Specific quinone reductase 2 inhibitors reduce metabolic burden and reverse Alzheimer's disease phenotype in mice.

Biological aging can be described as accumulative, prolonged metabolic stress and is the major risk factor for cognitive decline and Alzheimer's disease (AD). Recently, we identified and described a quinone reductase 2 (QR2) pathway in the brain, in which QR2 acts as a removable memory constraint and metabolic buffer within neurons. QR2 becomes overexpressed with age, and it is possibly a novel contributing factor to age-related metabolic stress and cognitive deficit. We found that, in human cells, genetic removal of QR2 produced a shift in the proteome opposing that found in AD brains while simultaneously reducing oxidative stress. We therefore created highly specific QR2 inhibitors (QR2is) to enable evaluation of chronic QR2 inhibition as a means to reduce biological age-related metabolic stress and cognitive decline. QR2is replicated results obtained by genetic removal of QR2, while local QR2i microinjection improved hippocampal and cortical-dependent learning in rats and mice. Continuous consumption of QR2is in drinking water improved cognition and reduced pathology in the brains of AD-model mice (5xFAD), with a noticeable between-sex effect on treatment duration. These results demonstrate the importance of QR2 activity and pathway function in the healthy and neurodegenerative brain and what we believe to be the great therapeutic potential of QR2is as first-in-class drugs.

Non-quaternary oximes detoxify nerve agents and reactivate nerve agent-inhibited human butyrylcholinesterase.

Government-sanctioned use of nerve agents (NA) has escalated dramatically in recent years. Oxime reactivators of organophosphate (OP)-inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) serve as antidotes toward poisoning by OPNAs. The oximes used as therapeutics are quaternary compounds that cannot penetrate the blood-brain barrier (BBB). There remains an urgent need for the development of next generation OPNA therapeutics. We have developed two high-throughput screening (HTS) assays using a fluorogenic NA surrogate, O-ethyl methylphosphonyl O-4-methyl-3-cyano-coumarin (EMP-MeCyC). EMP-MeCyC detoxification and EMP-BChE reactivation screening campaigns of ~155,000 small molecules resulted in the identification of 33 nucleophile candidates, including non-quaternary oximes. Four of the oximes were reactivators of both Sarin- and VX-inhibited BChE and directly detoxified Sarin. One oxime also detoxified VX. The novel reactivators included a non-quaternary pyridine amidoxime, benzamidoxime, benzaldoxime and a piperidyl-ketoxime. The VX-inhibited BChE reactivation reaction rates by these novel molecules were similar to those observed with known bis-quaternary reactivators and faster than mono-quaternary pyridinium oximes. Notably, we discovered the first ketoxime reactivator of OP-ChEs and detoxifier of OPNAs. Preliminary toxicological studies demonstrated that the newly discovered non-quaternary oximes were relatively non-toxic in mice. The discovery of unique non-quaternary oximes opens the door to the design of novel therapeutics and decontamination agents following OPNA exposure.

Catalytic Detoxification of Organophosphorus Nerve Agents by Butyrylcholinesterase-Polymer-Oxime Bioscavengers.

Organophosphorus nerve agents (OPNAs), used in chemical warfare, irreversibly inhibit essential cholinesterases (ChEs) in the cholinergic neurotransmission system. Several potent nucleophilic oximes have been approved for the treatment of acute poisoning by OPNAs, but they are rapidly cleared from blood circulation. Butyrylcholinesterase (BChE) stoichiometrically binds nerve agents, but because the molecular weight of a nerve agent is about 500-fold less than the enzyme, the bioscavenger has had limited utility. We synthesized BChE-polymer-oxime conjugates using atom transfer radical polymerization (ATRP) and azide-alkyne "click" chemistry. The activity of the BChE-polymer-oxime conjugates was dependent on the degree of oxime loading within the copolymer side chains. The covalent modification of oxime-containing copolymers prolonged the activity of BChE in the presence of the VX- and cyclosarin-fluorogenic analogues EMP-MeCyC and CMP-MeCyC, respectively. After complete inactivation by VX and cyclosarin fluorogenic analogues, the conjugates demonstrated efficient self-reactivation of up to 80% within 3-6 h. Repeated inhibition and high-level self-reactivation assays revealed that the BChE-polymer-oxime conjugates were excellent reactivators of OPNA-inhibited BChE. Recurring self-reactivation of BChE-polymer-oxime conjugates following repeated BChE inhibition by fluorogenic OPNAs (Flu-OPNAs) opens the door to developing the next generation of nerve agent "catalytic" bioscavengers.

Tuning Butyrylcholinesterase Inactivation and Reactivation by Polymer-Based Protein Engineering.

Organophosphate nerve agents rapidly inhibit cholinesterases thereby destroying the ability to sustain life. Strong nucleophiles, such as oximes, have been used as therapeutic reactivators of cholinesterase-organophosphate complexes, but suffer from short half-lives and limited efficacy across the broad spectrum of organophosphate nerve agents. Cholinesterases have been used as long-lived therapeutic bioscavengers for unreacted organophosphates with limited success because they react with organophosphate nerve agents with one-to-one stoichiometries. The chemical power of nucleophilic reactivators is coupled to long-lived bioscavengers by designing and synthesizing cholinesterase-polymer-oxime conjugates using atom transfer radical polymerization and azide-alkyne "click" chemistry. Detailed kinetic studies show that butyrylcholinesterase-polymer-oxime activity is dependent on the electrostatic properties of the polymers and the amount of oxime within the conjugate. The covalent coupling of oxime-containing polymers to the surface of butyrylcholinesterase slows the rate of inactivation of paraoxon, a model nerve agent. Furthermore, when the enzyme is covalently inhibited by paraoxon, the covalently attached oxime induced inter- and intramolecular reactivation. Intramolecular reactivation will open the door to the generation of a new class of nerve agent scavengers that couple the speed and selectivity of biology to the ruggedness and simplicity of synthetic chemicals.

Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening.

Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.

Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity.

The antidotal treatment of organophosphates (OP) nerve agents (NA) poisoning is based on anticholinergics (e.g. atropine) combined with oxime reactivators (e.g. 2PAM) of acetylcholinesterase (AChE). This treatment is symptomatic and does not degrade the OP. New small-molecule OP scavengers were developed as bifunctional hybrids. Their molecular design was based on combining a nucleophile that directly degrades OP with a moiety that reactivates OP-inhibited AChE. The OP degrading moiety is either benzhydroxamic acid (BHA) or 4-pyridinehydroxamic acid (4PHA) coupled via (CH2)n, (n = 1 or 3) to 2PAM. Three newly synthesized oxime-hydroxamate hybrids: 2PAMPr4PHA, 2PAMMeBHA and 2,4-DiPAMMeBHA were found to detoxify sarin, cyclosarin and soman in solution at 3-10-fold faster rate than 2PAM and to reactivate OP-AChE in vitro. 2PAMPr4PHA displayed 18-fold faster reactivation than 2-PAM of cyclosarin-inhibited HuAChE (kr = 3.6 × 102 vs. 0.2 × 102 M-1min-1, respectively, 37 °C). These hybrids inhibited AChE reversibly, IC50 = 16-48 µM, thereby decreasing the inhibition rates by OPs. The LD50 (im) of 2PAMPr4PHA, 2PAMMeBHA and 2,4DiPAMMeBHA are >568, 508 and >506 µmol/kg in rats and 144, 203 and >506 µmol/kg in guinea pigs. The rate of blood ChE recovery by the hybrids administered either pre- or post-exposure to 0.8xLD50 sarin was comparable or faster than 2PAM. Antidotal efficacy of 2PAMPr4PHA, 2PAMMeBHA and 2,4DiPAMMeBHA administered with atropine, as pre-treatment to sarin in rats (im), yielded protection ratios (PR) 11.6, 11.5 and 4.7, respectively, vs. 5.5 with 2PAM. Post-treatment against various OPs in rats and guinea-pigs yielded PRs higher or similar to that of 2 PAM. Our in vivo data indicates that some hybrids may serve as efficient small molecule scavengers for mitigating the toxicity of OP NAs.

Imidazole aldoximes effective in assisting butyrylcholinesterase catalysis of organophosphate detoxification.

Intoxication by organophosphate (OP) nerve agents and pesticides should be addressed by efficient, quickly deployable countermeasures such as antidotes reactivating acetylcholinesterase or scavenging the parent OP. We present here synthesis and initial in vitro characterization of 14 imidazole aldoximes and their structural refinement into three efficient reactivators of human butyrylcholinesterase (hBChE) inhibited covalently by nerve agent OPs, sarin, cyclosarin, VX, and the OP pesticide metabolite, paraoxon. Rapid reactivation of OP-hBChE conjugates by uncharged and nonprotonated tertiary imidazole aldoximes allows the design of a new OP countermeasure by conversion of hBChE from a stoichiometric to catalytic OP bioscavenger with the prospect of oral bioavailability and central nervous system penetration. The enhanced in vitro reactivation efficacy determined for tertiary imidazole aldoximes compared to that of their quaternary N-methyl imidazolium analogues is attributed to ion pairing of the cationic imidazolium with Asp 70, altering a reactive alignment of the aldoxime with the phosphorus in the OP-hBChE conjugate.

Mechanism of interaction of novel uncharged, centrally active reactivators with OP-hAChE conjugates.

A library of more than 200 novel uncharged oxime reactivators was used to select and refine lead reactivators of human acetylcholinesterase (hAChE) covalently conjugated with sarin, cyclosarin, VX, paraoxon and tabun. N-substituted 2-hydroxyiminoacetamido alkylamines were identified as best reactivators and reactivation kinetics of the lead oximes, RS41A and RS194B, were analyzed in detail. Compared to reference pyridinium reactivators, 2PAM and MMB4, molecular recognition of RS41A reflected in its Kox constant was compromised by an order of magnitude on average for different OP-hAChE conjugates, without significant differences in the first order maximal phosphorylation rate constant k(2). Systematic structural modifications of the RS41A lead resulted in several-fold improvement with reactivator, RS194B. Kinetic analysis indicated K(ox) reduction for RS194B as the main kinetic constant leading to efficient reactivation. Subtle structural modifications of RS194B were used to identify essential determinants for efficient reactivation. Computational molecular modeling of RS41A and RS194B interactions with VX inhibited hAChE, bound reversibly in Michaelis type complex and covalently in the pentacoordinate reaction intermediate suggests that the faster reactivation reaction is a consequence of a tighter RS194B interactions with hAChE peripheral site (PAS) residues, in particular with D74, resulting in lower interaction energies for formation of both the binding and reactivation states. Desirable in vitro reactivation properties of RS194B, when coupled with its in vivo pharmacokinetics and disposition in the body, reveal the potential of this oxime design as promising centrally and peripherally active antidotes for OP toxicity.

Catalytic detoxification of nerve agent and pesticide organophosphates by butyrylcholinesterase assisted with non-pyridinium oximes.

In the present paper we show a comprehensive in vitro, ex vivo and in vivo study on hydrolytic detoxification of nerve agent and pesticide OPs (organophosphates) catalysed by purified hBChE (human butyrylcholinesterase) in combination with novel non-pyridinium oxime reactivators. We identified TAB2OH (2-trimethylammonio-6-hydroxybenzaldehyde oxime) as an efficient reactivator of OP-hBChE conjugates formed by the nerve agents VX and cyclosarin, and the pesticide paraoxon. It was also functional in reactivation of sarin- and tabun-inhibited hBChE. A 3-5-fold enhancement of in vitro reactivation of VX-, cyclosarin- and paraoxon-inhibited hBChE was observed when compared with the commonly used N-methylpyridinium aldoxime reactivator, 2PAM (2-pyridinealdoxime methiodide). Kinetic analysis showed that the enhancement resulted from improved molecular recognition of corresponding OP-hBChE conjugates by TAB2OH. The unique features of TAB2OH stem from an exocyclic quaternary nitrogen and a hydroxy group, both ortho to an oxime group on a benzene ring. pH-dependences reveal participation of the hydroxy group (pKa=7.6) forming an additional ionizing nucleophile to potentiate the oxime (pKa=10) at physiological pH. The TAB2OH protective indices in therapy of sarin- and paraoxon-exposed mice were enhanced by 30-60% when they were treated with a combination of TAB2OH and sub-stoichiometric hBChE. The results of the present study establish that oxime-assisted catalysis is feasible for OP bioscavenging.

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

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

Oxime-assisted acetylcholinesterase catalytic scavengers of organophosphates that resist aging.

The cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase, are primary targets of organophosphates (OPs). Exposure to OPs can lead to serious cardiovascular complications, respiratory compromise, and death. Current therapy to combat OP poisoning involves an oxime reactivator (2-PAM, obidoxime, TMB4, or HI-6) combined with atropine and on occasion an anticonvulsant. Butyrylcholinesterase, administered in the plasma compartment as a bio-scavenger, has also shown efficacy but is limited by its strict stoichiometric scavenging, slow reactivation, and a propensity for aging. Here, we characterize 10 human (h) AChE mutants that, when coupled with an oxime, give rise to catalytic reactivation and aging resistance of the soman conjugate. With the most efficient human AChE mutant Y337A/F338A, we show enhanced reactivation rates for several OP-hAChE conjugates compared with wild-type hAChE when reactivated with HI-6 (1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4'-carbamoyl-1-pyridinium)). In addition, we interrogated an 840-member novel oxime library for reactivation of Y337A/F338A hAChE-OP conjugates to delineate the most efficient oxime-mutant enzyme pairs for catalytic bio-scavenging. Combining the increased accessibility of the Y337A mutation to oximes within the space-impacted active center gorge with the aging resistance of the F338A mutation provides increased substrate diversity in scavenging potential for aging-prone alkyl phosphate inhibitors.

New structural scaffolds for centrally acting oxime reactivators of phosphylated cholinesterases.

We describe here the synthesis and activity of a new series of oxime reactivators of cholinesterases (ChEs) that contain tertiary amine or imidazole protonatable functional groups. Equilibration between the neutral and protonated species at physiological pH enables the reactivators to cross the blood-brain barrier and distribute in the CNS aqueous space as dictated by interstitial and cellular pH values. Our structure-activity analysis of 134 novel compounds considers primarily imidazole aldoximes and N-substituted 2-hydroxyiminoacetamides. Reactivation capacities of novel oximes are rank ordered by their relative reactivation rate constants at 0.67 mm compared with 2-pyridinealdoxime methiodide for reactivation of four organophosphate (sarin, cyclosarin, VX, and paraoxon) conjugates of human acetylcholinesterase (hAChE). Rank order of the rates differs for reactivation of human butyrylcholinesterase (hBChE) conjugates. The 10 best reactivating oximes, predominantly hydroxyimino acetamide derivatives (for hAChE) and imidazole-containing aldoximes (for hBChE) also exhibited reasonable activity in the reactivation of tabun conjugates. Reactivation kinetics of the lead hydroxyimino acetamide reactivator of hAChE, when analyzed in terms of apparent affinity (1/K(ox)) and maximum reactivation rate (k(2)), is superior to the reference uncharged reactivators monoisonitrosoacetone and 2,3-butanedione monoxime and shows potential for further refinement. The disparate pH dependences for reactivation of ChE and the general base-catalyzed oximolysis of acetylthiocholine reveal that distinct reactivator ionization states are involved in the reactivation of ChE conjugates and in conferring nucleophilic reactivity of the oxime group.

Negative school perceptions and involvement in school bullying: a universal relationship across 40 countries.

Cross-national analyses explore the consistency of the relationship between negative school experiences and involvement in bullying across 40 European and North American countries, using the 2006 (40 countries n = 197,502) and 2002 (12 countries, n = 57,007) WHO-HBSC surveys. Measures include two Cumulative Negative School Perception (CNSP) scales, one based on 6 mandatory items (2006) and another including an additional 11 items (2002). Outcome measures included bullying perpetration, victimization and involvement as both bully and victim. Logistic regression analyses suggested that children with only 2-3 negative school perceptions, experience twice the relative odds of being involved in bullying as compared with children with no negative school perceptions. Odds Ratios (p < 0.001) increase in a graded fashion according to the CNSP, from about 2.2 to over 8.0. Similar consistent effects are found across gender and almost all countries. Further research should focus on the mechanisms and social context of these relationships.

Psychosocial outcomes related to subjective threat from armed conflict events (STACE): Findings from the Israeli-Palestinian cross-cultural HBSC study.

OBJECTIVE: This study investigates the relationship between exposure to armed conflict and terror events, and an array of mental and behavioral outcomes within a large cross-cultural scientifically representative sample of 24,935 Palestinian (7,430 West Bank and 7,217 Gaza) and Israeli (5,255 Jewish and 6,033 Arab) 11-, 13-, and 15-year-old school children. The children of the Middle East have been subjected to exposure from armed conflict and terrorism repeatedly with no adequate research or interventions aimed at shielding them from the hazards of such exposure to their mental and social well-being. METHOD: This paper studies the relationship between a newly developed scale (STACE) measuring levels of subjective perceptions of threat/fear due to exposure to armed conflict events and its predicting association with six psychosocial and behavioral outcomes covering (1) poor mental health, (2) positive well-being, and (3) risk behaviors. It also examines the role of parental support in "buffering" the effects of exposure to armed conflict events within the four target populations. RESULTS: Results showed that STACE has significant and strong effects on all six dependent variables representing (1) mental post trauma, (2) diminished well-being, and (3) elevated risk behaviors. STACE strongly affects all four populations with the greatest impact among the Jewish Israeli population and the least impact shown for the Arab Israeli youth. Parental support ("significant adult") has both a direct main effect on the outcomes of all six variables as well as a significant "buffering" effect on the impact of STACE on certain outcome variables (posttraumatic symptoms, life satisfaction, positive life perceptions, and tobacco use). CONCLUSIONS AND PRACTICE IMPLICATIONS: Regardless of the type of armed conflict events, the perception of threat and fear that a child experiences has a universal significant negative impact on mental, social, and behavioral well-being. The importance of the existence of a supporting significant adult in exposed children's lives is also emphasized. The findings show major implications for the development of community-based interventions focusing on enhancing parental, and other adult support in the lives of children living in armed conflict regions of the world.

Polyurethane-based leukocyte-inspired biocidal materials.

During the neutrophil respiratory burst myeloperoxidase uses hydrogen peroxide and chloride ion to generate hypochlorite which kills pathogens. Synthetic antimicrobial materials based on this chemistry are described herein. The oxidizing enzymes glucose oxidase (GOX) and horseradish peroxidase (HRP) catalyze two reactions in tandem using glucose, hydrogen peroxide and sodium halide (iodide or bromide). The final product of these two consecutive enzymatic reactions is either iodine or bromine. HRP, acting as haloperoxidase, utilizes the H(2)O(2) generated by GOX to oxidize halide ions into free halogens. Typically, 15 units/ml HRP and 25 units/ml GOX reacted with 0.8mm NaI and 5mm glucose to generate 5-7 ppm free iodine within 30 min. Medical grade polyurethane ChronoFlex AR (CF) was electrospun together with GOX and HRP. The electrospun fibers were collected as a uniform, water-insoluble, flexible elastomeric matrix with an average fiber diameter of 1+/-0.2 microm. Biocidal activity of CF/enzyme fibers resulted in >6-log unit reduction of both Escherichia coli and Staphylococcus aureus challenges. A time-course of biocidal activity displayed a 3-4 log reduction of E. coli and S. aureus within the first 5 min and complete kill (>6 logs) within 15 min. A dose-response study of fiber weight (0.5-30 mg/ml) exhibited complete kill of E. coli (>6 logs) and at least 99.99% S. aureus kill (>4 logs) with as little as 1mg fiber. The fibers were reusable with slightly less activity on the second use and significant activity after continuous soaking in buffer for up to 7 days. Electrospun CF/GOX/HRP fibers adhered to a thin film with embedded NaI and glucose caused a complete kill of E. coli (>7-log units) and MRSA (6-log unit reduction) within 1h at 37 degrees C.

Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase.

We addressed the ability of various organophosphorus (OP) hydrolases to catalytically scavenge toxic OP nerve agents. Mammalian paraoxonase (PON1) was found to be more active than Pseudomonas diminuta OP hydrolase (OPH) and squid O,O-di-isopropyl fluorophosphatase (DFPase) in detoxifying cyclosarin (O-cyclohexyl methylphosphonofluoridate) and soman (O-pinacolyl methylphosphonofluoridate). Subsequently, nine directly evolved PON1 variants, selected for increased hydrolytic rates with a fluorogenic diethylphosphate ester, were tested for detoxification of cyclosarin, soman, O-isopropyl-O-(p-nitrophenyl) methyl phosphonate (IMP-pNP), DFP, and chlorpyrifos-oxon (ChPo). Detoxification rates were determined by temporal acetylcholinesterase inhibition by residual nonhydrolyzed OP. As stereoisomers of cyclosarin and soman differ significantly in their acetylcholinesterase-inhibiting potency, we actually measured the hydrolysis of the more toxic stereoisomers. Cyclosarin detoxification was approximately 10-fold faster with PON1 mutants V346A and L69V. V346A also exhibited fourfold and sevenfold faster hydrolysis of DFP and ChPo, respectively, compared with wild-type, and ninefold higher activity towards soman. L69V exhibited 100-fold faster hydrolysis of DFP than the wild-type. The active-site mutant H115W exhibited 270-380-fold enhancement toward hydrolysis of the P-S bond in parathiol, a phosphorothiolate analog of parathion. This study identifies three key positions in PON1 that affect OP hydrolysis, Leu69, Val346 and His115, and several amino-acid replacements that significantly enhance the hydrolysis of toxic OPs. GC/pulsed flame photometer detector analysis, compared with assay of residual acetylcholinesterase inhibition, displayed stereoselective hydrolysis of cyclosarin, soman, and IMP-pNP, indicating that PON1 is less active toward the more toxic optical isomers.

Development of the bisquaternary oxime HI-6 toward clinical use in the treatment of organophosphate nerve agent poisoning.

The traditional therapeutic treatment of organophosphate cholinesterase inhibitor (nerve agents) poisoning consists of co-treatment with an antimuscarinic (atropine) and a reactivator of inhibited acetylcholinesterase (AChE), which contains a nucleophilic oxime function. Two oximes are presently widely available for clinical use, pralidoxime and obidoxime (toxogonin), but both offer little protection against important nerve agent threats. This has highlighted the real need for the development and availability of more effective oximes for human use, a search that has been going on for up to 30 years. However, despite the demonstration of more effective and safe oximes in animal experiments, no additional oximes have been licensed for human use. HI-6, (1-[[[4(aminocarbonyl)-pyridinio]methoxy]methyl]-2(hydroxyimino)pyridinium dichloride; CAS 34433-31-3) has been studied intensively and has been proved effective in a variety of species including non-human primates and appears from clinical experience to be safe in humans. These studies have led to the fielding of HI-6 for use against nerve agents by the militaries of the Czech republic, Sweden, Canada and under certain circumstances the Organisation for the Prohibition of Chemical Weapons. Nevertheless HI-6 has not been granted a license for clinical use, must be used only under restricted guidelines and is not available for civilian use as far as is known. This article will highlight those factors relating to HI-6 that pertain to the licensing of new compounds of this type, including the mechanism of action, the clinical and pre-clinical demonstration of safety and its efficacy against a variety of nerve agents particularly in non-human primates, since no relevant human population exists. This article also contains important data on the use of HI-6 in baboons, which has not been available previously. The article also discusses the possibility of successful therapy with HI-6 against poisoning in humans relative to doses used in non-human primates and relative to its ability to reactivate inhibited human AChE.