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.

The in silico identification of novel broad-spectrum antidotes for poisoning by organophosphate anticholinesterases.

Owing to their potential to cause serious adverse health effects, significant efforts have been made to develop antidotes for organophosphate (OP) anticholinesterases, such as nerve agents. To be optimally effective, antidotes must not only reactivate inhibited target enzymes, but also have the ability to cross the blood-brain barrier (BBB). Progress has been made toward brain-penetrating acetylcholinesterase reactivators through the development of a new group of substituted phenoxyalkyl pyridinium oximes. To help in the selection and prioritization of compounds for future synthesis and testing within this class of chemicals, and to identify candidate broad-spectrum molecules, an in silico framework was developed to systematically generate structures and screen them for reactivation efficacy and BBB penetration potential.

The in silico identification of novel broad-spectrum antidotes for poisoning by organophosphate anticholinesterases.

Because of their potential to cause serious adverse health effects, significant efforts have been made to develop antidotes for organophosphate (OP) anticholinesterases, such as nerve agents. To be optimally effective, antidotes must not only reactivate inhibited target enzymes, but also have the ability to cross the blood brain barrier (BBB). Progress has been made toward brain-penetrating acetylcholinesterase reactivators through the development of a new group of substituted phenoxyalkyl pyridinium oximes. To help in the selection and prioritization of compounds for future synthesis and testing within this class of chemicals, and to identify candidate broad-spectrum molecules, an in silico framework was developed to systematically generate structures and screen them for reactivation efficacy and BBB penetration potential.

Alterations of Systemic and Hepatic Metabolic Function Following Exposure to Trans-nonachlor in Low and High Fat Diet Fed Male Sprague Dawley Rats.

The overall prevalence of metabolic diseases such as type 2 diabetes (T2D) and associated co-morbidities have increased at an alarming rate in the United States and worldwide. There is a growing body of epidemiological evidence implicating exposure to persistent organic pollutants (POPs), including legacy organochlorine (OC) pesticides and their bioaccumulative metabolites, in the pathogenesis of metabolic diseases. Therefore, the goal of the present study was to determine if exposure to trans-nonachlor, a bioaccumulative OC pesticide contaminant, in concert with high fat diet intake induced metabolic dysfunction. Briefly, male Sprague Dawley rats were exposed to trans-nonachlor (.5 or 5 ppm) in either a low fat (LFD) or high fat diet (HFD) for 16 weeks. At 8 weeks of intake, trans-nonachlor decreased serum triglyceride levels in LFD and HFD fed animals and at 16 weeks compared to LFD fed animals. Interestingly, serum glucose levels were decreased by trans-nonachlor (5 ppm) in LFD fed animals at 16 weeks. Serum free fatty acids were increased by trans-nonachlor exposure (5 ppm) in LFD fed animals at 16 weeks. HFD fed animals displayed signs of hepatic steatosis including elevated liver triglycerides, liver enzymes, and liver lipid peroxidation which were not significantly altered by trans-nonachlor exposure. However, there was a trans-nonachlor mediated increase in expression of fatty acid synthase in livers of LFD fed animals and not HFD fed animals. Thus, the present data indicate exposure to trans-nonachlor in conjunction with LFD or HFD intake produces both diet and exposure dependent effects on lipid and glucose metabolism.

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.

In vitro P-glycoprotein activity does not completely explain in vivo efficacy of novel centrally effective oxime acetylcholinesterase reactivators.

Novel-substituted phenoxyalkyl pyridinium oxime acetylcholinesterase (AChE) reactivators (US patent 9,227,937) that showed convincing evidence of penetration into the brains of intact rats were developed by our laboratories. The oximes separated into three groups based on their levels of brain AChE reactivation following exposure of rats to the sarin surrogate nitrophenyl isopropyl methylphosphonate (NIMP). P-glycoprotein (P-gp) is a major blood-brain barrier (BBB) transporter and requires ATP for efflux. To determine if P-gp affinity screening could be used to reduce animal use, we measured in vitro oxime-stimulated ATPase activity to see if the in vivo reactivation efficacies related to the oximes' functions as P-gp substrates. High efficacy oximes were expected to be poor P-gp substrates, thus remaining in the brain longer. The high efficacy oximes (24-35% brain AChE reactivation) were worse P-gp substrates than the low efficacy oximes (0-7% brain AChE reactivation). However, the oxime group with medium in vivo reactivation of 10-17% were even worse P-gp substrates than the high efficacy group so their reactivation ability was not reflected by P-gp export. The results suggest that in vitro P-gp ATPase activity can remove the low efficacy oximes from in vivo testing, but is not sufficient to differentiate between the top two tiers.

Neuroprotection From Organophosphate-Induced Damage by Novel Phenoxyalkyl Pyridinium Oximes in Rat Brain.

The nerve agents are extremely toxic organophosphates which lead to massive inhibition of acetylcholinesterase (AChE) in both the central and peripheral nervous systems. The currently approved pyridinium oxime reactivators of organophosphate-inhibited AChE (eg, 2-PAM in the United States) cannot penetrate the blood-brain barrier because of the permanent positive charge in the pyridinium ring. Therefore these current oximes cannot rescue inhibited AChE in the brain. Our laboratories have invented and patented a platform of substituted phenoxyalkyl pyridinium oximes that have been tested for efficacy as therapy within the brains of adult male rats which were challenged with a high sublethal dosage of highly relevant surrogates of sarin (nitrophenyl isopropyl methylphosphonate, NIMP) and VX (nitrophenyl ethyl methylphosphonate, NEMP). The histochemical astrocyte marker glial fibrillary acidic protein (GFAP) was investigated as an indication of neuropathology in two brain regions, the piriform cortex and the dentate gyrus of the hippocampus, which are regions known to be damaged by nerve agent toxicity. Rats treated with either NIMP or NEMP without therapy or with NIMP or NEMP plus 2-PAM therapy showed similar increases in GFAP compared with vehicle controls. However, the rats challenged with NIMP or NEMP plus therapy with our novel Oxime 20 (either a bromide or a mesylate salt) showed GFAP levels statistically undistinguishable from controls. These data provide highly supportive functional evidence of novel oxime entry into the brain. These novel oximes have the potential to provide central neuroprotection from organophosphate anticholinesterase-induced damage, which is a characteristic not displayed by most pyridinium oximes.

A case-control study: The association of serum paraoxonase 1 activity and concentration with the development of type 2 diabetes mellitus.

BACKGROUND: A longitudinal study assessed serum paraoxonase 1 (PON1) activity and concentration as affected by age and as associated with the development of type 2 diabetes (T2D). PON1's recently established physiological function is the hydrolysis of lipolactones in oxidized LDL particles. METHODS: Serum samples and clinical data collected and stored at different time points over a 20-year interval in the Air Force Health Study were analysed. PON1 activity and concentration and C-reactive protein concentration in samples from the same individuals 20 years apart were compared using a paired t test (n = 159). A case-control study design and multivariable logistic regression analysis assessed the association of PON1's activity and concentration with the subsequent development of T2D (n = 222 and α = 0.10). RESULTS: No difference with age was found in PON1 activity assessed using 3 substrates, paraoxon (P = 0.897), phenyl acetate (P = 0.994), and dihydrocoumarin (P = 0.505), or PON1 serum concentration (P = 0.357). C-reactive protein concentration increased 0.7 mg/L (P = 0.004) over the 20-year interval. Lower PON1 activity assayed with phenyl acetate (P = 0.015, OR = 1.25 per 1000 U/L decrease) was associated with an increased risk of developing T2D as was a lower PON1 serum concentration (P = 0.004, OR = 1.72 per 2 µmol/L decrease). PON1 activity assayed with paraoxon (P = 0.681) or dihydrocoumarin (P = 0.136) was not associated with the development of T2D. CONCLUSIONS: Lower PON1 activity and concentration were associated with an increased risk of developing T2D when adjusted for many of the common risk markers for T2D previously identified. Thus, PON1 may have merit as a biomarker for the development of T2D.

Effects of Chlorpyrifos or Methyl Parathion on Regional Cholinesterase Activity and Muscarinic Receptor Subtype Binding in Juvenile Rat Brain.

The effects of developmental exposure to two organophosphorus (OP) insecticides, chlorpyrifos (CPF) and methyl parathion (MPS), on cholinesterase (ChE) activity and muscarinic acetylcholine receptor (mAChR) binding were investigated in preweanling rat brain. Animals were orally gavaged daily with low, medium, and high dosages of the insecticides using an incremental dosing regimen from postnatal day 1 (PND1) to PND20. On PND12, PND17 and PND20, the cerebral cortex, corpus striatum, hippocampus, and medulla-pons were collected for determination of ChE activity, total mAChR density, and the density of the individual mAChR subtypes. ChE activity was inhibited by the medium and high dosages of CPF and MPS at equal levels in all four brain regions at all three ages examined. Exposure to both compounds decreased the levels of the M1, M2/M4, and M3 subtypes and the total mAChR level in all brain regions, but the effects varied by dosage group and brain region. On PND12, only the high dosages induced receptor changes while on PND17 and PND20, greater effects became evident. In general, the effects on the M1 subtype and total receptor levels appeared to be greater in the cerebral cortex and hippocampus than in the corpus striatum and medulla-pons. This did not appear to be the case for the M2/M4 and M3 subtypes effects. The differences between CPF and MPS were minimal even though in some cases, CPF exerted statistically greater effects than MPS did. In general, repeated exposure to organophosphorus insecticides can alter the levels of the various mAChR subtypes in various brain regions which could induce perturbation in cholinergic neurochemistry during the maturation of the brain regions.

Efficacy of novel phenoxyalkyl pyridinium oximes as brain-penetrating reactivators of cholinesterase inhibited by surrogates of sarin and VX.

Pyridinium oximes are strong nucleophiles and many are effective reactivators of organophosphate-inhibited cholinesterase (ChE). However, the current oxime reactivators are ineffective at crossing the blood-brain barrier and reactivating brain ChE in the intact organism. Our laboratories have developed a series of substituted phenoxyalkyl pyridinium oximes (US patent 9,227,937 B2) with the goal of identifying reactivators effective in crossing the blood-brain barrier. The first 35 of the series were found to have similar in vitro efficacy as reactivators of ChE inhibited by a sarin surrogate (phthalimidyl isopropyl methylphosphonate, PIMP) or a VX surrogate (nitrophenyl ethyl methylphosphonate, NEMP) in bovine brain preparations as previously observed in rat brain preparations. A number of these novel oximes have shown the ability to decrease the level of ChE inhibition in the brains of rats treated with a high sublethal dosage of either a sarin surrogate (nitrophenyl isopropyl methylphosphonate, NIMP) or the VX surrogate NEMP. Levels of reactivation at 2 h after oxime administration were up to 35% while the currently approved therapeutic, 2-PAM, yielded no reduction in brain ChE inhibition. In addition, there was evidence of attenuation of seizure-like behavior with several of the more effective novel oximes, but not 2-PAM. Therefore these novel oximes have demonstrated an ability to reactivate inhibited ChE in brain preparations from two species and in vivo data support their ability to enter the brain and provide a therapeutic action. These novel oximes have the potential to be developed into improved antidotes for nerve agent therapy.

Novel brain-penetrating oximes for reactivation of cholinesterase inhibited by sarin and VX surrogates.

Current oxime reactivators for organophosphate-inhibited cholinesterase (ChE) do not effectively cross the blood-brain barrier and therefore cannot restore brain ChE activity in vivo. Our laboratories have studied highly relevant sarin and VX surrogates, which differ from their respective nerve agents only in the leaving group and thereby leave ChE phosphylated with the same chemical moiety as sarin and VX. Our laboratories have developed novel substituted phenoxyalkyl pyridinium oximes that lead to reduced ChE inhibition in the brains of rats challenged with a high sublethal dosage of the sarin surrogate, whereas 2-PAM did not, using a paradigm designed to demonstrate brain penetration. In addition, treatment of rats with these novel oximes is associated with attenuation of seizure-like behavior compared to rats treated with 2-PAM, providing additional evidence that the oximes penetrate the blood-brain barrier. Further, some of the oximes provided 24-h survival superior to 2-PAM, and shortened the duration of seizure-like behavior when rats were challenged with lethal dosages of the sarin and VX surrogates, providing additional support for the conclusion that these oximes penetrate the brain.

The association of serum trans-nonachlor levels with atherosclerosis.

Recent epidemiological studies suggest a strong association between exposure to environmental contaminants, including organochlorine (OC) insecticides or their metabolites, and development of pathologies, such as atherosclerosis, in which oxidative stress plays a significant etiological role. Biomarkers of systemic oxidative stress have the potential to link production of reactive oxygen species (ROS), which are formed as a result of exposure to xenobiotic toxicants, and underlying pathophysiological states. Measurement of F2-isoprostane concentrations in body fluids is the most accurate and sensitive method currently available for assessing in vivo steady-state oxidative stress levels. In the current study, urinary concentrations of F2-isoprostanes and serum levels of persistent OC compounds p,p'-dichlorodiphenyldichloroethene (DDE), trans-nonachlor (a component of the technical chlordane mixture), and oxychlordane (a chlordane metabolite) were quantified in a cross-sectional study sample and the association of these factors with a clinical diagnosis of atherosclerosis determined. Urinary isoprostane levels were not associated with atherosclerosis or serum concentrations of OC compounds in this study sample. However, occurrence of atherosclerosis was found to be associated with serum trans-nonachlor levels. DDE and oxychlordane were not associated with atherosclerosis. This finding supports current evidence that exposure to environmental factors is a risk factor for atherosclerosis, in addition to other known risk factors.