Antimicrobial susceptibility testing: currently used methods and devices and the near future in clinical practice.

Rapid identification of pathogen and its resistance to antimicrobial drugs, and subsequent appropriate antimicrobial treatment are essential for correct patient outcomes. Conventional detection methods of bacterial resistance, such as disc diffusion, broth microdilution and automated instruments, are constantly widely used and primarily standardized. Nevertheless, the results cannot be obtained earlier than 48 h after receiving a sample, which may lead to prolonged use or overuse of broad-spectrum antibiotics. Hence, there is a drive to develop and introduce novel, faster, standardized, sensitive and specific methods with reliable results into routine microbiological laboratory practice. Recently developed matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been introduced in recent years into laboratory practice, and methods based on microfluidics and microdroplets might be introduced in the near future. This review is focused on the methods and instruments in use both currently and in the foreseeable future, applicable to determine antimicrobial efficacy in clinical microbiology laboratories.

The summary on non-reactivation cholinergic properties of oxime reactivators: the interaction with muscarinic and nicotinic receptors.

Organophosphorus inhibitors (OP) of acetylcholinesterase (AChE) represent a group of highly toxic compounds. The treatment of OP intoxication is, however, insufficiently ensured. Currently, two main categories of drugs-anticholinergics and oxime reactivators- are employed as antidotes. Oximes have been reported to act at several levels of the cholinergic transmission, and among the non-reactivation effects, the interaction with cholinergic receptors stands out. This review addresses issues correlated with non-reactivating effects of oxime reactivators with a special focus on the muscarinic and nicotinic receptors, but involvement of other cholinergic structures such as AChE and choline uptake carriers are discussed too. It can be concluded that the oxime reactivators show a variation in their antagonistic effect on the muscarinic and nicotinic receptors, which is likely to be of significance in the treatment of OP poisoning. In vitro data reported oximes to exert higher efficacy on the muscarinic M2 subtype than on the AChE. However, this effect seemed to be subtype specific since the antagonistic M3 effect was lower. Also, and importantly, the antimuscarinic effect was larger than that on nicotinic receptors. Even though atropine showed a much higher muscarinic antagonism, it is supposed that non-reactivation properties of oxime reactivators play a significant role in the treatment of OP poisoning.

UHPLC-Orbitrap study of the first phase tacrine in vitro metabolites and related Alzheimer's drug candidates using human liver microsomes.

Tacrine was the first drug used in the therapy of Alzheimer's disease (AD) and is one of the leading structures frequently pursued in the drug discovery of novel candidates for tackling AD. However, because tacrine has been withdrawn from the market due to its hepatotoxicity, ascribed to specific metabolites, concerns are high about the toxicity profile of newly developed compounds related to tacrine. From the point of view of drug safety, the formation of metabolites must be uncovered and analyzed. Bearing in mind that the main culprit of tacrine hepatotoxicity is its biotransformation to hydroxylated metabolites, human liver microsomes were used as a biotransformation model. Our study aims to clarify phase I metabolites of three potentially non-toxic tacrine derivatives (7-methoxytacrine, 6-chlorotacrine, 7-phenoxytacrine) and to semi-quantitatively determine the relative amount of individual metabolites as potential culprits of tacrine-based hepatotoxicity. For this purpose, a new selective UHPLC-Orbitrap method has been developed. Applying UHPLC-Orbitrap method, two as yet unpublished tacrine and 7-methoxytacrine monohydroxylated metabolites have been found and completely characterized, and the separation of ten dihydroxylated tacrine and 7-methoxytacrine metabolites was achieved for the first time. Moreover, the structures of several new metabolites of 7-phenoxytacrine and 6-chlorotacrine have been identified. In addition, the relative amount of these newly observed metabolites was determined. Based on the results and known facts about the toxicity of tacrine metabolites published so far, it appears that 7-phenoxytacrine and 6-chlorotacrine could be substantially less hepatotoxic compared to tacrine, and could potentially pave the way for metabolically safe molecules applicable in AD therapy.

Pralidoxime--the gold standard of acetylcholinesterase reactivators--reactivation in vitro efficacy.

OBJECTIVE: In this work, we aim to summarize the universality of this compound, its reactivation potential when different cholinesterase inhibitors are used. BACKGROUND: Pralidoxime is considered as a gold standard of acetylcholinesterase reactivators--antidotes used in case of nerve agent poisonings. It has been commercially available for many years. However, several studies deem this oxime an old-fashion antidote. METHODS: Pralidoxime was synthesized at our department. The reactivating efficacy was tested on 10% (w/v) rat brain homogenate that had been incubated with appropriate inhibitor for 30 minutes to reach 96% inhibition of AChE. Then, pralidoxime was added for 10 minutes. Measurements were performed at 25 degrees C, pH 8, and 10(-3) and 10(-5) M concentrations of AChE reactivators. The activities of brain AChE were measured by a potentiostatic method. RESULTS: No sufficient reactivation was achieved at the concentration of 10(-5) M, which is a concentration that can be reached after administration of therapeutic doses. At a higher dose (10(-3) M), pralidoxime reactivated AChE inhibited by paraoxon, chlorpyrifos, Russian VX, VX and sarin. CONCLUSION: From the obtained results, it is clear that pralidoxime seems to be a poor reactivator of AChE inhibited by organophosphorous AChE inhibitors and thus cannot be labeled as a universal reactivator (Tab. 1, Fig. 3, Ref. 31).

The concept of hybrid molecules of tacrine and benzyl quinolone carboxylic acid (BQCA) as multifunctional agents for Alzheimer's disease.

Novel tacrine-benzyl quinolone carboxylic acid (tacrine-BQCA) hybrids were designed based on multi-target directed ligands (MTLDs) paradigm, synthesized and evaluated in vitro as inhibitors of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE). Tacrine moiety is represented herein as 7-methoxytacrine, 6-chlorotacrine or unsubstituted tacrine forming three different families of seven members, i.e. 21 compounds in overall. Introducing BQCA, a positive modulator of M1 muscarinic acetylcholine receptors (mAChRs), the action of novel compounds on M1 mAChRs was evaluated via Fluo-4 NW assay on the Chinese hamster ovarian (CHO-M1WT2) cell line. All the novel tacrine-BQCA hybrids were able to block the action of hAChE and hBChE in micromolar to nanomolar range. The hAChE kinetic profile of 5p was found to be mixed-type which is consistent with our docking experiments. Moreover, selected ligands were assessed for their potential hepatotoxicity on HepG2 cell line and presumable permeation through the blood-brain barrier by PAMPA assay. Expected agonistic profile towards M1 mAChRs delivered by BQCA moiety was not confirmed. From all the hybrids, 5o can be highlighted as non-selective cholinesterase inhibitor (hAChE IC50 = 74.5 nM; hBChE IC50 = 83.3 nM) with micromolar antagonistic activity towards M1 mAChR (IC50 = 4.23 µM). A non-selective pattern of cholinesterase inhibition is likely to be valuable during the onset as well as later stages of AD.

Assessment of DNA-binding affinity of cholinesterase reactivators and electrophoretic determination of their effect on topoisomerase I and II activity.

In this paper, we describe the biochemical properties and biological activity of a series of cholinesterase reactivators (symmetrical bisquaternary xylene-linked compounds, K106-K114) with ctDNA. The interaction of the studied derivatives with ctDNA was investigated using UV-Vis, fluorescence, CD and LD spectrometry, and electrophoretic and viscometric methods. The binding constants K were estimated to be in the range 1.05 × 10(5)-5.14 × 10(6) M(-1) and the percentage of hypochromism was found to be 10.64-19.28% (from UV-Vis titration). The used methods indicate that the studied samples are groove binders. Electrophoretic methods proved that the studied compounds clearly influence calf thymus Topo I (at 5 µM concentration, except for compounds K107, K111 and K114 which were effective at higher concentrations) and human Topo II (K110 partially inhibited Topo II effects even at 5 µM concentration) activity.

A 7-methoxytacrine-4-pyridinealdoxime hybrid as a novel prophylactic agent with reactivation properties in organophosphate intoxication.

Chemical warfare agents constitute an increasing threat to both military and civilian populations. Therefore, effective prophylactic approaches are urgently needed. Herein, we present a novel hybrid compound which is able not only to keep acetylcholinesterase resistant to organophosphate (OP) inhibitors, but also to serve as an enzyme reactivator in the case of OP intoxication.

The interaction of quaternary reversible acetylcholinesterase inhibitors with the nicotinic receptor.

Acetylcholinesterase inhibitors (AChEIs) are used in the treatment of myasthenia gravis (MG). We investigated the effects of AChEIs on peripheral nicotinic receptors (nAChR), which play a crucial role in the treatment of MG symptoms. The positive modulation of those receptors by AChE inhibitors could have an added value to the anti-AChE activity and might be useful in the therapy of MG. Furthermore, to estimate the potential drawbacks of the compounds, cytotoxicity has been assessed on various cell lines. The whole-cell mode of the patch-clamp method was employed. The experiments were performed on medulloblastoma/rhabdomyosarcoma cell line TE671 expressing human embryonic muscle-like receptor with subunits alpha2betagammadelta. The effect of the compounds on cell viability was measured by standard MTT assay (Sigma Aldrich) on ACHN (renal cell adenocarcinoma), HeLa (immortal cell line derived from a cervical carcinoma), HEPG2 (hepatocellular carcinoma) and BJ (skin fibroblasts) cell lines. No positive modulation by the tested AChE inhibitors was observed. Moreover, the compounds exhibited antagonistic activity on the peripheral nAChR. Standard drugs used in MG treatment were shown to be less potent inhibitors of muscle-type nAChR than the newly synthesized compounds. The new compounds showed very little effect on cell viability, and toxicities were comparable to standards. Newly synthesized AChEIs inhibited peripheral nAChR. Furthermore, the inhibition was higher than that of standards used for the treatment of MG. They could be used for the study of nAChR function, thanks to their high antagonizing potency and fast recovery of receptor activity after their removal. However, since no positive modulation was observed, the new compounds do not seem to be promising candidates for MG treatment, even though their cytotoxic effect was relatively low.

The interaction of standard oxime reactivators with hemicholinium-3 sensitive choline carriers.

Organophosphorus poisoning manifests as a cholinergic syndrome due to an inhibition of acetylcholinesterase. It is treated symptomatically by anticholinergics and oxime reactivators are used as causal antidotes. Reactivators possess a complex mechanism of action and interact at various levels of the cholinergic transmission. The aim of this study was to investigate the effect of standard oxime reactivators (HI-6, obidoxime, trimedoxime, methoxime and pralidoxime) on the hemicholinium-3 sensitive carriers, which are involved in the high-affinity choline uptake (HACU) transport, a key regulatory step in the synthesis of acetylcholine. The activity of the carriers was estimated in vitro on hippocampal synaptosomes using the substrate (3H)-choline and the competitive inhibitor (3H)-hemicholinium-3. Furthermore, the effect of the reactivators on the fluidity of hippocampal membranes was assessed. All tested compounds, except methoxime, showed an acute inhibitory effect on the carriers, however, only at µM concentrations. Trimedoxime showed the highest potency to inhibit HACU among all tested compounds (I(max) 62%, IC(50)=3 µM). All compounds, except HI-6, influenced also a membrane fluidity in the region of the hydrophilic heads of phospholipid bilayer, nevertheless, only methoxime was able to penetrate more deeply into the hydrocarbon core. We suggest that the direct interaction of oxime reactivators with the carrier protein (HI-6 and trimedoxime) and/or the changes in carrier conformation mediated by alterations in membrane fluidity (trimedoxime, obidoxime and pralidoxime) could occur here. The influence of reactivators on the carriers could be unfavorable in the case of their prolonged administration in vivo. From this point of view, the application of methoxime appears to be the best.

The effect of oxime reactivators on muscarinic receptors: functional and binding examinations.

The antidotal treatment of organophosphorus poisoning is still a problematic issue since no versatile antidote has been developed yet. In our study, we focused on an interesting property, which does not relate to the reactivation of inhibited acetylcholinesterase (AChE) of some oximes, but refers to their anti-muscarinic effects which may contribute considerably to their treatment efficacy. One standard reactivator (HI-6) and two new compounds (K027 and K203) have been investigated for their antimuscarinic properties. Anti-muscarinic effects were studies by means of an in vitro stimulated atrium preparation (functional test), the [(3)H]-QNB binding assay and G-protein coupled receptor assay (GPCR, beta-Arrestin Assay). Based on the functional data HI-6 demonstrates the highest anti-muscarinic effect. However, only when comparing [(3)H]-QNB binding results and GPCR data, K203 shows a very promising compound with regard to anti-muscarinic potency. The therapeutic impact of these findings has been discussed.

Oxime reactivators and their in vivo and in vitro effects on nicotinic receptors.

Current treatment of organophosphorus poisoning, resulting in overstimulation and desensitization of muscarinic and nicotinic receptors by acetylcholine (ACh), consists of the administration of atropine and oxime reactivators. However, no versatile oxime reactivator has been developed yet and some mortality still remains after application of standard atropine treatment, probably due to its lack of antinicotinic action. In our study, we focused on the interesting non-acetylcholinesterase property of oximes, i.e. antinicotinic effect of reactivators. Two standard reactivators (HI-6, obidoxime) and two new compounds (K027 and K203) were chosen for in vitro (patch clamp) and in vivo (nerve-evoked muscle contraction) testings. Both examinations showed antinicotinic effects of the reactivators. In vitro inhibition of acetylcholine-evoked currents by obidoxime, HI-6 and K203 was equivalent while K027 was less potent. Similar order of potency was observed by the in vivo examinations. We thus confirm previous in vitro results, which describe antinicotinic effects of oxime reactivators, and furthermore, we show in vivo antagonism of oxime reactivators exerted by the inhibition of ACh effect on the nicotinic receptor in the neuromuscular junction. Taking together, the effects of tested oxime reactivators indicate an antagonism on both embryonic and adult form of the muscle nicotinic receptors.

Novel acetylcholinesterase reactivator K112 and its cholinergic properties.

The oxime reactivator K112 is a member of the new group of xylene linker-containing AChE reactivators. Its cholinergic properties could be of importance at OP poisoning and are not related to the AChE reactivation that has been studied. It has been found that, despite of reactivating potency, this compound has additional effects. These cholinergic effects include a weak inhibition of AChE (IC(50)=43.8 ± 4.88 µM), inhibition of binding to the porcine muscarinic M2 receptor (IC(50)=4.36 µM) and finally, the inhibition of HACU (68.4 ± 9.9%), a key regulatory step in the synthesis of ACh. The inhibition of the binding of (3H)-HC-3 (64.7 ± 4.7%) and the influence on the membrane fluidity have also been observed. Blocking properties of K112 on the muscarinic receptors have been revealed in the in vitro experiment (rat urinary bladder) and in the in vivo experiment (rat heart BPM) as well. All these cholinergic properties could significantly contribute to the antidotal effect of K112 at the poisoning by the organophosphates.

Interaction of nerve agent antidotes with cholinergic systems.

The poisoning with organophosphorus compounds represents a life threatening danger especially in the time of terroristic menace. No universal antidote has been developed yet and other therapeutic approaches not related to reactivation of acetylcholinesterase are being investigated. This review describes the main features of the cholinergic system, cholinergic receptors, cholinesterases and their inhibitors. It also focuses on the organophosphorus nerve agents, their properties, effects and a large part describes various possibilities in treatments, mainly traditional oxime therapies based on reactivation of AChE. Furthermore, non-cholinesterase coupled antidotal effects of the oximes are thoroughly discussed. These antidotal effects principally include oxime interactions with muscarinic and nicotinic receptors.