The Journal of Toxicological Sciences (J

The Journal of Toxicological Sciences (J. Toxicol. Sci.) 511 Vol.41, No.4, 511-521, 2016

Original Article Toxicity and median effective doses of oxime therapies against percutaneous organophosphorus pesticide and nerve agent challenges in the Hartley guinea pig

Thomas H. Snider1, Michael C. Babin1, David A. Jett2, Gennady E. Platoff Jr.3 and David T. Yeung2

1Battelle, 505 King Avenue, JM-7, Columbus, Ohio 43201-2693, USA 2National Institutes of Health/National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892, USA 3National Institutes of Health/National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, USA

(Received March 4, 2016; Accepted April 22, 2016)

ABSTRACT — Anticholinesterases, such as organophosphorus pesticides and warfare nerve agents, present a significant health threat. Onset of symptoms after exposure can be rapid, requiring quick-acting, efficacious therapy to mitigate the effects. The goal of the current study was to identify the safest anti-

dote with the highest therapeutic index (TI = oxime 24-hr LD50/oxime ED50) from a panel of four oximes deemed most efficacious in a previous study. The oximes tested were pralidoxime chloride (2-PAM Cl),

MMB4 DMS, HLö-7 DMS, and obidoxime Cl2. The 24-hr median lethal dose (LD50) for the four by intra-

muscular (IM) injection and the median effective dose (ED50) were determined. In the ED50 study, male

guinea pigs clipped of hair received 2x LD50 topical challenges of undiluted Russian VX (VR), VX, or phorate oxon (PHO) and, at the onset of cholinergic signs, IM therapy of atropine (0.4 mg/kg) and vary- ing levels of oxime. Survival was assessed at 3 hr after onset clinical signs. The 3-hr 90th percentile dose

(ED90) for each oxime was compared to the guinea pig pre-hospital human-equivalent dose of 2-PAM Cl, 149 μmol/kg. The TI was calculated for each OP/oxime combination. Against VR, MMB4 DMS had a

higher TI than HLö-7 DMS, whereas 2-PAM Cl and obidoxime Cl2 were ineffective. Against VX, MMB4

DMS > HLö-7 DMS > 2-PAM Cl > obidoxime Cl2. Against PHO, all performed better than 2-PAM Cl.

MMB4 DMS was the most effective oxime as it was the only oxime with ED90 < 149 μmol/kg against all three topical OPs tested.

Key words: Nerve agent, Pesticide, Oxime, Therapy, ED50, Therapeutic index

INTRODUCTION es of brain tissue and peripheral nerves. If this increase is uncontrolled, clinical symptoms of intoxication such Organophosphorus compounds (OPs) are among as miosis, fasciculations, respiratory distress, seizures, the most toxic substances known to man (Dacre, 1984; convulsions, increased secretions, and death can quick- Taylor, 2001; Ballantyne and Marrs, 1992). Original- ly occur (Taylor, 2001; Bajgar, 2004, 2005). Although the ly developed as insecticides for agricultural pest control mechanism of toxicosis via cholinolergic hyperstimula- (Jansen, 2000), their extreme toxicity toward higher ver- tion is the same between OP insecticides/pesticides and tebrates quickly led to their adoption as potential weap- OP CWNAs, the relative potencies can be radically dif- ons of mass destruction, i.e., chemical warfare nerve ferent. In general, CWNAs such as sarin, soman, cyclosa- agents or CWNAs (Szinicz, 2005; Holstege et al., 1997; rin, VX and Russian VX (VR), have been shown to exhib- Loder, 2000; Ede, 2011). The primary mechanism of OP it greater toxicity than bioactivated OP pesticides such as toxicosis is anti-cholinesterase (anti-ChE) activity via paraoxon, chlorpyrifos-oxon and phorate oxon or PHO irreversible inhibition of ChE enzymes, especially ace- (Balali-Mood and Shariat, 1998; Wilhelm et al., 2014; tylcholinesterase (AChE). Inhibition of AChE results Snider et al., 2015; Misik et al., 2015). in increased acetylcholine (ACh) levels in the synaps- While OPs were never believed to have been deployed Correspondence: Thomas H. Snider (E-mail: [email protected])

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T.H. Snider et al. during the Second World War, recent nefarious use of ical countermeasures, it is important to note that these are these compounds against both civilian and military tar- approved for use only in emergency and pre-hospital set- gets have been documented, e.g., CWNAs have been tings. As such, no more than three injections should be deployed by Aum Shinrikyo in Tokyo (Vale, 2005), dur- administered without definitive supportive care, e.g., ven- ing the Iraq-Iran War (Haines and Fox, 2014), and tilator assistance. more recently during Syrian Civil War (Dolgin, 2013; Although 2-PAM Cl is efficacious to some extent Rosman et al., 2014). Additionally, the general availabil- against sarin and VX (Dawson, 1994), efforts to replace it ity of OP pesticides has also become more widespread with a broader-spectrum AChE reactivator have neverthe- due to higher global production, sales, and usages, espe- less been ongoing for decades. In a previously published cially in developing countries (Food and Agriculture work, we described an experimental model using topical

Organization of the United Nations Statistics website; OP challenges at the LD85 level on clipped guinea pigs Allied Markets Research, World Insecticides Market- to assess the relative efficacies of eight candidate oxi- Opportunities and Forecasts, 2013-2020 website). Con- mes, including 2-PAM Cl, in terms of 24-hr survivabili- sequently, this increase in the worldwide prevalence of ty and mitigation of blood cholinesterase (ChE) inhibition OP pesticides also raises overall poisoning risk to public (Snider et al., 2015). The objectives of the work presented health. Unintentional human exposure can occur if OPs herein were to determine the median effective dose (ED50) are released from transportation and storage facilities dur- for the four most efficacious oximes down-selected from ing industrial accidents, natural disasters, or inadvertent- our prior screen against each of three topical threat OPs: ly during occupational applications. Meanwhile, deliber- VR, VX, or PHO. The ED50 was determined from pro- ate exposure via release of OPs into ambient air and/or bit analysis of oxime dose/survival data. The therapeu- food and water supplies for the purpose of inducing mass tic index (TI = oxime 24-hr LD50/oxime ED50) was cal- terror and panic is also possible. As such, intoxication by culated for each OP/oxime combination. This required a either OP pesticides or CWNAs is now a very realistic 24-hr LD50 value in the guinea pig, and therefore, necessi- threat to both the military and more importantly, civilian tated oxime toxicity studies as the data were absent from populations today (National Institutes of Health Strategic the published literature. Additionally, the 3-hr 90th per-

Plan and Research Agenda for Medical Countermeasures centile dose (ED90) against a dermal 2x LD50 of the OPs Against Chemical Threats website; Katzman, 2000). was determined for each oxime and contrasted with the Based on the particular OP, route, duration, and level molar equivalent of 2-PAM Cl contained in three Duo- of exposure, the onset of clinical symptoms can be quite Dote® injectors (for a 70-kg person), 149 μmol/kg. The rapid, requiring a quick-acting, efficacious therapeutic general intent of this last aim of the study was to evaluate regimen to mitigate the pharmacologic effects (Eddleston the efficacy of these oximes within a 3-hr window direct- et al., 2008; Newmark, 2007). In the United States, the ly after treatment, where the therapy was administered primary components of the FDA-approved and current- only upon presentation of clinical effects. The goal of this ly fielded medical countermeasures to counter OP poi- approach was to simulate a mass casualty response where soning are a competitive muscarinic receptor antagonist emergency personnel may only be able to treat sympto- (atropine) to mitigate the excitotoxic effect at post-synap- matic patients, but evacuation to a fully equipped medical tic targets (Shutt and Bowes, 1979), and an AChE oxime facility would not be immediately possible. reactivator, pralidoxime chloride (2-PAM Cl), to augment OP dissociation from and reactivation of inhibited AChE MATERIALS AND METHODS (US FDA, 2006). The combination of these two therapeu- tic strategies aims to reduce the hyperstimulation of par- Identities of the OP challenge materials, atropine, and asympathetic nerves that results from the over-accumula- oximes are listed in Table 1, and chemical structures tion of ACh (Harris and Stitcher, 1983; Eyer, 2003). The are presented (Fig. 1). Challenge materials included VR US government currently fields these two drugs within a and VX, procured from the Edgewood Chemical and single two-chambered, self-propelled syringe via a com- Biological Center, Aberdeen Proving Ground, MD, mon needle intramuscular (IM) injection delivery sys- USA) and PHO (custom synthesized at Battelle, Colum- tem, the Antidote Treatment - Nerve Agent Autoinjec- bus, OH, USA). Atropine free base at 1.64 mg/mL in a tor (ATNAA; https://www.meridianmeds.com/products/ citrate buffered saline solution (batch RP-526-1) and atnaa) for military use only and the DuoDote® Autoinjec- 2-PAM Cl (batch number RP-526-2) both from King tor (https://www.meridianmeds.com/products/duodote) Pharmaceuticals, St Louis, MO, USA, and 1,1-methylene for civilian application. Per the drug labels for both med- bis(4(hydroxyimino-methyl)pyridinium) dimethanesul-

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Table 1. Identification of challenge materials and therapies.

Challenge material or therapy CAS RN† Synonym(s)

Russian VX (VR) 159939-87-4 S-[2-(diethylamino)ethyl] O-isobutyl methylphosphonothioate

VX 50782-69-9 S-[2-(diisopropylamino)ethyl] O-ethyl methylphosphonothioate phorate oxon (PHO) 2600-69-3 O,O-diethyl S-[(ethylsulfanyl)methyl] phosphorothioate atropine free base 51-55-8 (3-endo)-8-Methyl-8-azabicyclo[3.2.1]oct-3-yl tropate 2-hydroxyiminomethyl-1-methylpyridinium chloride 2-PAM Cl 51-15-0 in aqueous benzyl alcohol (20 mg/mL), pH 2.6 1-(((4-(aminocarbonyl) pyridinio)methoxy)methyl)-2,4- bis((hydroxyimino) HLö-7 DMS 120103-35-7 methyl)pyridinium dimethanesulfonate MMB-4 DMS - 1,1-methylene bis[4(hydroxyimino- methyl)pyridinium) dimethanesulfonate

Toxogonin®, or oxo-[[1-[[4-(oxoazaniumylmethylidene)pyridin-1-yl] Obidoxime Cl 114-90-9 2 methoxymethyl]pyridin-4-ylidene]methyl]azanium dichloride † Chemical Abstracts Service Registration Number - none given fonate (MMB4 DMS; lot number 1004) were government and the observations, including death, were recorded at furnished (Medical Countermeasure Systems Joint Project 1, 3, 6, and 24 hr. Probit dose-response models were fit- Management Office, Frederick, MD, USA). oxo-[[1-[[4- ted to lethality data using the method of maximum like- (oxoazaniumylmethylidene)pyridin-1-yl]methoxymethyl] lihood (Finney, 1971). Estimated parameters of probit pyridin-4-ylidene]methyl]azanium dichloride (Obidoxi- dose-response models were used to compute each LD01 me Cl2) was purchased from Sigma Aldrich (Lot Number and LD50. Fieller’s method or the delta method (Oehlert, 1446981V, St Louis, MO, USA). 1-(((4-(aminocarbonyl) 1992) was used to compute a 95 percent confidence limit pyridinio)methoxy)methyl)-2,4-bis((hydroxyimino) for each LD50. If experimental results were such that these methyl)pyridinium dimethanesulfonate (HLö-7 DMS) methods could not be used to calculate a confidence limit, was custom synthesized by Southwest Research Institute alternative methods such as the Spearman-Karber method (San Antonio, TX, USA). All oximes were formulated for (Spearman, 1908; Kärber, 1931) or the binomial method IM injection in normal (0.9%, w/v) saline. (Clopper and Pearson, 1934) were considered. STATA® A total of 769 specific pathogen-free male Dunkin- 11.0 (StataCorp, College Station, TX, USA) was used to Hartley guinea pigs (approximately 4 weeks old, mean analyze the lethality data. Test days of eight guinea pigs body weight = 346 g) were obtained from three Charles per oxime were repeated until statistical requirements for River facilities (Raleigh, NC, USA; Stone Ridge, NY, a valid probit analysis were met, that is, a probit slope USA; Saint Constant, QC, Canada). Guinea pigs were significantly (p < 0.05, one-sided) greater than zero. identified with ear tags and pair housed before challenge On the day prior to a topical challenge in the ED50 in polycarbonate cages labeled with cage cards. During study, 34 designated guinea pigs were anesthetized with 7-day quarantine, the guinea pigs were weighed and rand- a combination of 70 mg/kg ketamine and 10 mg/kg xyla- omized by body weight into test days, each comprised of zine, and clipped of hair on both thighs and on the dor- multiple treatment groups depending on the study design. sum between the scapulae. Each test day consisted of five In the oxime toxicity studies, the oximes, adminis- groups, an atropine-only, no-oxime control group (n = tered into the high limbs by IM injection, varied in injec- 2) and four groups (n = 8 each) per oxime within which tion volume between 0.88 and 3.00 mL/kg. Two or three was administered a fixed level of atropine and a vary- guinea pigs were used per group among the four groups ing level of the oxime designated for that group (Table included per oxime and test day (Table 2). Multiple injec- 3). On the day of challenge, a rubber “O” ring, 12-mm tion sites were used, when needed, to keep the injection ID/20-mm OD, was affixed around each dorsal test site volumes to less than 0.3 mL. Survivors were observed using cyanoacrylate adhesive. OP applications were

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Fig. 1. Chemical structures of challenge materials and therapeutics. placed in the center of the “O” ring using a 0.5-μL (VR able. and VX) or 100-μL (PHO) syringe (Hamilton, Reno, NV, The dermal OP dose employed in the experimental par-

USA). Percutaneous applications were semi-occluded by adigm was standardized at 2x LD50 under the assumption affixing a 20-mm disk of Durapore® (Millipore, Billeri- that in a real world scenario, any higher exposure lev- ca, MA, USA, Catalogue Number HVLP2932A) 0.45-μm el would be immediately lethal and therefore, rescue by filter paper cover onto each “O” ring using cyanoacrylate emergency responders will not be possible. Topical appli- adhesive. The cover did not impede evaporation of chal- cations of 2x LD50 OP challenges used in the ED50 study, lenge material but prevented disturbance of the test site by previously determined in similarly atropinized guin- the guinea pig. After receiving the challenge dose, guin- ea pigs, were VR at 0.365 mg/kg, VX at 0.240 mg/kg, ea pigs were singly housed in disposable plastic shoebox and PHO at 196 mg/kg. Therapy, administered at the cages in the dosing hood with food and water made avail- onset of clinical evidence of toxicosis, consisted of atro-

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ED50s for oxime therapies against organophosphates pine free base, administered at 0.4 mg/kg by IM injec- uous monitoring. Atropine in the DuoDote® autoinjector is tion in the right thigh, and a variable, non-toxic dose sufficient for reducing painful ciliary muscle spasm, mit-

(<¼ LD50) of oxime/saline solution in the left thigh. Atro- igating muscarinic hyperstimulation of secretory organs, pine was administered at the recommended human equiv- and correcting bradycardia from cholinergic poisonings. alent dose (HED) for a 70-kg person receiving three Duo- Survival was assessed at 3 hr after therapy. Dote® autoinjectors each containing 2.1 mg of atropine free base. The HED was calculated by multiplying the RESULTS human dose, (3 x 2.1 mg)/70 kg, by 4.6, the ratio of guin- ea pig body surface area/kg body mass to that for human Oxime Toxicity Studies: A summary of oxime toxici- beings (CDER, US FDA, 2002). An atropinization level ties is presented in Table 4. A reference for obidoxime Cl2 of 0.4 mg/kg in the guinea pigs was equivalent to what indicated a relatively low LD50 = 78.9 mg/kg by the IM a human victim of OP poisoning would be expected to route in guinea pigs but lacked other details (Fleisher et receive in a pre-hospital setting without extended support- al., 1970). In our laboratory, the LD50 (and LD01, 95% fidu- ive care, i.e., no more than three administrations of the cial limits, probit slope) determined were 179 (142, 157- ATNAA or DuoDote® IM autoinjectors. Sustained atropi- 191 mg/kg, 23) for 2-PAM Cl, 314 (277, 300-354 mg/kg, nization is expected only in a hospital setting with contin- 43) for HLö-7 DMS, and 679 (528, 635-944 mg/kg, 21) for MMB4 DMS. The toxicity data indicated that HLö-7 DMS, and especially MMB4 DMS, were safer (had high- Table 2. Oxime toxicity study design, treatment groups er LD s) than 2-PAM Cl, and all probit slopes exceeded on each test day. 50 20. Obidoxime Cl2 was less safe than 2-PAM Cl, and is a Intramuscular injection in hind leg(s) good example of why the TI is important to know. Target Group n Challenge Target ED Study: The geometric mean and range of time rel- concentration 50 material dose (mg/mL) ative to topical challenge for when signs began, and there- 12 fore, when treatment was given, are presented (Fig. 2). 232-PAM Cl/ The geometric mean (and range) onset time was 0.65 (0.2 600 Varied 33normal saline - 3.4) hr for VR, 1.3 (0.2 - 8.3) hr for VX, and 3.3 (0.3 - 42 7.6) hr for PHO. A summary of oxime efficacy parame- 52 ters is presented in Table 5. 63HLö-7 DMS/ VR-challenged guinea pigs that received an admin- 600 Varied 73normal saline istration of atropine and saline exhibited tremors, atax- 82 ia/lethargy, salivation, lacrimation, dyspnea, and death 92 (6/6 = 100%) with a geometric mean (range) in time to 10 3 MMB4 DMS / 400 Varied death after challenge of 1.5 (0.5-7.9) hr. 2-PAM Cl in the 11 3 normal saline 4-45 mg/kg range occasionally provided 3-hr survival 12 2 (Fig. 3, upper left). However, the probit regression was not

Table 3. ED50 iterative study design for each OP, treatment groups on each test day. Number of Nominal n per Oxime Number of Atropine free Oxime Group oxime therapy Oxime dose challenge dose therapy level animals needed base (right thigh) (left thigh) levels vehicle at 1 212 - 0.257 mL/kg 2 2 4 8 MMB4 DMS Varied

32x LD50 2 4 80.4 mg/kg HLö-7 DMS Varied

4 2 4 8 obidoxime Cl2 Varied

5 2 4 8 2-PAM Cl Varied

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Table 4. Summary of toxicities of four oximes by intramuscular injection in non-sedated guinea pigs. 24-hr Toxicity (mg/kg) Oxime n Probit slope 1 2 3 4 LD01 LFL LD50 UFL 2-PAM Cl 40 142 157 179 191 23

HLö-7 DMS 30 277 300 314 354 43

MMB4 DMS 26 528 635 679 944 21 79 Obidoxime Cl -- - -- 2 (Fleisher, 1970) 1 Oxime dose predicted to be lethal to 1% of animals challenged. 2 Lower 95% fiducial limit. 3 Oxime dose predicted to be lethal to 50% of animals challenged. 4 Upper 95% fiducial limit. - Not given.

Table 5. Median effective dose, 90th percentile effective dose, and therapeutic index for four oximes against topical challenges of VR, VX, and phorate oxon in atropinized guinea pigs.

Organophosphate Probit regression ED50 ED90 Oxime challenge at 2 x 24-hr significance1 Therapeutic (mg/kg) (μmol/kg) (mg/kg) (μmol/kg) 2 LD50 in atropinized index guinea pigs Equimolar to three DuoDotes®: 149 149 2-PAM Cl 0.4803 677 3922 9.9E+05 5.7E+06 0.3 VR, HLö-7 DMS 0.0344 37 72 86 165 8.4 0.365 mg/kg MMB4 DMS 0.0042 7.7 17 58 129 89

Obidoxime Cl2 0.9783 ----- 2-PAM Cl 0.0188 14 81 185 1072 13 VX, HLö-7 DMS 0.0854 14 27 223 428 22 0.240 mg/kg MMB4 DMS 0.0049 4.8 11 26 58 142

Obidoxime Cl2 0.0594 14 40 43 120 5.5 2-PAM Cl 0.0015 5.7 33 29 168 31 PHO, HLö-7 DMS 0.0252 2.9 5.5 6.2 12 109 196 mg/kg MMB4 DMS <0.0001 6.1 14 29 65 111

Obidoxime Cl2 0.0092 0.8 2.3 5.4 15 97 1 Regressions of p ≤ 0.05 are given in bold font. 2 The therapeutic index is the ratio of the LD50 to the ED50 for a given oxime. Thus, a larger TI indicates a wider window of safety,

whereas a smaller ED50 indicates that less drug is needed to protect half of the individuals challenged at a fixed level. significant (p = 0.4803) and indicated overall that 2-PAM VX-challenged guinea pigs that received an admin- Cl was not effective against VR. HLö-7 DMS offered sig- istration of atropine and saline exhibited ataxia/lethar- nificant 3-hr therapy (p = 0.0344) with ED50 = 37 mg/kg, gy, salivation, tremors, dyspnea, prostration, lacrimation,

ED90 = 86 mg/kg (165 μmol/kg), and TI = 8.4 with no fasciculation, and death (8/8 = 100%) with a geometric deaths associated with therapy levels exceeding 60 mg/ mean (range) in time to death after challenge of 3.6 (2.1- kg (Fig. 3, upper right). MMB4 DMS offered signifi- 4.7) hr. 2-PAM Cl provided therapy-related 3-hr survival cant 3-hr therapy (p = 0.0042) with ED50 = 7.7 mg/kg, (Fig. 4, upper left), and the probit regression was signifi-

ED90 = 58 mg/kg (129 μmol/kg), and TI = 89, with no cant (p = 0.0188) with ED50 = 14 mg/kg, ED90 = 185 mg/ deaths by 3 hr associated with therapy levels exceeding kg (1,072 μmol/kg), and TI = 13. HLö-7 DMS offered

45 mg/kg (Fig. 3, lower left). Obidoxime Cl2 offered no occasional 3-hr therapy, but the probit regression was significant 3-hr therapy against VR (Fig. 3, lower right). not significant (p = 0.0854; Fig. 4, upper right). MMB4

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DMS offered significant 3-hr therapy (p = 0.0049) with

ED50 = 4.8 mg/kg, ED90 = 26 mg/kg (58 μmol/kg), and TI = 142 (Fig. 4, lower left), with no deaths by 3 hr asso- ciated with therapy levels exceeding 90 mg/kg. The probit

regression for obidoxime Cl2 was equivocally significant (p = 0.0594) and offered occasional 3-hr therapy against VX (Fig. 4, lower right). PHO-challenged guinea pigs that received an admin- istration of atropine and saline exhibited fasciculation, tremors, salivation, lacrimation, prostration, and death (6/6 = 100%) with a geometric mean (range) in time to death after challenge of 5.5 (4.0-7.4) hr. 2-PAM Cl pro- vided therapy-related 3-hr survival (Fig. 5, upper left), and the probit regression was significant (p = 0.0015)

with ED50 = 5.7 mg/kg , ED90 = 29 mg/kg (168 μmol/kg), and TI = 31. HLö-7 DMS offered 3-hr therapy, signifi- Fig. 2. Onset times of clinical signs and initiating a single therapy treatment. cant (p = 0.0252) probit regression, with ED50 = 2.9 mg/

Fig. 3. Dose-survivability response for 2 x LD50 VR administered topically to awake, clipped guinea pigs treated at first sign with 0.4 mg/kg atropine free base and varying doses of 2-PAM Cl, HLö-7 DMS, MMB4 DMS, or obidoxime Cl2 in treatment groups of two per symbol; survival at 3 hr after treatment.

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Fig. 4. Dose-survivability response for 2 x LD50 VX administered topically to awake, clipped guinea pigs treated at first sign with 0.4 mg/kg atropine free base and varying doses of 2-PAM Cl, HLö-7 DMS, MMB4 DMS, or obidoxime Cl2 in treatment groups of two per symbol; survival at 3 hr after treatment.

kg, ED90 = 6.2 mg/kg (12 μmol/kg), and TI = 109 (Fig. 5, on-site therapy administration until a patient (a victim of upper right). MMB4 DMS offered significant 3-hr thera- a skin exposure incident, whether accidental or of mali- py (p < 0.0001) with ED50 = 6.1 mg/kg, ED90 = 29 mg/kg cious intent) could be evacuated from the scene of expo- (65 μmol/kg), and TI = 111 (Fig. 5, lower left), with no sure and hospitalized, which might then allow for con- deaths by 3 hr associated with therapy levels exceeding tinuous intravenous administration of atropine/oxime

50 mg/kg. The probit regression for obidoxime Cl2 was therapy, perhaps for days, until cholinergic signs abated. significant (p = 0.0092), as that oxime provided the most Thus, this paradigm provided an endpoint for measuring efficient 3-hr therapy against PHO, with ED50 = 0.8 mg/kg, oxime efficacy at the point in time when a patient might

ED90 = 5.4 mg/kg (15 μmol/kg), and TI = 97 (Fig. 5, low- be entering a hospital. Against 2x LD50 topical VR, the er right). MMB4 DMS was the most efficient on both a mass and molar basis to offer both 50% and 90% survivals at 3 hr DISCUSSION after therapy, followed by HLö-7 DMS. The therapeutic

index is the ratio of the LD50 to the ED50 for a given drug. Lethality in topically challenged guinea pigs was Thus, a larger TI indicates a wider window of safety, assessed at 3 hr after a single, immediate administration whereas a smaller ED50 indicates that less drug is need- of atropine/oxime therapy was given at the onset of clin- ed to protect half of the individuals challenged at a fixed ical signs. This therapy paradigm was meant to simulate level. MMB4 DMS was especially impressive in terms of

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Fig. 5. Dose-survivability response for 2 x LD50 PHO administered topically to awake, clipped guinea pigs treated at first sign with 0.4 mg/kg atropine free base and varying doses of 2-PAM Cl, HLö-7 DMS, MMB4 DMS, or obidoxime Cl2 in treatment groups of two per symbol; survival at 3 hr after treatment.

safety, as TI = 89 against 2x LD50 VR. 2-PAM Cl and obi- with the rim of the AChE gorge while the other end can doxime Cl2 were ineffective against VR. Also against 2x extend into the gorge (Harel et al., 1993). Such dual bind-

LD50 topical VX, MMB4 DMS was the most efficient and ing may help to shield AChE from OP attack, as well as safest (TI = 142). A dose of 2-PAM Cl equivalent to three outcompete and/or dislodge OP molecules interacting at ® DuoDotes (25.7 mg/kg) used against 2x LD50 topical VX sites within the gorge. Reactivation of AChE by an oxi- would approximate the ED65. HLö-7 DMS and obidoxi- me depends on (1) molecular spacing, as oximes tend to me Cl2 were equivocally effective. Against 2x LD50 topi- protect better against smaller alkyl substituents across cal PHO, all four oximes offered significant 3-hr survival structurally similar OPs, (2) relative absence of ster- therapy, with MMB4 DMS, HLö-7 DMS, and obidoxi- ic hindrance presented by an AChE-OP conjugate to an me Cl2 being more efficient on a TI basis than 2-PAM Cl. approaching oxime molecule, and (3) oxime nucleophilic-

A 25.7 mg/kg dose of 2-PAM Cl against 2x LD50 topical ity. As a possible example of the latter, the presence of

PHO would approximate the ED88. an isonicotinamide group on an oxime such as HLö-7 Figure 1 shows that MMB4, HLö-7, and obidoxime has been associated with enhanced efficacy against nerve are bis-quaternary oximes, whereas 2-PAM is mono-func- agents (Maxwell et al., 2008), but the exact mechanism tional. As discussed in Snider (2015), the bis nature of the for this remains unknown to the community. larger molecules gives them an advantage over 2-PAM in Although the guinea pig efficacy data presented here protecting AChE from OP attack, as one end can interact can only serve to be an illustrative prediction for humans,

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T.H. Snider et al. they present a valuable contribution demonstrating the and Human Services Biomedical Advance Research and potential benefits of several oxime therapy options over Development Authority (HHS BARDA); Washington what is currently approved and fielded in the USA For DC, USA), John H. McDonough and Irvin Koplovitz (US example, the 3-hr ED90 results support the concept that Army Medical Research Institute of Chemical Defense; medical intervention can be safely delayed until the onset DoD USAMRICD; Aberdeen Proving Ground, Maryland, of clinical symptoms of poisoning to avoid unnecessary USA) for their expertise and guidance in the design of medical countermeasure treatments and even in that sce- this study. nario, there may still be enough time to properly decon- This work was supported by the NIH Office of the taminate and evacuate the patient to a medical care facil- Director through an interagency agreement (OD#: Y1- ity. Additionally, if immediate evacuation is not feasible, OD-0387-01) between the National Institute of Aller- lethality may still be preventable if lower (and presum- gy and Infectious Diseases (NIAID) and Department of ably even safer) dose of the therapy, particularly with Defense (DoD) and prepared under the auspices of the MMB4-DMS, can somehow be sustained in the field. NIH, NIAID, NINDS, and the DoD Defense Technical Collating across the topical OP threats tested, the Information Center (DTIC) under the Chemical, Biolog-

ED90 for MMB4 DMS was ≤ 129 μmol/kg while offer- ical, Radiological & Nuclear Defense Information Analy- ing the largest margin of safety relative to the other oxi- sis Center (CBRNIAC) program, Contract No. SP0700-00 mes tested (TI ≥ 89). Interestingly, while this same oxime -D-3180, Delivery Order Number 0687, CBRNIAC Task completely prevented lethality against both percutaneous- 832/CB-10-0832 and SP0700-00-D-3180, Delivery Order ly (Snider et al., 2015) or subcutaneously (Wilhelm et al., Number 0794, CBRNIAC Task 689/CB-13-0689. 2014) administered VX, it did not significantly promote overall survival against PHO in either previous reports. Conflict of interest---- The authors declare that there Efficacy in those studies was evaluated at 24-hr after a is no conflict of interest. The views expressed in this arti-

LD85 challenge followed by a single administration of cle are those of the authors and do not reflect the official therapy that was given either 1 min or at onset of clinical policy of the NIH, HHS, DoD, or the U.S. Government. toxic signs. Based on the efficacy observed here (against No official support or endorsement of this article by the a higher level of PHO), it is possible that sustained oxime NIAID, NINDS, or NIH is intended or should be inferred. therapy with MMB4 DMS - even at levels below what The experimental protocol was approved by the Institu- was used in those prior studies –may offer more protec- tional Animal Care and Use Committee at Battelle. All tion than those previously reported 24-hr lethality results. procedures were conducted in accordance with the prin- A low level therapy could, in theory, be sustained by sev- ciples stated in the Guide for the Care and Use of Labo- eral means, e.g., repeated injections, or reformulation ratory Animals and the Animal Welfare Act of 1966 (P.L. for slower release after a bolus administration. In sum- 89-544), as amended. The sponsor developed the concept mary, the oxime that offered the most consistent protec- of the study, contributed to its design, and the interpre- tion in the 3-hr survival paradigm against the spectrum tation of the data as well as the preparation of the man- of 2x LD50 OPs evaluated here -VX, VR, and PHO - was uscript and the decision to submit it for publication. The MMB4 DMS. sponsor also made similar contributions to other studies occurring at Battelle during the same time frame. ACKNOWLEDGMENTS REFERENCES The authors wish to recognize the excellent tech- nical assistance of Jennifer Webb, Ashley Robertson, Allied Markets Research, World Insecticides Market - Opportunities Richard Morosco, Beth Reed, Kevin McGarry, Ernest and Forecasts, 2013 - 2020 website, https://www.alliedmarketre- search.com/insecticides-market (Accessed 24 Feb., 2016). Johnson, and Benjamin Carper. We wish to express a spe- Bajgar, J. (2004): Organophosphates/nerve agent poisoning: mecha- cial thanks to the Medical Countermeasure Systems Joint nism of action, diagnosis, prophylaxis, and treatment. Adv. Clin. Project Management Office, Department of Defense, for Chem., 38, 151-216. providing the oxime MMB-4 DMS through an agency to Bajgar, J. (2005): Complex view on poisoning with nerve agents agency material transfer. and organophosphates. Acta Medica (Hradec Kralove), 48, 3-21. Balali-Mood, M. and Shariat, M. (1998): Treatment of organophos- The authors also wish to thank National Institutes of phate poisoning. Experience of nerve agents and acute pesticide Health (NIH) Countermeasures Against Chemical Threats poisoning on the effects of oximes. J. Physiol. Paris., 92, 375- (CounterACT) Program Steering Committee (CPSC) 378. members, Drs. Judith W. Laney (Department of Health Ballantyne, B. and Marrs, T.C. (1992): Overview of the biological

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