Pharmacokinetics of K117 and K127, two novel antidote candidates to treat Tabun poisoning.

AIMS: K117 and K127 are bis-pyridinium aldoximes but K117 is a bis-pyridinium bis-aldoxime while K127 has only one single aldoxime in addition to its amide substituent. Is there any difference in pharmacokinetics in these compounds that otherwise have the same chemical structure? Both K117 and K127 are developed as antidotes in acetylcholinesterase and butyrylcholinesterase poisoning in terrorist attacks or intoxication with other organophosphorous compounds. Their distributions have been scouted in the bodies of rats. MAIN METHODS: White male Wistar rats were intramuscularly injected. The animals were sacrificed, tissue samples were homogenized, and either K117 or K127 concentrations were determined using reversed-phase high-performance liquid chromatography. KEY FINDINGS: Both K117 and K127 were present in all tissues that were analyzed including blood (serum), the brains, cerebrospinal fluid, the eyes, livers, kidneys, lungs and testes. Their pharmacokinetics and body distributions are similar. SIGNIFICANCE: Either K117 or K127 meets the essential requirements for antidotes. Dose dependence and kinetics of their distribution were compared to that of other pyridinium aldoximes.

Some Possibilities to Study New Prophylactics against Nerve Agents.

Nerve agents belong to the most dangerous chemical warfare agents and can be/were misused by terrorists. Effective prophylaxis and treatment is necessary to diminish their effect. General principles of prophylaxis are summarized (protection against acetylcholinesterase inhibition, detoxification, treatment "in advance" and use of different drugs). They are based on the knowledge of mechanism of action of nerve agents. Among different examinations, it is necessary to test prophylactic effectivity in vivo and compare the results with protection in vitro. Chemical and biological approaches to the development of new prophylactics would be applied simultaneously during this research. Though the number of possible prophylactics is relatively high, the only four drugs were introduced into military medical practice. At present, pyridostigmine seems to be common prophylactic antidote; prophylactics panpal (tablets with pyridostigmine, trihexyphenidyl and benactyzine), transant (transdermal patch containing HI-6) are other means introduced into different armies as prophylactics. Scavenger commercionally available is Protexia®. Future development will be focused on scavengers, and on other drugs either reversible cholinesterase inhibitors (e.g., huperzine A, gallantamine, physostigmine, acridine derivatives) or other compounds.

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.

Study on medicinal chemistry of K203 in wistar rats and beagle dogs.

K203 is an experimental bis-pyridinium mono-aldoxime type cholinesterase reactivator of potential use in organophosphate/ organophosphonate poisoning. Pharmacokinetics of K203 were examined in Wistar rats and beagle dogs using ion-pair HPLC. Serum and cerebrospinal fluid concentrations of K203 were determined using ion-pair reversedphase chromatography on octadecyl silica column. HPLC with ultraviolet detection was used for determination of serum concentration of K203 higher than 0.1 µg/mL while its low concentrations in cerebrospinal fluid required electrochemical detection (0.015 through 4 µg/mL range). In rats the serum levels of K203 followed zero order pharmacokinetics from 15 to 120 minutes post administration. Zero order pharmacokinetics was also observed in beagle dogs after low dose (15 µmol/kg) of K203 administration. High dose administration (250 µmol/kg) led to subsequent hindered elimination from both cerebrospinal fluid and serum.

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).

Structure-activity relationship of quaternary acetylcholinesterase inhibitors - outlook for early myasthenia gravis treatment.

Myasthenia gravis is a rare autoimmune neuromuscular junction disorder mainly caused by antibodies being targeted against the muscle acetylcholine receptors (AChRs). The loss of AChRs leads to a defect in neuromuscular transmission resulting in muscle weakness and fatigue. Although once an often fatal illness, Myasthenia gravis can now be well managed with relatively safe and effective treatments. However, the severe myasthenic cases associated with thymus tumors remain often fatal exception in the management of the disease. The early treatment includes the use of acetylcholinesterase inhibitors (AChEI) which enhance neuromuscular transmission. To ensure a peripheral effect, charged molecules are used, particularly quaternary ammonium salts. The structure of AChEIs has been continuously modified to obtain the optimal ratio between AChE inhibition and potential side-effects. This review summarizes progress in the use of quaternary compounds as AChE inhibitors in vitro with respect to their structure and inhibitory ability. Namely, carbamic acid esters, piperidinium and pyridinium salts, bisquaternary pyridinium salts and heterogeneous quaternary inhibitors are all discussed. Among data found in the literature, many compounds have shown promising inhibition of AChE when compared to commercial standards (pyridostigmine, neostigmine). Besides a promising inhibitory ability, selectivity for AChE versus butyrylcholinesterase (BChE) for the most potent compounds (sub-nanomolar IC(50)) was also identified.

Efficacy of eight experimental bispyridinium oximes against paraoxon-induced mortality: comparison with the conventional oximes pralidoxime and obidoxime.

Recently, several experimental K-oximes with two functional aldoxime groups have been synthesized that show excellent in vitro efficacy in protecting acetylcholinesterase (AChE) from inhibition by a broad variety of organophosphorus compounds (OPCs). However, oximes themselves are also AChE inhibitors, albeit at higher concentrations, which is a major cause of their toxicity and may be a dose-limiting factor in oxime therapy. To assess the efficacy of the experimental K-oximes in vivo, the extent of oxime-conferred protection from mortality induced by paraoxon was quantified. Rats received paraoxon in a dosage of 1, 5, or 10 mumol, and immediately thereafter intraperitoneal injections of the respective oxime at a dosage of half the LD(01). The relative risk of death (RR) over time was estimated by Cox survival analysis for treatment with experimental K-oximes (K-53, K-74, K-75, K-107, K-108, and K-113), with the clinically available oximes pralidoxime (2-PAM) and obidoxime, and with the well-characterized K-oximes K-27 and K-48, comparing results with the no-treatment group. Best protection was conferred by K-27, reducing the RR to 20% of controls (P

Eight new bispyridinium oximes in comparison with the conventional oximes pralidoxime and obidoxime: in vivo efficacy to protect from diisopropylfluorophosphate toxicity.

In search for more efficacious reactivators of acetylcholinesterase (AChE) inhibited by organophosphorus compounds, experimental K-oximes have been synthesized which show good in vitro efficacy. However, AChE inhibition by oximes themselves (as quantified by their intrinsic IC50) is the major cause of oxime toxicity and the dose-limiting factor. To assess K-oxime efficacy in vivo, the extent of protection from mortality induced by diisopropylfluorophosphate (DFP) was quantified by Cox survival analysis and compared with that of the clinically available oximes. Oximes were administered in an equitoxic dosage, i.e. half the LD01. Best protection was conferred by K-27, reducing the relative risk of death (RR) to 16% of control RR (P < or = 0.05), which was statistically significantly better (P < or = 0.05) than all other tested oximes, except obidoxime, K-53 and K-75. The efficacy of obidoxime (RR = 0.19), K-48 (RR = 0.28), K-53 (RR = 0.22), K-74 (RR = 0.38) and K-75 (RR = 0.29) was significantly (P < or = 0.05) better than that of 2-PAM (RR = 0.62) and K-113 (RR = 0.73). No significant protective effect was observed for K-107 and K-108. Our LD50 data show that K-107, K-108 and K-113 (which strongly inhibit AChE in vitro) are in vivo markedly more toxic than all other oximes tested and can therefore only be safely administered at a low dosage which is insufficient to protect from DFP-induced mortality. Dosage calculations based on in vitro IC50 measurements may therefore in future replace in vivo LD50 determinations, thereby reducing the number of animals required.

In vitro oxime protection of human red blood cell acetylcholinesterase inhibited by diisopropyl-fluorophosphate.

Oximes are enzyme reactivators used in treating poisoning with organophosphorus cholinesterase (AChE) inhibitors. The oxime dose which can be safely administered is limited by the intrinsic toxicity of the substances such as their own AChE-inhibiting tendency. Clinical experience with the available oximes is disappointing. To meet this need, new AChE reactivators of potential clinical utility have been developed. The purpose of the study was to estimate in vitro both the intrinsic toxicity and the extent of possible protection conferred by established (pralidoxime, obidoxime, HI-6, methoxime, trimedoxime) and experimental (K-type) oximes, using diisopropyl-fluoro-phosphate (DFP) as an AChE inhibitor. The IC50 of DFP against human red blood cell AChE was determined ( approximately 120 nm). Measurements were then repeated in the presence of increasing oxime concentrations, leading to an apparent increase in DFP IC50. Calculated IC50 values were plotted against oxime concentrations to obtain an IC50 shift curve. The slope of this shift curve (tan alpha) was used to quantify the magnitude of the protective effect (nm IC50 increase per microm oxime). We show that, in the case of a linear relationship between oxime concentration and IC50, the binding constant K, determined using the Schild equation, equals IC50/DFP/tan alpha. Based on the values of tan alpha and of the binding constant K, some of the new K-oxime reactivators are far superior to pralidoxime (tan alpha = 0.8), obidoxime (1.5), HI-6 (0.8), trimedoxime (2.9) and methoxime (5.9), with K-107 (17), K-108 (20), and K-113 (16) being the outstanding compounds.

[Synthesis of reactivators of phosphorylated acetylcholinesterase of bis-pyridiniumdialdoxime type with a 3-oxapentane connecting chain and their testing in vitro on a model of the enzyme inhibited by chlorpyrifos and methylchlorpyrifos]. / Syntéza reaktivátoru fosforylované acetylcholinesterasy bis-pyridiniumdialdoximového typu s 3-oxapentanovým spojovacím retezcem a jejich testování in vitro na modelu enzymu inhibovaného chlorpyrifosem a methylchlorpyrifosem.

Insecticides (e.g., parathion, chlorpyrifos, methylchlorpyrifos) and nerve agents (e.g.. soman, sarin, tabun, VX) belong to the group of organophosphates. They are able to irreversibly inhibit the enzyme acetylcholinesterase (AChE). Three new reactivators with a 3-oxapentane connecting chain were prepared. The ability of the new compounds to reactivate AChE inhibited by pesticides was tested in vitro and compared to known oxime 10(-3) M which is unfortunately not applicable to in vivo experiments. All tested compounds are practically ineffective for methylchlorpyrifos-inhibited AChE at the physiological concentration (10(-5) M). On the other hand, the known reactivators surpass new substances in the case of chlorpyrifos-inhibited AChE at both concentrations.

Synthesis of a novel series of bispyridinium compounds bearing a xylene linker and evaluation of their reactivation activity against chlorpyrifos-inhibited acetylcholinesterase.

Nine potential AChE reactivators were synthesized using a modification of currently known synthetic pathways. Their potency to reactivate AChE inhibited by insecticide chlorpyrifos was tested in vitro. 2,2'-Bis(hydroxyiminomethyl)-1,1'-(1,4-phenylenedimethyl)-bispyridinium dibromide seems to be the most potent AChE reactivator. The reactivation potency of these compounds depends on structural factors such as length of the linking chain between both pyridinium rings and position of the oxime moiety on the pyridinium ring.