Assessing the Therapeutic and Toxicological Profile of Novel Acetylcholinesterase Reactivators: Value of In Silico And In Vitro Data.

Organophosphorus compounds (OP) make up an important class of inhibitors, mostly employed as pesticides, even as chemical weapons. These toxic substances act through the inhibition of the acetylcholinesterase (AChE) enzyme, which results in elevated synaptic acetylcholine (ACh) levels, leading to serious adverse effects under the cholinergic syndrome. Many reactivators have been developed to combat the toxic effects of these AChE inhibitors. In this line, the oximes highlight because of their good reactivating power of cholinesterase enzymes. To date, no universal antidotes can reactivate AChE inhibited by any OP agent. This review summarizes the intoxication process by neurotoxic OP agents, along with the development of reactivators capable of reversing their effects, approaching aspects like the therapeutic and toxicological profile of these antidotes. Computational methods and conscious in vitro studies, capable of significantly predicting the toxicological profile of these drug candidates, might support the process of development of these reactivators before entering in vivo studies in animals, and then clinical trials. These approaches can assist in the design of safer and more effective molecules, reducing related cost and time for the process.

Interaction of Cucurbit[7]uril with Oxime K027, Atropine, and Paraoxon: Risky or Advantageous Delivery System?

Antidotes against organophosphates often possess physicochemical properties that mitigate their passage across the blood-brain barrier. Cucurbit[7]urils may be successfully used as a drug delivery system for bisquaternary oximes and improve central nervous system targeting. The main aim of these studies was to elucidate the relationship between cucurbit[7]uril, oxime K027, atropine, and paraoxon to define potential risks or advantages of this delivery system in a complex in vivo system. For this reason, in silico (molecular docking combined with umbrella sampling simulation) and in vivo (UHPLC-pharmacokinetics, toxicokinetics; acetylcholinesterase reactivation and functional observatory battery) methods were used. Based on our results, cucurbit[7]urils affect multiple factors in organophosphates poisoning and its therapy by (i) scavenging paraoxon and preventing free fraction of this toxin from entering the brain, (ii) enhancing the availability of atropine in the central nervous system and by (iii) increasing oxime passage into the brain. In conclusion, using cucurbit[7]urils with oximes might positively impact the overall treatment effectiveness and the benefits can outweigh the potential risks.

Identification of metabolite biomarkers in serum of rats exposed to chlorpyrifos and cadmium.

Chlorpyrifos (CPF) and cadmium (Cd) are widespread environmental pollutants, which are often present in drinking water and foods. However, the combined effects of CPF and Cd were not entirely clear at present. There was also no biomarker available to diagnose the poisoning of the two chemicals at low dose for long-term exposures. In this study, we investigated the change of serum metabolites of rats with subchronic exposure to CPF, Cd, and CPF plus Cd using gas chromatography-mass spectrometer-based metabolomics approach. We performed a stepwise optimization algorithm based on receiver operating characteristic to identify serum metabolite biomarkers for toxic diagnosis of the chemicals at different doses after 90-day exposure. We found that aminomalonic acid was the biomarker for the toxicity of Cd alone administration, and serine and propanoic acid were unique biomarkers for the toxicities of CPF plus Cd administrations. Our results suggest that subchronic exposure to CPF and Cd alone, or in combination at their low doses, could cause disturbance of energy and amino acid metabolism. Overall, we have shown that analysis of serum metabolomics can make exceptional contributions to the understanding of the toxic effects following long-term low-dose exposure of the organophosphorus pesticide and heavy metal.

Encapsulation of oxime K027 into cucurbit[7]uril: In vivo evaluation of safety, absorption, brain distribution and reactivation effectiveness.

Oxime-based acetylcholinesterase reactivators (briefly oximes) regenerate organophosphate-inactivated acetylcholinesterase and restore its function. Poor blood-brain-barrier passage and fast elimination from blood limit their actual use in treatment of patients exposed to organophosphates. Previous in vitro results implicated further testing of cucurbit[7]uril as a delivery vehicle for bisquaternary oximes. The present paper focuses on cell toxicity, in vivo safety and influence of cucurbit[7]uril on oxime pharmacokinetics and pharmacodynamics. Neither the K027 nor the complex caused any cell toxicity, changes in blood biochemistry or hepato- or nephrotoxicity in tested concentrations. The encapsulation of K027 increased and accelerated the blood-brain-barrier penetration. The peripheral oxime exposure also increased, supporting the suggestion that cucurbit[7]uril protects the circulating oxime from rapid renal clearance. Contrary to the comparable in vitro reactivation power of K027 and the encapsulated K027, we failed to confirm this in vivo. In theory, this might result from the non-specific binding of molecules to the cucurbit[7]uril or the interaction of K027 with cucurbit[7]uril being too strong for acetylcholinesterase reactivation. Precise explanation requires additional in silico, in vitro and also in vivo experiments.

Pharmacological and toxicological in vitro and in vivo effect of higher doses of oxime reactivators.

The major function of compounds with an oxime moiety attached to a quarternary nitrogen pyridinium ring is to reactivate acetylcholinesterase inhibited by organophosphorus agent (OP). However, other oxime mechanisms (e.g. modulation of cholinergic or glutamatergic receptor) may be involved in the recovery. The main disadvantage of positively charged reactivators is their low ability to penetrate into the brain although crossing the blood brain barrier could be supported via increasing the dose of administered oxime. Thus, this study presents maximal tolerated doses (MTD) for marketed oximes (TMB-4, MMB-4, LüH-6, HI-6, 2-PAM) and the most promising K-oximes (K027, K048, K203) which can be used in OP therapy in the future. No signs of sarin intoxication were observed in mice treated with 100% MTD of HI-6 in contrast to those treated with atropine and only 5% LD50 of HI-6. 100% MTD of HI-6 resulted in levels of 500 µM and 12 µM in plasma and brain, respectively. This concentration is by a far margin safe with respect to direct effects on neuronal cell viability and, on the other hand, does not have any effects on central NMDA receptors or central nACh receptors. However, a weak antimuscarinic activity in case of LüH-6 and a weak peripheral antinicotinic action in case of TMB-4 and 2-PAM could be observed at their respective 100% MTD dose. These high doses, represented by MTD, are, however, irrelevant to clinical practice since they led to mild to moderate toxic side effects. Therefore, we conclude that clinically used doses of marketed oxime reactivators have no significant direct pharmacological effect on the tested receptors.

Interspecies and intergender differences in acute toxicity of K-oximes drug candidates.

K-oximes were developed as modern drug candidates acting as AChE reactivators. In this study, it has been investigated which interspecies and intergender differences changes could be observed in Wistar rats and Swiss mice, both genders, after the treatment with increasing doses of selected acetylcholinesterase reactivators - asoxime, obidoxime, K027, K048, and K075. After the 24 h, a number of died animals was counted and the median lethal dose (LD50) for each oxime was calculated. By using the intramuscular route of administration, asoxime and K027 had the least toxicity in female rats (640.21 mg/kg and 686.08 mg/kg), and in female mice (565.75 mg/kg and 565.74 mg/kg), respectively. Moreover, asoxime and K027 showed 3, 4 or 8 times less acute toxicity in comparison to K048, obidoxime and K075, respectively. Beyond, K075 had the greatest toxicity in male rats (81.53 mg/kg), and in male mice (57.34 mg/kg), respectively. Our results can help to predict likely adverse toxic effects, target organ systems and possible outcome in the event of massive human overexposure, and in establishing risk categories or in dose selection for the initial repeated dose toxicity tests to be conducted for each oxime.

Cytotoxicity of acetylcholinesterase reactivators evaluated in vitro and its relation to their structure.

The development of acetylcholinesterase reactivators, i.e., antidotes against organophosphorus poisoning, is an important goal of defense research. The aim of this study was to compare cytotoxicity and chemical structure of five currently available oximes (pralidoxime, trimedoxime, obidoxime, methoxime, and asoxime) together with four perspective oximes from K-series (K027, K074, K075, and K203). The cytotoxicity of tested substances was measured using two methods - colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and impedance based real-time cytotoxicity assay - in three different cell lines (HepG2, ACHN, and NHLF). Toxicity was subsequently expressed as toxicological index IC50. The tested compounds showed different cytotoxicity ranging from 0.92 to 40.06 mM. In HepG2 cells, K027 was the least and asoxime was the most toxic reactivator. In ACHN and NHLF cell lines, trimedoxime was the compound with the lowest adverse effects, whereas the highest toxicity was found in methoxime-treated cells. The results show that at least five structural features affect the reactivators' toxicity such as the number of oxime groups in the molecule, their position on pyridinium ring, the length of carbon linker, and the oxygen substitution or insertion of the double bond into the connection chain. Newly synthetized oximes with IC50 ≥ 1 mM evaluated in this three cell lines model might appear suitable for further testing.

Behavioral, Pharmacokinetic, and Cardiovascular Evaluation of Candidate Oximes in African Green Monkeys.

Oxime reactivators are critical antidotes after organophosphate pesticide or nerve agent poisoning, directly restoring the function of inhibited acetylcholinesterase. In the continuing search for more broad-spectrum acetylcholinesterase reactivators, this study evaluated one of the leading next-generation oxime reactivators: methoxime, (1,1'-trimethylene bis[4-(hydroxyimino)methyl]pyridinium dichloride (MMB-4). The pharmacokinetics of both salts of MMB-4 (dichloride [2Cl] and dimethanesulphonate [DMS]) were characterized across a range of relevant doses (19, 58, and 116 µmol/kg, intramuscular) in a nonhuman primate model (male African green monkeys), and only subtle differences were observed between the salts. Additionally, the behavioral and cardiovascular safety of these MMB-4 salts was compared directly to other available oximes (HI-6 2Cl, HI-6 DMS, and pyridine-2-aldoxime chloride (2-PAM Cl)) at comparable projected doses. Automated operant behavioral tests were used to examine attention, motivation, visual discrimination, concept execution, and fine motor coordination after high doses of all oxime salts, and of all oximes studied, only the highest dose of 2-PAM Cl (447 µmol/kg) disrupted behavioral performance. Likewise, the effects of a range of doses of MMB-4 2Cl or DMS, HI-6 2Cl or DMS, or 2-PAM Cl on cardiovascular parameters were measured in African green monkeys implanted with telemetry devices. Only a small transient decrease in pulse pressure was observed following administration of the highest dose of MMB-4 DMS (116 µmol/kg). Thus, MMB-4 salts, up to the 9× equivalent of a projected autoinjector dose in humans, did not produce behavioral or cardiovascular toxicity in African green monkeys in the current study, and the pharmacokinetic parameters were orderly and predictable.

In vitro and in silico Evaluation of Non-Quaternary Reactivators of AChE as Antidotes of Organophosphorus Poisoning - a New Hope or a Blind Alley?

BACKGROUND: In the last decade, the concept of uncharged reactivators potentially able to penetrate the CNS has been introduced as an alternative to the classic charged oxime reactivators. However, this concept brings with it several associated drawbacks such as higher lipophilicity, difficulty in administration, lower affinity to cholinesterases, and higher toxicity risk. OBJECTIVE: In this study, we compare data obtained for a set of five classic charged reactivators and a set of three recently published uncharged oximes supplemented by two novel ones. METHODS: This time, we used only in silico prediction and in vitro approaches. RESULTS: Our data showed that tested uncharged oximes have low affinity for cholinesterases, do not possess high reactivation potency, and certainly represent a greater toxicity risk due to higher lipophilicity. We assume that balanced physicochemical properties will be required for the successful treatment of OP poisoning. Nevertheless, the compound meeting such criteria and pinpointed in silico (K1280) failed in this particular case. CONCLUSION: From the presented data, it seems that the concept of uncharged reactivators will have to be modified, at least to improve the bioavailability and to satisfy requirements for in vivo administration.

Toxicity and median effective doses of oxime therapies against percutaneous organophosphorus pesticide and nerve agent challenges in the Hartley guinea pig.

Anticholinesterases, such as organophosphorus pesticides and warfare nerve agents, present a significant health threat. Onset of symptoms after exposure can be rapid, requiring quick-acting, efficacious therapy to mitigate the effects. The goal of the current study was to identify the safest antidote with the highest therapeutic index (TI = oxime 24-hr LD50/oxime ED50) from a panel of four oximes deemed most efficacious in a previous study. The oximes tested were pralidoxime chloride (2-PAM Cl), MMB4 DMS, HLö-7 DMS, and obidoxime Cl2. The 24-hr median lethal dose (LD50) for the four by intramuscular (IM) injection and the median effective dose (ED50) were determined. In the ED50 study, male guinea pigs clipped of hair received 2x LD50 topical challenges of undiluted Russian VX (VR), VX, or phorate oxon (PHO) and, at the onset of cholinergic signs, IM therapy of atropine (0.4 mg/kg) and varying levels of oxime. Survival was assessed at 3 hr after onset clinical signs. The 3-hr 90th percentile dose (ED90) for each oxime was compared to the guinea pig pre-hospital human-equivalent dose of 2-PAM Cl, 149 µmol/kg. The TI was calculated for each OP/oxime combination. Against VR, MMB4 DMS had a higher TI than HLö-7 DMS, whereas 2-PAM Cl and obidoxime Cl2 were ineffective. Against VX, MMB4 DMS > HLö-7 DMS > 2-PAM Cl > obidoxime Cl2. Against PHO, all performed better than 2-PAM Cl. MMB4 DMS was the most effective oxime as it was the only oxime with ED90 < 149 µmol/kg against all three topical OPs tested.

Brain cholinesterase reactivation as a marker of exposure to anticholinesterase pesticides: a case study in a population of yellow-legged gull Larus michahellis (Naumann, 1840) along the northern coast of Portugal.

Between late 2010 to early 2011, an increased mortality in gulls was observed along the northern coast of Portugal, with individuals exhibiting neurologic disorders consistent with an eventual anticholinesterase pesticide poisoning event. To clarify if this mortality was related to organophosphate (OP) and/or carbamate (CB) poisoning, chemical and spontaneous cholinesterase (ChE) reactivation was tested in the brain of the yellow-legged gull (Larus michahellis). Initial brain ChE activity in L. michahellis was 40.92 ± 5.23 U/mg of protein (average ± SE). Following chemical and spontaneous reactivation, ChE activity increased in average 70.38 ± 48.59% and 131.95 ± 92.64%, respectively. ChE reactivation was found to decrease at increasing concentrations of the oxime pyridine-2-aldoxime methochloride and dilution factor, underscoring the importance of first optimizing the assay conditions prior to its use on bird species. These results suggest that birds analysed could have been exposed to OP and CB pesticide compounds and that in most cases CB exposure appeared to be the main cause of birds poisoning. These results are an important contribution to environmental monitoring as it demonstrates the suitability of L. michaellis as sentinel species of OP and CB pesticides within an urban environment.

Mediation of a glutamate antagonist, a NOS inhibitor and antioxidants with - SH groups on striatal dopamine release induced by clothianidin

Is Clothianidin is a neonicotinoid insecticide with selective action on nicotinic acetylcholine receptors. The aim of this study was to determine if the administration of a glutamate antagonist (APV), a NOS inhibitor (L-NAME) or two antioxidants (glutathione, and dithiothreitol,) prevent the increase in the striatal dopamine levels induced by clothianidin, using the microdialysis technique in freely moving and conscious rats. Intrastriatal administration of clothianidin (3.5 mM) produced an increase in striatal dopamine levels of 2462 ± 627%, with respect to basal levels. Coadministration of 0.65 mM APV and 3.5 mM clothianidin generated an increase in extracellular dopamine levels of 1089 ± 243.5%, being this increase 55.7% lower than the generated by clothianidin alone. Coadministration of 0.1 mM L-NAME and 3.5 mM clothianidin generated a significant increase in extracellular dopamine levels of 836.5 ± 150.6%., this increase is 70% lower than the generated by clothianidin alone. Coadministration of 3.5 mM clothianidin in combination with 0.4 mM glutathione induced an increase in striatal dopamine levels of 465.6 ± 126.8% , indicating that the administration of glutathione results in an inhibition of 81% of the effect generated by the infusion of clothianidin alone. Administration of 3.5 mM clothianidin associated with 0.005 mM dithiothreitol induced an increase in extracellular dopamine levels in the striatum of 693.8 ± 117.8% with respect to basal levels, being this increase 72% lower that the generated by clothianidin alone. Our results suggest that the effect of clothianidin on striatal dopamine release can be reduced by the administration of a glutamate antagonist, a NOS inhibitor or antioxidants with –SH groups, which suppose a simple protection mechanism against the damage caused for clothianidin (AU)

La clotianidina es un insecticida neonicotinoide con actividad selectiva sobre los receptores de acetilcolina. El objetivo de este estudio es comprobar si un inhibidor de los receptores glutamatérgicvos (APV), un inhibidor de la óxido nítrico sintetasa (L-NAME) y dos antioxidantes como el glutatión y el dithiotreitol previene la liberación de dopamina inducida por la clotianidina, usando la técnica de microdiálisis en ratas conscientes y en libre movimiento. La administración intraestriatal de clothianidina (3.5 mM) produce un aumento de 2462 ± 627%, de los niveles estriatales de dopamina respecto a los niveles basales. La coadministracion de 0.65 mM de APV y 3.5 mM d clothianidina genera un a aumento de 1089 ± 243.5% de los niveles estriatales de dopamina, siendo este incremento 55.7% más bajo que el generado por la clotianidina sola. La Coadministration de.0,1 mM de L-NAMEy3.5 mM de clotianidina genera un aumento de 836.5 ± 150.6% de los nivelesextracelulares de dopamina, siendo este aumento un 55.7% más bajo que el generado por la clotianidina sola. La coadministracion of 3.5 mM clothianidina en combinación con 0.4 mM de glutatión induce un aumento de 465.6 ± 126.8% de los niveles estriatales de dopamina, indicando que la administración de glutatión provoca una inhibición del 81% del efecto generado por la infusión de clotianidina sola. La administración de 3.5 mM de clothianidinajunto con 0.005 mM de diithiothreitol induce un aumento de 693.8 ± 117.8% en los niveles extracelulares de dopamina en el estriado, siendo este incremento 72% más bajo que el generado por la clotianidina sola. Nuestros resultados sugieren que el efecto de la clotianidina sobre la liberación estriatal de dopamina pueden ser reducidos por la administración de un antagonista glutamatérgico, un ihibibidor de la NOS o por antioxidantes con grupo –SH, lo cualsupone un simple mecanismo de protección contra el daño causadopor la clotianidina (AU)

Biological significance of the overexpression of HSP70 and alpha B-crystallin in rat substantia nigra exposed to different doses of permethrin.

The aim of this study was to investigate the possible role of the Heat Shock Protein 70 (HSP70) and Alpha B-crystallin (αBC) in the substantia nigra of rats exposed to permethrin at different doses on the apoptotic cell status. The orogastric gavage method was used to administer the different doses of permethrin (75 mg kg-1 in Group I, 150 mg kg-1 in group II, 300 mg kg-1 in group III) to the rats. Using the Western blot test, all the permethrin-treated groups showed a dose-dependent increase in the expression of HSP70 and αBC when compared to the control group. TUNEL positive apoptotic cells were not detected in the dopaminergic neurons of the substantia nigra after treatment with permethrin; however, upon immunofluorescent staining, intense positive reactions for HSP70 and αBC were observed in all of the treated groups. No immunopositive cells were detected in the tissue sections of the control group. These results suggest that the different administered doses of permethrin did not cause apoptotic cell death in the substantia nigra dopaminergic neurons; however, they did induce an increase in HSP70 and αBC expression. Thus, it appears that HSP70 and αBC could play a neuroprotective role in permethrin-induced neurotoxicity.

The evaluation of the reactivating and therapeutic efficacy of three novel bispyridinium oximes (K454, K456, K458) in comparison with the oxime K203 and trimedoxime in tabun-poisoned rats and mice.

The potency of three newly developed bispyridinium compounds (K454, K456, K458) to reactivate tabun-inhibited acetylcholinesterase and reduce tabun-induced lethal toxic effects was compared with the oxime K203 and trimedoxime using in vivo methods. The study determining percentage of reactivation of tabun-inhibited diaphragm and brain acetylcholinesterase in poisoned rats showed that the reactivating efficacy of all newly developed oximes is comparable with K203 but lower than the reactivating potency of trimedoxime in diaphragm. In the brain, their potency to reactivate tabun-inhibited acetylcholinesterase is lower compared with trimedoxime and the oxime K203. All three newly developed oximes were also found to be relatively effective in reducing lethal toxic effects in tabun-poisoned mice. Their therapeutic efficacy is consistent with the therapeutic potency of the oxime K203. On the other hand, their potency to reduce acute toxicity of tabun is significantly lower compared with trimedoxime. In conclusion, the reactivating and therapeutic potency of all three newly developed oximes does not prevail the effectiveness of the oxime K203 and trimedoxime and, therefore, they are not suitable for their replacement of commonly used oximes for the treatment of acute tabun poisoning.

Evaluation and computational characterization of the facilitated transport of Glc carbon C-1 oxime reactivators across a blood brain barrier model.

We are evaluating a facilitative transport strategy to move oximes across the blood brain barrier (BBB) to reactivate inhibited brain acetylcholinesterase (AChE). We selected glucose (Glc) transporters (GLUT) for this purpose as these transporters are highly represented in the BBB. Glc conjugates have successfully moved drugs across the BBB and previous work has shown that Glc-oximes (sugar-oximes, SOxs) can reduce the organophosphonate induced hypothermia response. We previously evaluated the reactivation potential of Glc carbon C-1 SOxs. Here we report the reactivation parameters for VX- and GB-inhibited human (Hu) AChE of the best SOx (13c) and our findings that the kinetics are similar to those of the parent oxime. Although crystals of Torpedo californica AChE were produced, neither soaked or co-crystallized experiments were successful at concentrations below 20mM 13c, and higher concentrations cracked the crystals. 13c was non-toxic to neuroblastoma and kidney cell lines at 12-18 mM, allowing high concentrations to be used in a BBB kidney cell model. The transfer of 13c from the donor side was asymmetric with the greatest loss of 13c from the apical- or luminal-treated side. There was no apparent transfer from the basolateral side. The 13cP(app) results indicate a 'low' transport efficiency; however, mass accounting revealed only a 20% recovery from the apical dose in which high concentrations were found in the cell lysate fraction. Molecular modeling of 13c through the GLUT-1 channel demonstrated that transport of 13c was more restricted than Glc. Selected sites were compared and the 13c binding energies were greater than two times those of Glc.

Cytotoxicity and genotoxicity evaluation of antidote oxime HI-6 tested on eight cell lines of human and rodent origin.

Oxime HI-6 is an efficient reactivator of the acetylcholinesterase inhibited by organophosphorous nerve agents. In this study we have estimated cytotoxicity of HI-6 by the colony forming assay and genotoxicity by the comet assay on human and rodent cell lines. IC50 of HI-6 assessed by the colony forming capacity was 3.59 mM for HeLa cells and 5.18 mM for a mouse cell line L929. Small difference in cytotoxicity was found among other cell lines tested: IC50 was 1.61 mM for human A549 cells, 1.14 mM for UROtse line, 1.96 mM and 1.71 mM for Chinese hamster cells AA8 and UV-20, respectively. The A549 cell viability measured with the MTT test was 5 times decreased comparing 2 and 24 hours of HI-6 oxime treatment. The 5 mM HI-6 concentration reduced the viability within 2 hours to 95% only, however, it induced a significant number of DNA breaks in mouse cells L929, and also in human UROtse and HepG2 cells. 1-ß-D-arabinofuranosylcytosine (10(-4) M) and hydroxyurea (10(-2) M), supplemented to the cultivation medium, did not cause any significant accumulation of DNA breaks during treatment, which indicated that the nucleotide excision repair was not acting on the induced DNA damage.

Refinement of structural leads for centrally acting oxime reactivators of phosphylated cholinesterases.

We present a systematic structural optimization of uncharged but ionizable N-substituted 2-hydroxyiminoacetamido alkylamine reactivators of phosphylated human acetylcholinesterase (hAChE) intended to catalyze the hydrolysis of organophosphate (OP)-inhibited hAChE in the CNS. Starting with the initial lead oxime RS41A identified in our earlier study and extending to the azepine analog RS194B, reactivation rates for OP-hAChE conjugates formed by sarin, cyclosarin, VX, paraoxon, and tabun are enhanced severalfold in vitro. To analyze the mechanism of intrinsic reactivation of the OP-AChE conjugate and penetration of the blood-brain barrier, the pH dependence of the oxime and amine ionizing groups of the compounds and their nucleophilic potential were examined by UV-visible spectroscopy, (1)H NMR, and oximolysis rates for acetylthiocholine and phosphoester hydrolysis. Oximolysis rates were compared in solution and on AChE conjugates and analyzed in terms of the ionization states for reactivation of the OP-conjugated AChE. In addition, toxicity and pharmacokinetic studies in mice show significantly improved CNS penetration and retention for RS194B when compared with RS41A. The enhanced intrinsic reactivity against the OP-AChE target combined with favorable pharmacokinetic properties resulted in great improvement of antidotal properties of RS194B compared with RS41A and the standard peripherally active oxime, 2-pyridinealdoxime methiodide. Improvement was particularly noticeable when pretreatment of mice with RS194B before OP exposure was combined with RS194B reactivation therapy after the OP insult.

[Drug testing with use of POCT].

Drug testing with the use of point of care testing (POCT) has been widely used in Japan, especially in the field of drug abuse, poisoning, and anticoagulant therapy with warfarin. For evidence-based medicine of POCT, an interesting report was presented by the National Academy of Clinical Biochemistry in the United States as the guideline in 2006. Users of POCT devices should understand all limitations of the devices. This strength/consensus recommendation is strong and the level of evidence is high. In this field, cyan, arsenic, paraquat, organic phosphate, methanol, acetaminophen, barbiturates, benzodiazepines, antidepressants, and some other drugs were detected by POCT devices such as Triage DOA and a detector tube system and others in Japan. The usefulness of the organophosphorus pesticide detection kit in the accident of GYOZA POISONING from china was noteworthy. In the case of toluene intoxication, the detector tube system was useful as a screening test for the gas phase test of a 2-year-old patient's vomit and excreta without any information from his parents. In warfarin treatment, a POCT device was useful for small hospitals and clinics. Although the cost is not covered by the health insurance system in Japan, the emergency centers of hospitals use these POCT devices for clinical decision-making. This is the most important problem.

Effects of K074 and pralidoxime on antioxidant and acetylcholinesterase response in malathion-poisoned mice.

The organophosphorus (OP) pesticide malathion is a highly neurotoxic compound and its toxicity is primarily caused by the inhibition of acetylcholinesterase (AChE), leading to cholinergic syndrome. Although oximes have been used as potential antidotal treatments in malathion poisoning because of their potential capability to reactivate the inhibited enzyme, the clinical experience with the clinically available oximes (e.g. pralidoxime) is disappointing and their routine use has been questioned. In the present study, we investigated the potency of pralidoxime and K074 in reactivating AChE after acute exposure to malathion, as well as in preventing malathion-induced changes in oxidative-stress related parameters in mice. Malathion (1.25 g/kg, s.c.) induced a significant decrease in cortico-cerebral, hippocampal and blood AChE activities at 24h after exposure. Oxime treatments (1/4 of LD(50), i.m., 6h after malathion poisoning) showed that pralidoxime significantly reversed malathion-induced blood AChE inhibition, although no significant effects were observed after K074 treatment. Interestingly, both oximes tested were unable to reactivate the cortico-cerebral and hippocampal enzymes after intramuscular or intracerebroventricular injection (1/4 of LD(50), 6h after malathion poisoning). Biochemical parameters related to oxidative stress (cerebro-cortical and hippocampal glutathione peroxidase, glutathione reductase and catalase activities, as well as lipid peroxidation) were not affected in animals treated with malathion, oximes or atropine alone. However, pralidoxime and K074, administered intramuscularly 6h after malathion poisoning, were able to increase the endogenous activities of these antioxidant enzymes in the prefrontal cortex and hippocampus. Taken together, the results presented herein showed that pralidoxime (the most common clinically used oxime) and the recently developed oxime K074, administered 6h after malathion poisoning, were unable to reactivate the inhibited AChE in mouse prefrontal cortex and hippocampus. However, only pralidoxime significantly reversed the blood AChE inhibition induced by malathion poisoning. This indicates that peripheral and central AChE activities are not necessarily correlated after the treatment of OP compounds and/or oximes, which should be taken into account in the diagnosis and management of OP-exposed humans. In addition, considering that the available treatments to malathion poisoning appear to be ineffective, the present study reinforce the need to search for potential new AChE reactivators able to efficiently reactivate the brain and blood AChEs after malathion poisoning.