Pro-2-PAM therapy for central and peripheral cholinesterases

Abstract

Novel therapeutics to overcome the toxic effects of organophosphorus (OP) chemical agents are needed due to the documented use of OPs in warfare (e.g. 1980-1988 Iran/Iraq war) and terrorism (e.g. 1995 Tokyo subway attacks). Standard OP exposure therapy in the United States consists of atropine sulfate (to block muscarinic receptors), the acetylcholinesterase (AChE) reactivator (oxime) pralidoxime chloride (2-PAM), and a benzodiazepine anticonvulsant to ameliorate seizures. A major disadvantage is that quaternary nitrogen charged oximes, including 2-PAM, do not cross the blood brain barrier (BBB) to treat brain AChE. Therefore, we have synthesized and evaluated pro-2-PAM (a lipid permeable 2-PAM derivative) that can enter the brain and reactivate CNS AChE, preventing seizures in guinea pigs after exposure to OPs. The protective effects of the pro-2-PAM after OP exposure were shown using a) surgically-implanted radiotelemetry probes for electroencephalogram (EEG) b) neurohistopathology of brain, c) cholinesterase activities in the PNS and CNS, and d) survivability. The PNS oxime 2-PAM was ineffective at reducing seizures/ status epilepticus (SE) in diisopropyl-fluorophosphate (DFP)-exposed animals. In contrast, pro-2-PAM significantly suppressed and then eliminated seizure activity. In OP-exposed guinea pigs, there was a significant reduction in neurological damage with pro-2-PAM, but not 2-PAM. Distinct regional areas of the brains showed significantly higher AChE activity 1.5 h after OP exposure in pro-2-PAM treated animals compared to the 2-PAM treated ones. However, blood and diaphragm showed similar AChE activities in animals treated with either oxime, as both 2-PAM and pro 2-PAM are PNS active oximes. In conclusion, pro-2-PAM can cross the BBB, is rapidly metabolized inside the brain to 2-PAM, and protects against OP-induced SE through restoration of * Corresponding author. Tel: +1 301 319 9987; fax: +1 301 319 9070. Richard.Gordon@amedd.army.mil.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

Oxidative mechanisms for the biotransformation of 1-methyl-1,6-dihydropyridine-2-carbaldoxime to pralidoxime chloride

“…With these results, we conclude that pro-2-PAM can be a potential candidate for protection against seizure induced by OP inhibited AChE, and that BBB oxime therapeutics are needed as next generation treatments for OP. In contrast (DeMar et al, 2010;Shih et al, 2011) 2-PAM did not show protection against seizures induced by OP compounds in these studies.…”

“…Nanoparticles, which penetrate the BBB are also another attractive carrier to deliver the quaternary oximes to the CNS, but to date has not been shown to enter the CNS in a pharmacologically relevant time (Wagner et al, 2010). Our group have (DeMar et al, 2010) clearly demonstrated that pro-2-PAM, unlike 2-PAM, protected guinea pigs from DFP induced seizure, restored CNS AChE activity, and protected the animal from neuronal death in hippocampus. When pro-2-PAM treatment occurred immediately after OP exposure, pro-2-PAM provided protection against soman.…”

“…In addition, there is a low affinity active transport mechanism for riboflavin in choroid plexus that transports riboflavin from the CSF into the blood, to remove excess riboflavin from the CSF-ECS and the brain (Spector, 1989). It is likely that riboflavinoxidized pro-2-PAM to 2-PAM in the extracellular environment of the brain permitted reactivation of OP-inhibited extracellular AChE at synapses (DeMar et al, 2010). In vitro results show that μM levels of FAD, FMN, and riboflavin are able to convert pro-2-PAM to 2-PAM, and this biotransformation is complete within~5 min (Fig.…”

Post-exposure treatment with the oxime RS194B rapidly reverses early and advanced symptoms in macaques exposed to sarin vapor

“…Of many novel oximes synthesized and evaluated to date, only a few types have shown the potential to cross the BBB. These include pro-drug analogs of PAM (21), phenoxyalkyl pyridinium, detergent-like oximes (22), conjugation of various pyridinium aldoximes to a glucose C-6 Sox transporter (23), various nucleophiles that encompass association with the active center and peripheral site on AChE (24–26) and low molecular weight zwitterions, whose neutral species enables rapid blood-brain barrier passage (27,28). …”

The biodistribution and pharmacokinetics of the oxime acetylcholinesterase reactivator RS194B in guinea pigs

“…Although 2-PAM is an effective reactivator, one major deficiency is its inability to cross the blood-brain-barrier (BBB), thus preventing it from reactivating vital OP-inhibited AChE in the central nervous system (CNS). Over the last decade efforts have focused on screening and identifying other reactivating oximes that have a higher BBB permeability while still retaining a high reactivation profile (DeMar et al, 2010; García et al, 2010; Sit et al, 2011; Radic et al, 2012; Radic et al, 2013; Okolotowicz et al, 2014). In recent work by Radic et al, a promising centrally active oxime reactivator (RS194B, 4, Fig 1.)…”