently available therapeutic agents to reactivate inhibited acetylcholinesterase (AChE) in case of intoxications with organophosphorus (OP) compounds. However, each oxime has characteristic agent-dependent reactivating efficacy, and therefore the combined administration of complementary oximes might be a promising approach to improve therapy. Accordingly, a new high-performance liquid chromatography method with diode-array detection (HPLC-DAD) was developed and validated allowing for simultaneous or single quantification of 2-PAM, Obi, and HI-6 in human plasma. Plasma was precipitated using 5% w/v aqueous zinc sulfate solution and subsequently acetonitrile yielding high recoveries of 94.2%-101.0%. An Atlantis T3 column (150 × 2.1mm I.D., 3 µm) was used for chromatographic separation with a total run time of 15 min. Quantification was possible without interferences within a linear range from 0.12 to 120 µg/mL for all oximes. Excellent intra-day (accuracy 91.7%-98.6%, precision 0.5%-4.4%) and inter-day characteristics (accuracy 89.4%-97.4%, precision 0.4%-2.2%) as well as good ruggedness were found. Oximes in processed samples were stable for at least 12 h in the autosampler at 15°C as well as in human plasma for at least four freeze-thaw cycles. Finally, the method was applied to plasma samples of a clinical case of pesticide poisoning.

Screening of chiral shift reagents suitable to generically separate the enantiomers of V-agents by 31P-NMR spectroscopy.

Fourteen amino acids protected at the N-terminal and at their side chains were screened for resolving the enantiomers of V-agents by NMR. While none of the shift reagents tested showed really effective separation in proton NMR, two of them (BOC-Gln(Xan)-OH, 16, and Z-Arg(Z)2-OH), 21, with 16 superior to 21) were found suitable to separate the enantiomers of all V-agent homologues involved in the test by 31P-NMR. Molar ratios investigated were 1:0.5, 1:1, 1:1.5, 1:2, and 1:3 with the V-agent set to 1 throughout the experiments. All these ratios were more or less effective, but 1:3 was found to separate the V-agents the most reliable way. It is postulated that three chiral solvating molecules are then coordinated around the organophosphate: ion pair formation with the amino nitrogen of the V agent side chain, hydrogen bonding provided by the PO unit, and extension of coordination at the phosphorus atom itself. After chiral separation of VX by semi-preparative LC-MS the enantiomers were examined with both configurations of 16 releasing four different 31P NMR peaks which correspond to four different complexes: R-S3, R-R3, S-R3, and S-S3. Comparing these results with literature data it is assumed that (+)-VX corresponds to the RP configuration and (-)-VX to the SP-configuration.

New Resensitizers for the Nicotinic Acetylcholine Receptor by Ligand-Based Pharmacophore Modeling.

INTRODUCTION: Irreversible inhibition of the acetylcholinesterase upon intoxication with organophosphorus compounds leads to an accumulation of acetylcholine in the synaptic cleft and a subsequent desensitization of nicotinic acetylcholine receptors which may ultimately result in respiratory failure. A direct intervention at the nicotinic acetylcholine receptor (nAChR) was proposed as an alternative therapeutic approach to the treatment with atropine and oximes. METHODS: The bispyridinium compound MB327 has been found to recover functional activity of nAChR thus representing a promising starting point for the development of new drugs for the treatment of organophosphate poisoning. Recent solid-supported membrane-based electrophysiological experiments have identified symmetrically substituted bispyridinium compounds e.g. MB327, MB583, and PTM0001 that are able to resensitize nAChR of Torpedo californica. In addition, six compounds have been found not to show any resensitizing potential and were thus classified as inactive. This set of active and inactive bispyridinium compounds was taken to develop a pharmacophore model and in silico screening of a virtual database of bispyridinium compounds to identify new compounds that are able to restore the functional activity of desensitized nAChR. RESULTS: Screening of a virtual compound database of symmetrically substituted bispyridinium compounds with the derived pharmacophore yielded several promising compounds which satisfy the pharmacophore and ought to have the same or even better resensitizing effect on nAChR as the parent compound MB327.

Simultaneous quantification of atropine and scopolamine in infusions of herbal tea and Solanaceae plant material by matrix-assisted laser desorption/ionization time-of-flight (tandem) mass spectrometry.

RATIONALE: Atropine (Atr) and scopolamine (Scp) are toxic secondary plant metabolites of species within the Solanaceae genus that can accidentally or intentionally reach the food store chain by inaccurate harvesting of any plant material, e.g. for herbal tea infusions. Ingestion may cause severe anticholinergic poisoning thus requiring risk-oriented determination in food and beverages. The suitability of matrix-assisted laser desorption/ionization time-of-flight (tandem) mass spectrometry, MALDI-TOF MS(/MS), should be characterized for simultaneous analysis. METHODS: We herein present the first MALDI-TOF MS(/MS) procedure for quantitative determination of both alkaloids in herbal tea infusions and Solanaceae plant material. A standard additions procedure using triply deuterated Atr as internal standard was developed and validated. RESULTS: Tropane alkaloids were detected without interferences and the standard additions procedure allowed reliable quantification. Intraday and interday precision were less than 17% and corresponding accuracies were between 77% and 112%. Limits of detection in the spotting solution were found at 5 ng/mL (Atr) and 0.5 ng/mL (Scp). The assay was applied to diverse tea infusions as well as to berries and leaves of deadly nightshade and angel's trumpet. CONCLUSIONS: The usefulness of MALDI-TOF MS(/MS) for investigations of plant-derived samples to prove contaminations by small basic compounds was demonstrated. The elaborate procedure is reliable but quite laborious to obtain quantitative results, but MALDI-TOF MS(/MS) was also shown to be a valuable tool for rapid qualitative screening for Atr and Scp in plant extracts.

Effects of succinylcholine in an organotypic spinal cord-skeletal muscle coculture of embryonic mice.

Intoxication with organophosphorus compounds is an important clinical problem worldwide. Although the core treatments - atropine, oximes and diazepam - are defined, high case fatalities were reported for intoxication with organophosphorus insecticides. In particular the role of oximes is not completely understood since they might benefit only patients poisoned by specific pesticides or patients with moderate poisoning and few randomised trials of such poisoning have been performed. This justifies the need for new in vitro test-systems like cocultures of spinal cord and muscle tissue, which have been recently introduced. However this test-system is not yet fully characterized. In order to estimate the applicability of cocultures of spinal cord and muscle tissue their sensitivity to succinylcholine (di-acetylcholine), a depolarizing muscle relaxant in clinical use, was tested. The test-system evaluated in the current study showed sensitivity to succinylcholine with an EC50 as low as 1µM thereby being close to the EC50 value in adult human patients (2.6µM). Furthermore, action potential activity of spinal ventral horn neurons was not altered by succinylcholine. The latter observations strongly suggest that our preparation well predicts the qualitative and quantitative actions of novel drugs targeting the neuromuscular system in vivo. In summary, cocultures of spinal cord and muscle tissue seem to be a valid test-system for the development and investigation of new oximes. Moreover, practical aspects like transport over long distances to further laboratories, the opportunity to conduct long-term studies and the reduction of animal usage display further advantages of its use.

Silibinin as a potential therapeutic for sulfur mustard injuries.

Sulfur mustard (SM) is a vesicating chemical warfare agent causing skin blistering, ulceration, impaired wound healing, prolonged hospitalization and permanent lesions. Silibinin, the lead compound from Silybum marianum, has also been discussed as a potential antidote to SM poisoning. However, its efficacy has been demonstrated only with regard to nitrogen mustards. Moreover, there are no data on the efficacy of the water-soluble prodrug silibinin-bis-succinat (silibinin-BS). We investigated the effect of SIL-BS treatment against SM toxicity in HaCaT cells with regard to potential reduction of necrosis, apoptosis and inflammation including dose-dependency of any protective effects. We also demonstrated the biotransformation of the prodrug into free silibinin. HaCaT cells were exposed to SM (30, 100, and 300µM) for 30min and treated thereafter with SIL-BS (10, 50, and 100µM) for 24h. Necrosis and apoptosis were quantified using the ToxiLight BioAssay and the nucleosome ELISA (CDDE). Pro-inflammatory interleukins-6 and -8 were determined by ELISA. HaCaT cells, incubated with silibinin-BS were lysed and investigated by LC-ESI MS/MS. LC-ESI MS/MS results suggest that SIL-BS is absorbed by HaCaT cells and biotransformed into free silibinin. SIL-BS dose-dependently reduced SM cytotoxicity, even after 300µM exposure. Doses of 50-100µM silibinin-BS were required for significant protection. Apoptosis and interleukin production remained largely unchanged by 10-50µM silibinin-BS but increased after 100µM treatment. Observed reductions of SM cytotoxicity by post-exposure treatment with SIL-BS suggest this as a promising approach for treatment of SM injuries. While 100µM SIL-BS is most effective to reduce necrosis, 50µM may be safer to avoid pro-inflammatory effects. Pro-apoptotic effects after high doses of SIL-BS are in agreement with findings in literature and might even be useful to eliminate cells irreversibly damaged by SM. Further investigations will focus on the protective mechanism of silibinin and its prodrug and should establish an optimum concentration for treatment.

Structural requirements for effective oximes--evaluation of kinetic in vitro data with phosphylated human AChE and structurally different oximes.

Treatment of poisoning by various organophosphorus (OP) nerve agents with established acetylcholinesterase (AChE) reactivators (oximes) is insufficient. In consequence, extensive research programs have been undertaken in various countries in the past decades to identify more effective oximes. The efficacy of new compounds has been investigated with different in vitro and in vivo models which hamper the comparison of results from different laboratories. The crucial mechanism of action of oximes is the reactivation of phosphylated AChE. The kinetic properties of these compounds can be quantified in vitro with isolated AChE from different origin. It was tempting to evaluate the reactivation kinetics of a series of oximes with various OP inhibitors performed under identical experimental conditions in order to get insight into structural requirements for adequate affinity and reactivity towards inhibited AChE. The determination of reactivation rate constants with bispyridinium oximes having different linkers, bearing oxime group(s) at different positions and having in part additional substituents revealed that (a) the reactivating potency was dependent on the position of the oxime groups and of additional substituents, (b) small modifications of the oxime structure had an in part marked effect on the kinetic properties and (c) no single oxime had an adequate reactivating potency with AChE inhibited by structurally different OP. These and previous studies underline the necessity to investigate in detail the kinetic properties of novel oximes and that the identification of a single oxime being effective against a broad range of structurally different OP will remain a major challenge.

Long-term evaluation of organophosphate toxicity and antidotal therapy in co-cultures of spinal cord and muscle tissue.

Victims of nerve agents basically require antidotal treatment. There is need for novel antidotes and for therapeutic procedures that are specifically adapted to these patients. To cope with this challenge, in vitro test systems which are easy to handle and allow for conducting long-term studies would be of great benefit. The present work introduces co-cultures of spinal cord and muscle tissue as ex vivo testing systems meeting these criteria. Cell cultures in which functional neuromuscular synapses formed ex vivo were prepared from embryonic mice. Spontaneous muscle activity was recorded by video microscopy. Muscle contractions involved intact neuromuscular transmission as indicated by the effect of succinylcholine, a muscle relaxant that completely abolished muscle activity. At a concentration of 0.75 µM the nerve agent VX reduced the frequency of spontaneous muscle contractions by about 75%. Subsequent application of obidoxime re-established muscle movements. After 24 h of antidotal treatment, muscle activity approached the level of sham-treated cultures and remained stable over the following week. In summary, co-cultures of spinal cord and muscle tissue are promising tools for evaluating the success of antidotal treatment following organophosphate intoxication over a period of at least seven days.