Reactivation by novel pyridinium oximes of rat serum and skeletal muscle acetylcholinesterase inhibited by organophosphates.

The treatment of organophosphate (OP) anticholinesterases currently lacks an effective oxime reactivator of OP-inhibited acetylcholinesterase (AChE) which can penetrate the blood-brain barrier (BBB). Our laboratories have synthesized novel substituted phenoxyalkyl pyridinium oximes and tested them for their ability to promote survival of rats challenged with lethal doses of nerve agent surrogates. These previous studies demonstrated the ability of some of these oximes to promote 24-h survival to rats challenged with a lethal level of highly relevant surrogates for sarin and VX. The reactivation of OP-inhibited AChE in peripheral tissues was likely to be a major contributor to their efficacy in survival of lethal OP challenges. In the present study, twenty of these novel oximes were screened in vitro for reactivation ability for AChE in rat skeletal muscle and serum using two nerve agent surrogates: phthalimidyl isopropyl methylphosphonate (PIMP, a sarin surrogate) and 4-nitrophenyl ethyl methylphosphonate (NEMP, a VX surrogate). The oximes demonstrated a range of 23%-102% reactivation of AChE in vitro across both tissue types. Some of the novel oximes tested in the present study demonstrated the ability to more effectively reactivate AChE in serum than the currently approved oxime, 2-PAM. Therefore, some of these novel oximes have the potential to reverse AChE inhibition in peripheral target tissues and contribute to survival efficacy.

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.

Inhibition Kinetics of 16 Organophosphorus Pesticides or Their Active Metabolites on Erythrocyte Acetylcholinesterase From Humans and Rats.

Inhibition kinetics assays were conducted with 16 commercial organophosphate (OP) pesticides or their metabolites on acetylcholinesterase (AChE) in erythrocyte "ghost" preparations from 18 individual humans (both sexes; adults, juveniles, and cord blood samples; mixed races/ethnicities) and pooled samples from adult rats (both sexes). A well-established spectrophotometric assay using acetylthiocholine as substrate and a chromogen was employed. The kinetic parameters bimolecular rate constant (ki), dissociation constant (KI), and phosphorylation constant (kp) were calculated for each compound. As expected, a wide range of potencies were displayed among the tested compounds. Statistical analysis of the resultant data indicated no differences in sex, age, or race/ethnicity among the human samples that are unexpected based on chance (4.2% statistically significant out of 48 parameters calculated) and no differences between the sexes in rats. The bimolecular rate constants for 10 of the compounds were not statistically different between rats and humans. The data indicate that, consistent with the high level of conservation of AChE among species and the fact that AChE at different locations within a species arises from the same gene, the inhibition kinetic parameters calculated from rat erythrocyte ghost preparations should be useful in estimating potencies of OP compounds on target AChE in humans. Additionally, the data indicate that differences in sensitivities among individual humans were not apparent.

In vitro age-related differences in rats to organophosphates.

The mechanism of toxic action for organophosphates (OPs) is the persistent inhibition of acetylcholinesterase (AChE) resulting in accumulation of acetylcholine and subsequent hyperstimulation of the nervous system. Organophosphates display a wide range of acute toxicities. Differences in the OP's chemistries results in differences in the compound's metabolism and toxicity. Acute toxicities of OPs appear to be principally dependent on compound specific efficiencies of detoxication, and less dependent upon efficiencies of bioactivation and sensitivity of AChE. Serine esterases, such as carboxylesterase (CaE) and butyrylcholinesterase (BChE), play a prominent role in OP detoxication. Organophosphates can stoichiometrically inhibit these enzymes, removing OPs from circulation thus providing protection for the target enzyme, AChE. This in vitro study investigated age-related sensitivity of AChE, BChE and CaE to twelve structurally different OPs in rat tissues. Sensitivity of esterases to these OPs was assessed by inhibitory concentration 50s (IC50s). The OPs displayed a wide range of inhibitory potency toward AChE with IC50s in the low nM-µM range with no differences among ages; however, the CaE IC50s generally increased with age reflecting greater protection in adults. These results suggest age-related differences in acute toxicities of OPs in mammals are primarily a result of their detoxication capacities.

Novel pyridinium oximes enhance 24-h survivability against a lethal dose of nerve agent surrogate in adult female rats.

Our laboratory has developed novel substituted phenoxyalkyl pyridinium oximes (US Patent 9,227,937) designed to more efficiently penetrate the central nervous system to enhance survivability and attenuate seizure-like signs and neuropathology. Previous studies with male Sprague-Dawley rats indicated that survivability was enhanced against the nerve agent (sarin) surrogate, 4-nitrophenyl isopropyl methylphosphonate (NIMP). In this study, female adult Sprague-Dawley rats, tested specifically in diestrus, were challenged subcutaneously with lethal concentrations of NIMP (0.6 mg/kg). After development of seizure-like behavior and other signs of cholinergic toxicity, human equivalent dosages of atropine (0.65 mg/kg) and one of four oximes (2-PAM, or novel oxime 15, 20, or 55; 0.146 mmol/kg) or Multisol vehicle was administered alone or in binary oxime combinations intramuscularly. Animals were closely monitored for signs of cholinergic toxicity and 24 h survivability. Percentages of animals surviving the 24 h NIMP challenge dose were 35 % for 2-PAM and 55 %, 70 %, and 25 % for novel oximes 15, 20, and 55, respectively. Improvements in survival were also observed over 2-PAM alone with binary combinations of 2-PAM and either oxime 15 or oxime 20. Additionally, administration of novel oximes decreased the duration of seizure-like behavior as compared to 2-PAM suggesting that these oximes better penetrate the blood-brain barrier to mitigate central nervous system hypercholinergic activity. Efficacies were similar between females and previously reported males. These data indicate that the novel pyridinium oximes enhance survivability against lethal OP toxicity as compared to 2-PAM in adult female rats.

Oxime-mediated reactivation of organophosphate-inhibited acetylcholinesterase with emphasis on centrally-active oximes.

This review provides an overview of the global research leading to the large number of compounds developed as reactivators of acetylcholinesterase inhibited by a variety of organophosphate compounds, most of which are nerve agents but also some insecticides. A number of these organophosphates are highly toxic and effective therapy by reactivators contributes to saving lives. Two major challenges for more effective therapy with reactivators are identification of a broad spectrum reactivator efficacious against a variety of organophosphate structures, and a reactivator that can cross the blood-brain barrier to protect the brain. The most effective of the reactivators developed are the nucleophilic pyridinium oximes, which bear a permanent positive charge from the quaternary nitrogen in the pyridinium ring. The permanent positive charge retards the oximes from crossing the blood-brain barrier and therefore restoration of normal cholinergic function in the brain is unlikely. A number of laboratories have developed nucleophiles, mostly oximes, that are theorized to cross the blood-brain barrier by several strategies. At the present time, no reactivator is optimally broad spectrum across the wide group of organophosphate chemistries. Some oximes, including the substituted phenoxyalkyl pyridinium oximes invented by our laboratories, have the potential to provide neuroprotection in the brain and show evidence of efficacy against both nerve agent and insecticidal chemistries, so these novel oximes have promise for future development. This article is part of the special issue entitled 'Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield'.

Central neuroprotection demonstrated by novel oxime countermeasures to nerve agent surrogates.

Oximes remain a long-standing element of the therapy for nerve agents, organophosphates (OPs) that poison by inhibiting the enzyme acetylcholinesterase (AChE), resulting in hypercholinergic activity both centrally and peripherally. Oximes, such as the pyridinium oxime pralidoxime (2-PAM) in the United States, can reactivate the inhibited AChE and restore cholinergic function. However, there are several drawbacks to the current oximes; one of them, the inability of these oximes to effectively enter the brain, is the subject of study by several laboratories, including ours. Our laboratory invented a platform of substituted phenoxyalkyl pyridinium oximes that were tested against highly relevant surrogates of the nerve agents, sarin and VX. Using high sublethal dosages of the OPs, the novel oximes were observed to attenuate seizure-like behavior in rats and to reduce the levels of glial fibrillary acidic protein (an indicator of glial scarring) to control levels, in contrast to levels observed with 2-PAM or no oxime therapy. Using lethal levels of surrogates, some novel oximes protected against lethality compared with 2-PAM, shortened the time to cessation of seizure-like behavior (from 8+ to 6 h), and protected the brain neurons. Therefore, some of these novel oximes are showing exceptional promise alone or in combination with 2-PAM as therapeutics against nerve agent toxicity.

Novel centrally active oxime reactivators of acetylcholinesterase inhibited by surrogates of sarin and VX.

A novel oxime platform, the substituted phenoxyalkyl pyridinium oximes (US patent 9,227,937), was invented at Mississippi State University with an objective of discovering a brain-penetrating antidote to highly potent organophosphate anticholinesterases, such as the nerve agents. The goal was reactivation of inhibited brain acetylcholinesterase to attenuate the organophosphate-induced hypercholinergic activity that results in glutamate-mediated excitotoxicity and neuropathology. The currently approved oxime antidote in the US, 2-PAM, cannot do this. Using highly relevant surrogates of sarin and VX that leave acetylcholinesterase phosphylated with the same chemical moiety as their respective nerve agents, in vitro screens and in vivo tests in rats were conducted to identify the most efficacious members of this platform. The most promising novel oximes provided 24-h survival of lethal level surrogate exposure better than 2-PAM in almost all cases, and two of the oximes shortened the time to cessation of seizure-like behavior while 2-PAM did not. The most promising novel oximes attenuated neuropathology as indicated by immunohistochemical stains for both glia and neurons, while 2-PAM did not protect either glia or neurons. These results strongly suggest that these novel oximes can function within the brain to protect it, and therefore show great promise as potential future nerve agent antidotes.

Association of serum levels of p,p'- Dichlorodiphenyldichloroethylene (DDE) with type 2 diabetes in African American and Caucasian adult men from agricultural (Delta) and non-agricultural (non-Delta) regions of Mississippi.

Epidemiological associations were reported in several studies between persistent organochlorine organic pollutants and type 2 diabetes mellitus (T2D). Mississippi is a highly agricultural state in the USA, particularly the Delta region, with previous high usage of organochlorine (OC) insecticides such as p,p'- dichlorodiphenyltrichloroethane (DDT). In addition, there is a high proportion of African Americans who display elevated prevalence of T2D. Therefore, this State provides an important dataset for further investigating any relationship between OC compounds and metabolic diseases. The aim of this study was to assess whether soil and serum levels of OC compounds, such as p,p'- dichlorodiphenyldichloroethylene (DDE), arising from the heavy historical use of legacy OC insecticides, might serve as an environmental public health indicator for T2D occurrence. Soil samples from 60 Delta and 60 non-Delta sites randomly selected were analyzed for the presence of OC compounds. A retrospective cohort study of adult men (150 from each region) was recruited to provide a blood sample for OC compound quantitation and select demographic and clinical information including T2D. Using multivariable logistic regression, an association was found between increasing serum DDE levels and T2D occurrence in non-Delta participants (those subjects with lower serum DDE levels), as opposed to Delta participants (individuals with higher serum DDE levels). Thus, while there was a relationship between serum DDE levels and T2D in those with lower burdens of DDE, the lack of association in those with higher levels of DDE indicates a complex non-monotonic correlation between serum DDE levels and T2D occurrence complicating the goal of finding a public health marker for T2D. Abbreviations: BMI, body mass index; CVD, cardiovascular disease; CDC, Center for Disease Control, United States of America; DDE, p,p'- dichlorodiphenyldichloroethylene; DDT, p,p'- dichlorodiphenyltrichloroethane; GC/MS, gas chromatography/mass spectrometry; GIS, geographic information system; GPS, global positioning system; HDL, high-density lipoprotein; HTN, hypertension; IDW, inverse distance weighting; IRB, Institutional Review Board; LDL, low-density lipoprotein; LOQ, limit of quantitation; NHANES, National Health and Nutrition Examination Surveys; POPs, persistent organic pollutants; OC, organochlorine; PCB, polychlorinated biphenyl; SIM, single-ion monitoring; T2D, type 2 diabetes mellitus; USA, United States of America.

Novel Brain-Penetrating Oxime Acetylcholinesterase Reactivators Attenuate Organophosphate-Induced Neuropathology in the Rat Hippocampus.

Organophosphate (OP) anticholinesterases cause excess acetylcholine leading to seizures which, if prolonged, result in neuronal damage in the rodent brain. Novel substituted phenoxyalkyl pyridinium oximes have previously shown evidence of penetrating the rat blood-brain barrier (BBB) in in vivo tests with a sarin surrogate (nitrophenyl isopropyl methylphosphonate, NIMP) or the active metabolite of the insecticide parathion, paraoxon (PXN), by reducing the time to cessation of seizure-like behaviors and accumulation of glial fibrillary acidic protein, whereas 2-PAM did not. The neuroprotective ability of our lead oximes (15, 20, and 55) was tested using NeuN, Nissl, and Fluoro-Jade B staining in the rat hippocampus. Following lethal-level subcutaneous challenge with NIMP or PXN, rats were intramuscularly administered a novel oxime or 2-PAM plus atropine and euthanized at 4 days. There were statistically significant increases in the median damage scores of the NeuN-stained NIMP, NIMP/2-PAM, and NIMP/Oxime 15 groups compared with the control whereas the scores of the NIMP/Oxime 20 and NIMP/Oxime 55 were not significantly different from the control. The same pattern of statistical significance was observed with PXN. Nissl staining provided a similar pattern, but without statistical differences. Fluoro-Jade B indicated neuroprotection from PXN with novel oximes but not with 2-PAM. The longer blood residence times of Oximes 20 and 55 compared with Oxime 15 might have contributed to their greater efficacy. These results suggest that novel oximes 20 and 55 were able to penetrate the BBB and attenuate neuronal damage after NIMP and PXN exposure, indicating potential broad-spectrum usefulness.