UHPLC-HRMS study of anti-Alzheimer's drug candidates: metabolism of 7-MEOTA-tryptophan hybrids hampers their passage into brain.

Being among the top five causes of death in the developed world, Alzheimer's disease represents a major socio-economic issue. We administered a single intramuscular dose of two new hybrid anti-Alzheimer's compounds, with 7-methoxytacrine (7-MEOTA; acetylcholinesterase inhibitor) and tryptophan (inhibitor of amyloid accumulation) in their structure, to rats. Using validated ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) methods, we uncovered their inability to enter the site of action - the brain. We discuss four possible explanations: i) physico-chemical properties, ii) lack of active/facilitated transport, iii) effective efflux and/or iv) extensive metabolism. High-resolution mass spectrometric analyses proved that the compounds are easily hydrolysed at amide bond between tryptophan and the linker both in vitro and in vivo. Contrary to the parent compounds these metabolites - analogues of 7-MEOTA - can enter the brain in significant amounts.

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.

Two possibilities how to increase the efficacy of antidotal treatment of nerve agent poisonings.

Highly toxic organophosphorus inhibitors of acetylcholinesterase referred as nerve agents are considered to be among the most dangerous chemical warfare agents. The oximes represent very important part of medical countermeasures of nerve agent poisonings. They are used to reactivate the nerve agent-inhibited acetylcholinesterase. Despite long-term research activities, there is no single, broad-spectrum oxime suitable for the antidotal treatment of poisoning with all organophosphorus agents. There are two approaches how to increase and broaden the effectiveness of antidotal treatment of poisoning with nerve agents - to develop new structural analogues of currently available oximes and/or to combine currently available or newly developed oximes. The review describes the evaluation of the potency of newly developed oximes (especially the oxime K203) or combinations of oximes to reactivate nerve agent-inhibited acetylcholinesterase and to counteract the acute toxicity of nerve agents in comparison with single commonly used oxime (obidoxime, trimedoxime or HI-6).

Ascorbic acid: an old player with a broad impact on body physiology including oxidative stress suppression and immunomodulation: a review.

Ascorbic acid is a low molecular weight antioxidant well known as anti-scorbut acting vitamin C in humans, primates and guinea pigs. This review summarizes basic data about ascorbic acid in its physiological action point of view. It is divided into biochemistry of ascorbic acid synthesis, mechanism of antioxidant action and participation in anabolism, pharmacokinetics and excretion, exogenous ascorbic acid immunomodulatory effect and participation in infectious diseases, impact on irradiation and intoxication pathogenesis, and supplementary demands. The primary intention was to consider ascorbic acid not only as an antioxidant but also as a chemical compound affecting multiple pathways with a potential beneficial impact in many diseases and processes in human body.

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).

Inhibition of blood cholinesterases following intoxication with VX and its derivatives.

Nerve agents can be divided into G-agents (sarin, soman, tabun, cyclosarin etc.) and V-agents. The studies dealing with V-agents (O-alkyl S-2-dialkylaminoethyl methyl phosphonothiolates) are limited to one or two representatives only (VX, Russian VX). Anticholinesterase properties of 11 V-agents were studied in rats in vivo. Following intoxication with these agents in doses of 1 x LD(50) (intramuscular administration), activities of cholinesterases in the blood were continuously monitored and half-lives (t(0.5)) of inhibition were determined. These values varied from 3 min (VX and some other agents) to 10-14 min (derivatives substituted on the phosphorus head by O-ethyl- or O-isopropyl-, and by dimethyl-, diethyl- and dibutyl- on the nitrogen). Acetylcholinesterase activities in selected parts of the brain and diaphragm (30 min after the intoxication) were also detected. A correlation between toxicities and rates of inhibition of the blood enzymes was demonstrated. A similar relationship between acetylcholinesterase inhibition in vitro (from literature data) and half-lives of the blood cholinesterases was also observed. Though the chemical similarity of V compounds is evident, marked differences were observed among different derivatives; however, all agents examined had high inhibition potency corresponding to their toxicities.

Levels of energy-related metabolites in intact and isolated perfused-superfused rat skeletal muscles.

Adenosine 5'-triphosphate (ATP), phosphocreatine (PCr), creatine (Cr), inorganic phosphate (Pi), lactate (LAC), pyruvate (PYR) and glycogen as glucose (GLU) were determined and free adenosine 5'-diphosphate (ADP) was calculated from ATP:creatine phosphokinase (CPK) reaction in the gracilis muscle of cold-acclimated rats in vivo, and in completely isolated muscles under medium perfusion and superfusion in vitro, using the freeze-clamping method. The mean in vivo levels (mumol/g w.w.) were: ATP 4.8, PCr 12.0, Cr 7.8, Pi 16.1, LAC 1.6, PYR 0.09, GLU 22.9, ADP 0.62 x 10(-3). Isolation of the muscle (about 11 min of anoxia followed by perfusion in the air with a high pO2 medium) decreased macroergic phosphate levels (ATP 3.0, PCr 8.3). In isolated muscles perfused with a high pO2 medium (99 kPa O2, perfusion rate 70 microliters/min) and simultaneously superfused with a low pO2 medium (6.2 kPa O2, 2.3 ml/min) at 28 degrees C in vitro the levels of metabolites were (mumol/g w.w.): ATP 3.1, PCr 8.5, Cr 5.6, Pi 9.2, LAC 2.1, PYR 0.19, GLU 6.6, ADP 0.44 x 10(-3). The mean steady oxygen uptake of the isolated muscle was 97 nmol O2 x min-1 x g-1 w.w. Thus, the levels of macroergic phosphates and their derivatives are lower after isolation and perfusion of the muscle, but the creatine charge [PCr]/([PCr]+[Cr]) remains stable (0.61 in vivo versus 0.60 in the isolated muscle). This indicates that the steady-state and high energy status of the isolated perfused-superfused gracilis muscle is maintained [corrected].