Knockout of the intellectual disability-linked gene Hs6st2 in mice decreases heparan sulfate 6-O-sulfation, impairs dendritic spines of hippocampal neurons, and affects memory.

Heparan sulfate (HS) is a linear polysaccharide that plays a key role in cellular signaling networks. HS functions are regulated by its 6-O-sulfation, which is catalyzed by three HS 6-O-sulfotransferases (HS6STs). Notably, HS6ST2 is mainly expressed in the brain and HS6ST2 mutations are linked to brain disorders, but the underlying mechanisms remain poorly understood. To determine the role of Hs6st2 in the brain, we carried out a series of molecular and behavioral assessments on Hs6st2 knockout mice. We first carried out strong anion exchange-high performance liquid chromatography and found that knockout of Hs6st2 moderately decreases HS 6-O-sulfation levels in the brain. We then assessed body weights and found that Hs6st2 knockout mice exhibit increased body weight, which is associated with abnormal metabolic pathways. We also performed behavioral tests and found that Hs6st2 knockout mice showed memory deficits, which recapitulate patient clinical symptoms. To determine the molecular mechanisms underlying the memory deficits, we used RNA sequencing to examine transcriptomes in two memory-related brain regions, the hippocampus and cerebral cortex. We found that knockout of Hs6st2 impairs transcriptome in the hippocampus, but only mildly in the cerebral cortex. Furthermore, the transcriptome changes in the hippocampus are enriched in dendrite and synapse pathways. We also found that knockout of Hs6st2 decreases HS levels and impairs dendritic spines in hippocampal CA1 pyramidal neurons. Taken together, our study provides novel molecular and behavioral insights into the role of Hs6st2 in the brain, which facilitates a better understanding of HS6ST2 and HS-linked brain disorders.

A-series agent A-234: initial in vitro and in vivo characterization.

A-series agent A-234 belongs to a new generation of nerve agents. The poisoning of a former Russian spy Sergei Skripal and his daughter in Salisbury, England, in March 2018 led to the inclusion of A-234 and other A-series agents into the Chemical Weapons Convention. Even though five years have already passed, there is still very little information on its chemical properties, biological activities, and treatment options with established antidotes. In this article, we first assessed A-234 stability in neutral pH for subsequent experiments. Then, we determined its inhibitory potential towards human recombinant acetylcholinesterase (HssAChE; EC 3.1.1.7) and butyrylcholinesterase (HssBChE; EC 3.1.1.8), the ability of HI-6, obidoxime, pralidoxime, methoxime, and trimedoxime to reactivate inhibited cholinesterases (ChEs), its toxicity in rats and therapeutic effects of different antidotal approaches. Finally, we utilized molecular dynamics to explain our findings. The results of spontaneous A-234 hydrolysis showed a slow process with a reaction rate displaying a triphasic course during the first 72 h (the residual concentration 86.2%). A-234 was found to be a potent inhibitor of both human ChEs (HssAChE IC50 = 0.101 ± 0.003 µM and HssBChE IC50 = 0.036 ± 0.002 µM), whereas the five marketed oximes have negligible reactivation ability toward A-234-inhibited HssAChE and HssBChE. The acute toxicity of A-234 is comparable to that of VX and in the context of therapy, atropine and diazepam effectively mitigate A-234 lethality. Even though oxime administration may induce minor improvements, selected oximes (HI-6 and methoxime) do not reactivate ChEs in vivo. Molecular dynamics implies that all marketed oximes are weak nucleophiles, which may explain the failure to reactivate the A-234 phosphorus-serine oxygen bond characterized by low partial charge, in particular, HI-6 and trimedoxime oxime oxygen may not be able to effectively approach the A-234 phosphorus, while pralidoxime displayed low interaction energy. This study is the first to provide essential experimental preclinical data on the A-234 compound.

Synthesis and in vitro assessment of the reactivation profile of clinically available oximes on the acetylcholinesterase model inhibited by A-230 nerve agent surrogate.

The risk of the use of toxic chemicals for unlawful acts has been a matter of concern for different governments and multilateral agencies. The Organisation for the Prohibition of Chemical Weapons (OPCW), which oversees the implementation of the Chemical Weapons Convention (CWC), considering recent events employing chemical warfare agents as means of assassination, has recently included in the CWC "Annex on Chemicals" some organophosphorus compounds that are regarded as acting in a similar fashion to the classical G- and V-series of nerve agents, inhibiting the pivotal enzyme acetylcholinesterase. Therefore, knowledge of the activity of the pyridinium oximes, the sole class of clinically available acetylcholinesterase reactivators to date, is plainly justified. In this paper, continuing our research efforts in medicinal chemistry on this class of toxic chemicals, we synthesized an A-230 nerve agent surrogate and applied a modified Ellman's assay to evaluate its ability to inhibit our enzymatic model, acetylcholinesterase from Electrophorus eel, and if the clinically available antidotes are able to rescue the enzyme activity for the purpose of relating the findings to the previously disclosed in silico data for the authentic nerve agent and other studies with similar A-series surrogates. Our experimental data indicates that pralidoxime is the most efficient compound for reactivating acetylcholinesterase inhibited by A-230 surrogate, which is the opposite of the in silico data previously disclosed.

Pharmacokinetics of three novel pyridinium aldoxime acetylcholinesterase reactivators in female rats.

A platform of novel lipophilic substituted phenoxyalkyl pyridinium oximes was invented to reactivate organophosphate-inhibited acetylcholinesterase. This platform has provided superior efficacy in rats to the current standard of care, 2-PAM, for survival of lethal doses of nerve agent surrogates as well as evidence of brain penetration and neuroprotection. The pharmacokinetics of three of these novel oximes in female rats was studied for comparison to previous data in male rats. Compared to the published half-life of 2-PAM (less than 2 h), the lead novel oxime, Oxime 20, displayed a plasma half-life of about 5 h in both sexes of rats following intramuscular administration. Very few sex differences in pharmacokinetic parameters were apparent. Oxime 20 displayed an increase in brain concentration to plasma concentration over the initial 2 h following intramuscular administration in male rats, with a plateau at 1 h; there were no differences in brain concentrations between the sexes at 2 h. Hepatic metabolism of Oxime 20 was higher in rat microsomes than in human microsomes. The relatively long plasma half-life is likely an important factor in both the enhanced survival and the neuroprotection previously observed for Oxime 20. The metabolism data suggest that the clearance of Oxime 20 could be slower in humans than was observed in rats, which might allow less frequent administration than 2-PAM for therapy of organophosphate acute toxicity. Therefore, the pharmacokinetic data combined with our earlier efficacy data suggest that Oxime 20 has potential as a superior therapeutic for nerve agent poisoning.

Loading and Sustained Release of Pralidoxime Chloride from Swellable MIL-88B(Fe) and Its Therapeutic Performance on Mice Poisoned by Neurotoxic Agents.

Maintaining a long-term continuous and stable reactivator blood concentration to treat organophosphorus nerve agent poisoning using acetylcholinesterase (AChE) reactivator pralidoxime chloride (2-PAM) is very important yet difficult. Because the flexible framework of MIL-88B(Fe) nanoparticles (NPs) can swell in polar solvents, pralidoxime chloride (2-PAM) was loaded in MIL-88B(Fe) NPs (size: ca. 500 nm) by stirring and incubation in deionized water to obtain 2-PAM@MIL-88B(Fe), which had a maximum drug loading capacity of 12.6 wt %. The as-prepared composite was characterized by IR, powder X-ray diffraction (P-XRD), scanning electron microscopy (SEM), ζ-potential, Brunauer-Emmett-Teller (BET), and thermogravimetry/differential thermal analysis (TG/DTA). The results showed that under constant conditions, the maximum drug release rates of 2-PAM@MIL-88B(Fe) in absolute ethanol, phosphate-buffered saline (PBS) solution (pH = 7.4), and PBS solution (pH = 4) at 150 h were 51.7, 80.6, and 67.1%, respectively. This was because the composite showed different swelling behaviors in different solvents. In PBS solution with pH = 2, the 2-PAM@MIL-88B(Fe) framework collapsed after 53 h and released 100% of 2-PAM. For mice after intragastric poisoning with sarin (a neurotoxic agent), an atropine-assisted 2-PAM@MIL-88B(Fe) treatment experiment revealed that 2-PAM@MIL-88B(Fe) continuously released 2-PAM for more than 72 h so that poisoned AChE was continuously and steadily reactivated. The reactivation rate of AChE was 56.7% after 72 h. This composite is expected to provide a prolonged, stable therapeutic drug for the mid- and late-stage treatment of neurotoxic agent poisoning.

Enhancing Target Tissue Levels and Diminishing Plasma Clearance of Ionizing Zwitterionic Antidotes in Organophosphate Exposures.

Inhibition of acetylcholinesterase (AChE) by certain organophosphates (OPs) can be life-threatening and requires reactivating antidote accessibility to the peripheral and central nervous systems to reverse symptoms and enhance survival parameters. In considering dosing requirements for oxime antidotes in OP exposures that inactivate AChE, clearance of proton ionizable, zwitterionic antidotes is rapid and proceeds with largely the parent antidotal compound being cleared by renal transporters. Such transporters may also control disposition between target tissues and plasma as well as overall elimination from the body. An ideal small-molecule antidote should access and be retained in primary target tissues-central nervous system (brain), skeletal muscle, and peripheral autonomic sites-for sufficient periods to reactivate AChE and prevent acute toxicity. We show here that we can markedly prolong the antidotal activity of zwitterionic antidotes by inhibiting P-glycoprotein (P-gp) transporters in the brain capillary and renal systems. We employ the P-gp inhibitor tariquidar as a reference compound and show that tissue and plasma levels of RS194B, a hydroxyl-imino acetamido alkylamine reactivator, are elevated and that plasma clearances are reduced. To examine the mechanism, identify the transporter, and establish the actions of a transport inhibitor, we compare the pharmacokinetic parameters in a P-glycoprotein knockout mouse strain and see dramatic enhancements of short-term plasma and tissue levels. Hence, repurposed transport inhibitors that are candidate or Food and Drug Administration-approved drugs, should enhance target tissue concentrations of the zwitterionic antidote through inhibition of both renal elimination and brain capillary extrusion. SIGNIFICANCE STATEMENT: We examine renal and brain capillary transporter inhibition as means for lowering dose and frequency of dosing of a blood-brain barrier permanent reactivating antidote, RS194B, an ionizable zwitterion. Through a small molecule, tariquidar, and gene knockout mice, CNS antidote concentrations are enhanced, and total body clearances are concomitantly diminished. RS194B with repurposed transport inhibitors should enhance reactivation of central and peripheral OP-inhibited acetylcholinesterase. Activities at both disposition sites are a desired features for replacing the antidote, pralidoxime, for acute OP exposure.

The tertiary oxime monoisonitrosoacetone penetrates the brain, reactivates inhibited acetylcholinesterase, and reduces mortality and morbidity following lethal sarin intoxication in guinea pigs.

The brain is a critical target for the toxic action of organophosphorus (OP) inhibitors of acetylcholinesterase (AChE) such as the nerve agent sarin. However, the available oxime antidote 2-PAM only reactivates OP-inhibited AChE in peripheral tissues. Monoisonitrosoacetone (MINA), a tertiary oxime, reportedly reactivates AChE in the central nervous system (CNS). The current study investigated whether MINA would be beneficial as a supplemental oxime treatment in preventing lethality and reducing morbidity following lethal sarin exposure, MINA supplement would improve AChE recovery in the body, and MINA would be detectable in the CNS. Guinea pigs were exposed to sarin and treated with atropine sulfate and 2-PAM at one minute. Additional 2-PAM or MINA was administered at 3, 5, 15, or 30 min after sarin exposure. Survival and morbidity were assessed at 2 and 24 h. AChE activity in brain and peripheral tissues was evaluated one hour after MINA and 2-PAM treatment. An in vivo microdialysis technique was used to determine partitioning of MINA into the brain. A liquid chromatography-tandem mass spectrometry method was developed for the analysis of MINA in microdialysates. MINA-treated animals exhibited significantly higher survival and lower morbidity compared to 2-PAM-treated animals. 2-PAM was significantly more effective in reactivating AChE in peripheral tissues, but only MINA reactivated AChE in the CNS. MINA was found in guinea pig brain microdialysate samples beginning at ~10 min after administration in a dose-related manner. The data strongly suggest that a centrally penetrating oxime could provide significant benefit as an adjunct to atropine and 2-PAM therapy for OP intoxication.

A delayed treatment model for the evaluation of scopolamine for VX nerve agent intoxication.

Most research on medical countermeasures for nerve agent exposure assumes a military scenario, in which (autoinjector) treatment is envisaged to be available immediately. In a civilian setting however, treatment is delayed until arrival of first-aid responders. This may significantly affect treatment efficacy and the requirements for secondary intensive care. The aim of the current study was to develop a guinea pig model to evaluate the efficacy of delayed treatment following nerve agent exposure. We identified a trigger-to-treat based on a progressive stage of the toxidrome following VX exposure, which was associated with the subsiding of clonic movements. This paradigm resulted in treatment consistently being administered between 15 and 25 min post-exposure. Using the model, we investigated the potential for the anticholinergic scopolamine to act as a delayed treatment either as a standalone treatment, or as an adjunct to delayed treatment with Standard of Care (SOC), containing atropine, 2-PAM, and midazolam. The study provides a framework for a small animal model for evaluating the efficacy of treatment administered at a specific stage of the toxidrome, when immediate treatment is absent. As an adjunct, scopolamine treatment did not result in improved survival, but did show a beneficial effect on recovery, in terms of general posture. As a standalone treatment, scopolamine showed a significant, dose-responsive, beneficial effect on survival and recovery. These promising results warrant additional studies to investigate which observed physiological improvements are relevant for the recovery process and residual injury.

The antidotes atropine and pralidoxime distinctively recover cardiorespiratory components impaired by acute poisoning with chlorpyrifos in rats.

In a previous work we showed that the organophosphate pesticide (OP) chlorpyrifos (CPF) reduces the protective chemoreflex and baroreflex responses in rats. However, whether the antidotes atropine (ATR) and pralidoxime (2-PAM) are capable of restoring these reflex functions remains unexplored. Rats were poisoned with CPF (30 mg.kg-1, i.p.) and one hour after the intoxication, ATR (10 mg.kg-1, i.p.) and 2-PAM (40 mg.kg-1, i.p.) were administrated separately or in combination. Cardiorespiratory parameters were recorded in awake rats 24 h after CPF. Systolic blood pressure (SBP) and heart rate (HR) variability and spontaneous baroreflex sensitivity (sBRS) were derived from undisturbed recordings (30 min), while chemoreflex was assessed through potassium cyanide (KCN) i.v. injections (10, 20, 40, 80 µg/rat). CPF poisoning increased SBP variability and low frequency/high frequency (LF/HF) ratio of the HR variability spectrum, indicating autonomic imbalance with increased cardiac sympathetic tone. sBRS was not changed. Treatment with 2-PAM restored SBP variability, whilst both antidotes increased LF/HF ratio. CPF poisoning reduced the hypertensive, bradycardic and tachypneic chemoreflex responses. Chemoreflex-induced hypertensive response was restored by 2-PAM treatment, while ATR recovered the bradycardic response. Both antidotes restored the chemoreflex tachypneic response. Our data show distinct effects of ATR and 2-PAM on cardiorespiratory parameters affected by OP poisoning. While 2-PAM rescued the chemoreflex hypertensive response, ATR reversed chemoreflex bradycardic dysfunction. Although 2-PAM clinical use is questioned in some countries, our data indicate that summation of effects of both antidotes appears beneficial on the cardiorespiratory system and peripheral chemoreflex function.

Pralidoxime-Induced Potentiation of the Pressor Effect of Adrenaline and Hastened Successful Resuscitation by Pralidoxime in a Porcine Cardiac Arrest Model.

PURPOSE: Pralidoxime potentiated the pressor effect of adrenaline and facilitated restoration of spontaneous circulation (ROSC) after prolonged cardiac arrest. In this study, we hypothesised that pralidoxime would hasten ROSC in a model with a short duration of untreated ventricular fibrillation (VF). We also hypothesised that potentiation of the pressor effect of adrenaline by pralidoxime would not be accompanied by worsening of the adverse effects of adrenaline. METHODS: After 5 min of VF, 20 pigs randomly received either pralidoxime (40 mg/kg) or saline, in combination with adrenaline, during cardiopulmonary resuscitation (CPR). Coronary perfusion pressure (CPP) during CPR, and ease of resuscitation were compared between the groups. Additionally, haemodynamic data, severity of ventricular arrhythmias, and cerebral microcirculation were measured during the 1-h post-resuscitation period. Cerebral microcirculatory blood flow and brain tissue oxygen tension (PbtO2) were measured on parietal cortices exposed through burr holes. RESULTS: All animals achieved ROSC. The pralidoxime group had higher CPP during CPR (P = 0.014) and required a shorter duration of CPR (P = 0.024) and smaller number of adrenaline doses (P = 0.024). During the post-resuscitation period, heart rate increased over time in the control group, and decreased steadily in the pralidoxime group. No inter-group differences were observed in the incidences of ventricular arrhythmias, cerebral microcirculatory blood flow, and PbtO2. CONCLUSION: Pralidoxime improved CPP and hastened ROSC in a model with a short duration of untreated VF. The potentiation of the pressor effect of adrenaline was not accompanied by the worsening of the adverse effects of adrenaline.

Pralidoxime administered during cardiopulmonary resuscitation facilitates successful resuscitation in a pig model of cardiac arrest.

Pralidoxime is a common antidote for organophosphate poisoning; however, studies have also reported pralidoxime's pressor effect, which may facilitate the restoration of spontaneous circulation (ROSC) after cardiac arrest by improving coronary perfusion pressure (CPP). We investigated the immediate cardiovascular effects of pralidoxime in anaesthetised normal rats and the effects of pralidoxime administration during cardiopulmonary resuscitation (CPR) in a pig model of cardiac arrest. To evaluate the immediate cardiovascular effects of pralidoxime, seven anaesthetised normal rats received saline or pralidoxime (20 mg/kg) in a randomised crossover design, and the responses were determined using the conductance catheter technique. To evaluate the effects of pralidoxime administration during CPR, 22 pigs randomly received either 80 mg/kg of pralidoxime or an equivalent volume of saline during CPR. In the rats, pralidoxime significantly increased arterial pressure than saline (P = .044). The peak effect on arterial pressure was observed in the first minute. In a pig model of cardiac arrest, CPP during CPR was higher in the pralidoxime group than in the control group (P = .002). ROSC was attained in three animals (27.3%) in the control group and nine animals (81.8%) in the pralidoxime group (P = .010). Three animals (27.3%) in the control group and eight animals (72.2%) in the pralidoxime group survived the 6-hour period (P = .033). In conclusion, pralidoxime had a rapid onset of pressor effect. Pralidoxime administered during CPR led to significantly higher rates of ROSC and 6-hour survival by improving CPP in a pig model.

Functional basis for dose-dependent antagonism of rat and rabbit neuromuscular transmission by the bis-pyridinium oxime MMB4.

Organophosphorus (OP) compounds inhibit central and peripheral acetylcholinesterase (AChE) activity, overstimulating cholinergic receptors and causing autonomic dysfunction (e.g., bronchoconstriction, excess secretions), respiratory impairment, seizure and death at high doses. Current treatment for OP poisoning in the United States includes reactivation of OP-inhibited AChE by the pyridinium oxime 2-pyridine aldoxime (2-PAM). However, 2-PAM has a narrow therapeutic index and its efficacy is confined to a limited number of OP agents. The bis-pyridinium oxime MMB4, which is a more potent reactivator than 2-PAM with improved pharmaceutical properties and therapeutic range, is under consideration as a potential replacement for 2-PAM. Similar to other pyridinium oximes, high doses of MMB4 lead to off-target effects culminating in respiratory depression and death. To understand the toxic mechanisms contributing to respiratory depression, we evaluated the effects of MMB4 (0.25-16 mM) on functional and neurophysiological parameters of diaphragm and limb muscle function in rabbits and rats. In both species, MMB4 depressed nerve-elicited muscle contraction by blocking muscle endplate nicotinic receptor currents while simultaneously prolonging endplate potentials by inhibiting AChE. MMB4 increased quantal content, endplate potential rundown and tetanic fade during high frequency stimulation in rat but not rabbit muscles, suggesting species-specific effects on feedback mechanisms involved in sustaining neurotransmission. These data reveal multifactorial effects of MMB4 on cholinergic neurotransmission, with the primary toxic modality being reduced muscle nicotinic endplate currents. Evidence of species-specific effects on neuromuscular function illustrates the importance of comparative toxicology when studying pyridinium oximes and, by inference, other quaternary ammonium compounds.

Solvent Deuterium Oxide Isotope Effects on the Reactions of Organophosphorylated Acetylcholinesterase.

Organophosphates (OPs) are esters of substituted phosphates, phosphonates or phosphoramidates that react with acetylcholinesterase (AChE) by initially transferring the organophosphityl group to a serine residue in the enzyme active site, concomitant with loss of an alcohol or halide leaving group. With substituted phosphates, this transfer is followed by relatively slow hydrolysis of the organophosphoryl AChE, or dephosphorylation, that is often accompanied by an aging reaction that renders the enzyme irreversibly inactivated. Aging is a dealkylation that converts the phosphate triester to a diester. OPs are very effective AChE inhibitors and have been developed as insecticides and chemical warfare agents. We examined three reactions of two organophosphoryl AChEs, dimethyl- and diethylphosphorylated AChE, by comparing rate constants and solvent deuterium oxide isotope effects for hydrolysis, aging and oxime reactivation with pralidoxime (2-PAM). Our study was motivated (1) by a published x-ray crystal structure of diethylphosphorylated AChE, which showed severe distortion of the active site that was restored by the binding of pralidoxime, and (2) by published isotope effects for decarbamoylation that decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE. We previously reconciled these results by proposing a shift in the rate-limiting step from proton transfer for the small carbamoyl group to a likely conformational change in the distorted active site of the large carbamoyl enzyme. This proposal was tested but was not supported in this report. The smaller dimethylphosphoryl AChE and the larger diethylphosphoryl AChE gave similar isotope effects for both oxime reactivation and hydrolysis, and the isotope effect values of about two indicated that proton transfer was rate limiting for both reactions.

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.

Oximes as pretreatment before acute exposure to paraoxon.

Organophosphates, useful agents as pesticides, also represent a serious danger due to their high acute toxicity. There is indication that oximes, when administered before organophosphate exposure, can protect from these toxic effects. We have tested at equitoxic dosage (25% of LD01 ) the prophylactic efficacy of five experimental (K-48, K-53, K-74, K-75, K-203) and two established oximes (pralidoxime and obidoxime) to protect from mortality induced by the organophosphate paraoxon. Mortalities were quantified by Cox analysis and compared with those observed after pretreatment with a strong acetylcholinesterase inhibitor (10-methylacridine) and after the FDA-approved pretreatment compound pyridostigmine. All nine tested substances statistically significantly reduced paraoxon-induced mortality. Best protection was conferred by the experimental oxime K-48, reducing the relative risk of death (RR) to 0.10, which was statistically significantly superior to pyridostigmine (RR = 0.31). The other oximes reduced the RR to 0.13 (obidoxime), 0.20 (K-203), 0.21 (K-74), 0.24 (K-75) and 0.26 (pralidoxime), which were significantly more efficacious than 10-methylacridine (RR = 0.65). These data support the hypothesis that protective efficacy is not primarily due to cholinesterase inhibition and indicate that the tested experimental oximes may be considered promising alternatives to the established pretreatment compound pyridostigmine.

Molecular modeling studies on the interactions of 7-methoxytacrine-4-pyridinealdoxime, 4-PA, 2-PAM, and obidoxime with VX-inhibited human acetylcholinesterase: a near attack conformation approach.

7-methoxytacrine-4-pyridinealdoxime (7-MEOTA-4-PA, named hybrid 5C) is a compound formerly synthesized and evaluated in vitro, together with 4-pyridine aldoxime (4-PA) and commercial reactivators of acetylcholinesterase (AChE). This compound was designed with the purpose of being a prophylactic reactivator, capable of interacting with different subdomains of the active site of AChE. To investigate these interactions, theoretical results from docking were first compared with experimental data of hybrid 5C, 4-PA, and two commercial oximes, on the reactivation of human AChE (HssAChE) inhibited by VX. Then, further docking studies, molecular dynamics simulations, and molecular mechanics Poisson-Boltzmann surface area calculations, were carried out to investigate reactivation performances, considering the near attack conformation (NAC) approach, prior to the nucleophilic substitution mechanism. Our results helped to elucidate the interactions of such molecules with the different subdomains of the active site of HssAChE. Additionally, NAC poses of each oxime were suggested for further theoretical studies on the reactivation reaction.

Effect of six oximes on acutely anticholinesterase inhibitor-induced oxidative stress in rat plasma and brain.

Beside the key inhibition of acetylcholinesterase (AChE), involvement of oxidative stress in organophosphate (OP)-induced toxicity has been supported by experimental and human studies. On the other hand, according to our best knowledge, possible antioxidant properties of oximes, the only causal antidotes to OP-inhibited AChE, have been examined only by a few studies. Thus, we have determined the effect of four conventional (obidoxime, trimedoxime, pralidoxime, asoxime) and two promising experimental oximes (K027, K203) on dichlorvos (DDVP)-induced oxidative changes in vivo. Wistar rats (5/group) were treated with oxime (5% LD50 i.m) immediately after DDVP challenge (75% LD50 s.c). Oxidative stress biomarkers were determined in plasma and brain 60 min after the treatment: prooxidative-superoxide anion (O2·-) and total oxidative status (TOS); antioxidative-superoxide dismutase (SOD), total thiol (SH) groups, total antioxidant status (TAS) and paraoxonase (PON1); tissue oxidative stress burden-prooxidative-antioxidative balance (PAB) and oxidative stress index (OSI); oxidative tissue damage-malondialdehyde (MDA) and advanced oxidation protein products (AOPP). All oximes were able to attenuate DDVP-induced oxidative stress in rat plasma and brain. Changes of determined parameters in brain were not as prominent as it was seen in plasma. Based on OSI, better abilities of oxime K027, K203 and obidoxime to maintain DDVP-induced oxidative stress in rat brain were shown as compared to trimedoxime, pralidoxime and asoxime. Oximes can influence the complex in vivo redox processes that might contribute to their overall therapeutic efficacy. Further research is needed to understand the underlying molecular mechanisms involved in this phenomenon.

Comparative evaluation of antidotal efficacy of 2-PAM and HNK-102 oximes during inhalation of sarin vapor in Swiss albino mice.

Efficacy of two oximes treatments evaluated during inhalation of sarin vapor (LCt50, 755.9 mg/min/m3) in simulated real scenario in vivo. Majority of mice either became moribund or died within 1-2 min during exposure to multifold-lethal concentrations of sarin vapor. Protection indices were determined by exposing to sarin vapor in two sessions, 1 min exposure followed by treatments with or without HNK-102 (56.56 mg/kg, im) or 2-PAM (30 mg/kg, im) and atropine (10 mg/kg, ip), and again exposed for remaining 14 min. Protection offered by HNK-102 was found to be four folds higher compared to 2-PAM in the same toxic environment. Secondly, sub-lethal concentration of sarin vapor (0.8 × LCt50 or 605 mg/min/m3), 24 h post investigations revealed that the oximes could not reactivate brain and serum acetylcholinesterase (AChE) activity. The treatments prevented increase in protein concentration (p < .05) and macrophages infiltration compared to sarin alone group in broncho-alveolar lavage fluid. Lung histopathology showed intense peribronchial infiltration and edema with desquamating epithelial lining and mild to moderate alveolar septal infiltration in sarin and atropine groups, respectively. Noticeable peeling-off observed in epithelial lining and sporadic mild infiltration of epithelial cells at bronchiolar region in 2-PAM and HNK-102 groups, respectively. The oximes failed to reactivate AChE activity; however, the mice survived up to 6.0 × LCt50, proved involvement of non-AChE targets in sarin toxicity. Atropine alone treatment was found to be either ineffective or increased the toxicity. HNK-102, exhibited better survivability with lung protection, can be considered as a better replacement for 2-PAM to treat sarin inhalation induced poisoning.

The First Use of Pralidoxime in a Child With Rivastigmine Poisoning.

The aim of this report is to describe the successful use of pralidoxime in a pediatric patient who accidentally ingested 12 mg of rivastigmine and presented to the emergency department with weakness, drowsiness, hyporeactivity to environmental stimuli, and full cholinergic syndrome. CASE: The patient presented to the emergency department 2 hours after a suspected ingestion of rivastigmine. He was sleepy but oriented and cooperative, hypotonic, and hyporeflexic and has a Glasgow Coma Scale score of 13 (E3M6V4). Laboratory tests showed a low plasma cholinesterase levels of 2141 U/L (reference range, 5300-12 900 U/L), hyperglycemia (251 mg/dL), and leukocytosis with neutrophilia (21 900/mL, 75.2% neutrophils). CONCLUSIONS: Only 2 pediatric cases of rivastigmine poisoning have been reported in the literature, and there are no previous reports of using pralidoxime in the management of this poisoning. In the present case, intravenous pralidoxime (30 mg/kg) was administered twice at the fifth and sixth hours of ingestion for nicotinic and central effects. There is reasonable theoretical science to suggest pralidoxime in case of acetylcholinesterase inhibitor toxicity. We conclude that observed clinical improvement in weakness temporally associated with pralidoxime administration. Increased plasma cholinesterase activity after pralidoxime administration also makes it useful in this type of poisoning.

In silico and in vitro evaluation of two novel oximes (K378 and K727) in comparison to K-27 and pralidoxime against paraoxon-ethyl intoxication.

Organophosphate (OP) poisoning is a major global health issue; while compounds from this group have been used intensively over the last century, an effective antidote is still lacking. Oxime-type acetylcholinesterase (AChE) reactivators are used to reactivate the OP inhibited AChE. Pralidoxime is the only US Food and Drug Administration approved oxime for therapeutic use but its efficacy has been disappointing. Two novel oximes (K378 and K727) were investigated in silico and in vitro and compared with an experimental oxime (kamiloxime; K-27) and pralidoxime. In silico the molecular interactions between AChE and oximes were examined and binding energies were assessed. LogP (predicted log of the octanol/water partition coefficient) was estimated. In vitro the intrinsic ability of the oximes to inhibit AChE (IC50) and their reactivation potency (R50) when used in paraoxon inhibited human RBC-AChE was determined. Molecular docking revealed that K378 and K727 bind to the peripheral site(s) with high binding energies in contrast to the central binding of K-27 and pralidoxime. LogP values indicating that the novel compounds are significantly less hydrophilic than K-27 or pralidoxime. IC50 of K378 and K727 were comparable (0.9 and 1 µM, respectively) but orders of magnitude lower than comparators. R50 values revealed their inability to reactivate paraoxon inhibited AChE. It is concluded that the novel oximes K378 and K727 are unlikely to be clinically useful. The in silico and in vitro studies described allow avoidance of unnecessary in vivo animal work and contribute to the reduction of laboratory animal use.