Percutaneous exposure to the nerve agent VX: Efficacy of combined atropine, obidoxime and diazepam treatment

doi:10.1016/j.cbi.2010.06.010

Chemico-Biological Interactions

Volume 188, Issue 1, 6 October 2010, Pages 255-263

https://doi.org/10.1016/j.cbi.2010.06.010 Get rights and content

Abstract

The nerve agent VX is most likely to enter the body via liquid contamination of the skin. After percutaneous exposure, the slow uptake into the blood, and its slow elimination result in toxic levels in plasma for a period of several hours. Consequently, this has implications for the development of toxic signs and for treatment onset. In the present study, clinical signs, toxicokinetics and effects on respiration, electroencephalogram and heart rate were investigated in hairless guinea pigs after percutaneous exposure to 500 μg/kg VX.

We found that full inhibition of AChE and partial inhibition of BuChE in blood were accompanied by the onset of clinical signs, reflected by a decline in respiratory minute volume, bronchoconstriction and a decrease in heart rate. Furthermore, we investigated the therapeutic efficacy of a single dose of atropine, obidoxime and diazepam, administered at appearance of first clinical signs, versus that of repetitive dosing of these drugs on the reappearance of signs. A single shot treatment extended the period to detrimental physiological decline and death for several hours, whereas repetitive administration remained effective as long as treatment was continued. In conclusion, percutaneous VX poisoning showed to be effectively treatable when diagnosed on time and when continued over the entire period of time during which VX, in case of ineffective decontamination, penetrates the skin.

Introduction

Organophosphorous compounds (OPs) are potent irreversible acetylcholinesterase (AChE) inhibitors and widely used as insecticides. The most toxic OPs might be used as chemical warfare agents. Low volatility nerve agents like VX (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate) are likely to enter the body via the skin rather than via the respiratory route. Effective treatment of skin intoxications is difficult because of unpredictable toxicokinetics [1], [2].

Previous studies demonstrated that after percutaneous exposure of anesthetized hairless guinea pigs to VX, maximum blood levels of VX were not reached until several hours after exposure, followed by a slow elimination [1]. The variability in toxicokinetics has shown to lead to a delayed and variable onset of toxic signs [3], [4]. In addition, effective decontamination is hampered by the unawareness of time of intoxication and skin location of exposure, causing a long duration of the toxic effects. This has important implications for military or civilian first responders with respect to triage, decontamination approach and therapeutic drug regimens [5], [6]. The slow absorption of VX by the skin into the circulation does not parallel the short biological half-lives of the current medical countermeasures, likely making a single injection treatment insufficient.

Relatively little is known about the physiological consequences of the persistent action of VX. To investigate the toxicity mechanisms following percutaneous exposure to VX, we developed a hairless guinea pig model, in which toxicokinetics of VX, acetylcholinesterase and butyrylcholinesterase inhibition, and a broad range of physiological parameters can be measured. The hairless guinea pig skin closely reflects that of human skin in vitro, which justifies the use of this animal model to study toxicokinetics after VX exposure [7]. In this model, three characteristic target organs can be evaluated: the brain by measuring seizure activity by electroencephalogram (EEG) and acetylcholine (ACh) levels using microdialysis, the lungs by monitoring respiratory function using whole body plethysmography, and autonomic control of the heart by telemetric recording of ECG. Measuring physiological consequences is of importance as they might be independent from AChE inhibition and therefore may need separate treatment.

In the present study, we investigated the toxicokinetics of percutaneously administered VX and the development of typical signs of poisoning at different physiological levels after exposure to dose levels considered relevant for human poisoning. In previous experiments, a percutaneous dose of approximately 4 LD50 (500 μg/kg), yielded a reproducible development of signs of toxicity in hairless guinea pigs [3]. The human equivalent dose, calculated by extrapolation of this dose using basal metabolic rates (BMR) or surface area corrections, is approximately 120 μg/kg, which is 1.5 times the human LDL₀[8]. After a thorough examination of all model parameters affected during VX poisoning, the effect of the treatment with the anticholinergic atropine, the AChE reactivator obidoxime, and the anticonvulsant diazepam, was investigated following single treatment or after repeated administration.

Section snippets

Experimental design

Five groups of hairless guinea pigs were challenged percutaneously with 500 μg/kg of VX. Levels of unbound VX and obidoxime in treated animals were determined in plasma. AChE and BuChE activity was determined in blood. Simultaneously, the physiological parameters heart rate and respiration were registered in these animals. The guinea pigs were either untreated, treated with atropine, obidoxime and diazepam doses at the guidance of appearance and recurrence of clinical signs or treated at first

Toxicokinetics and clinical signs

The toxicokinetics of VX after percutaneous application was studied in hairless guinea pigs that either received no treatment, a single shot treatment or repetitive treatment (atropine 10; obidoxime 8.2 and diazepam 0.5 mg/kg im per injection). During the experiment the animals were scored for clinical signs. The average onset times of these signs are shown in Fig. 1A. The first clinical signs appeared in a gradual fashion, starting with chewing and shivering at 15–220 min and 55–374 min,

Discussion

The toxicokinetics of VX after percutaneous exposure to 500 μg/kg and the consequent physiological effects were investigated in hairless guinea pigs. The guinea pigs were either untreated, treated with atropine, obidoxime and diazepam doses at the guidance of appearance and recurrence of clinical signs or treated at first clinical signs with one single dose. The use of a small animal, such as the hairless guinea pig, requires the use of diluted VX to obtain a relevant level of exposure. It might

Conflict of interest statement

The authors declare that there are no conflicts of interest.

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Towards catch-up therapy: evaluation of nucleophilic active pharmaceutical ingredients for the treatment of percutaneous VX poisoning, in-vial and in-vitro studies
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Dermal exposure to low volatility organophosphorus chemical warfare agents (OP CWA) poses a great risk to the exposed person. Due to their lipophilic nature, these compounds rapidly absorb into the skin, leading to the formation of a “dermal reservoir” from which they slowly enter the bloodstream causing prolonged intoxication. Traditionally, strategies to counter the toxicity of such substances consist of chemical decontamination/physical removal of the residual agent from the skin surface (preferably as soon as possible following the exposure) and administration of antidotes in the case of intoxication signs. Hence, these strategies are unable to counter a substantial amount of the agent, which accumulates inthe dermal reservoir. More than a decade ago, the concept of a “catch-up therapy” intended to neutralize the dermal reservoir was suggested. Herein, we describe examples of potential “catch-up therapy” lotions - vehicles designed to deliver small nucleophilic molecules into the skin and potentially decompose the remaining CWA before it reaches the blood stream. Eleven nucleophilic compounds, based on approved drugs, were initially screened. They were then tested in various binary solutions, for their detoxification efficacy and degradation ability towards lipophilic OP CWA models such as dibutylphosphofluoridate and o-nitro-phenyl diphenyl phosphate, as well as the nerve agent VX, by means of kinetic ³¹P NMR and UV–Vis spectroscopy. Of these, the potassium and diethyl ammonium salts of acetohydroxamic acid (AHAK and AHA DEA) in (DMSO/H₂O 1:4) were found to be the most active nucleophiles, hydrolyzing VX in practical time scales (t_1/2 = 5.28 and 6.78 min, respectively). The vehicle solution DMSO/H₂O 1:4 promoted the penetration of substantial amounts of AHA K and AHA DEA through excised pig skin in in-vitro studies, suggesting that such formulations may serve as useful CWA nucleophilic scavengers for both on and within -skin detoxification. These findings may pave the way to a more efficacious treatment against low volatility OP CWA percutaneous poisoning.
Pharmacokinetics and efficacy of atropine sulfate/obidoxime chloride co-formulation against VX in a guinea pig model
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Nerve agent exposure is generally treated by an antidote formulation composed of a muscarinic antagonist, atropine sulfate (ATR), and a reactivator of acetylcholinesterase (AChE) such as pralidoxime, obidoxime (OBI), methoxime, trimedoxime or HI-6 and an anticonvulsant. Organophosphates (OPs) irreversibly inhibit AChE, the enzyme responsible for termination of acetylcholine signal transduction. Inhibition of AChE leads to overstimulation of the central and peripheral nervous system with convulsive seizures, respiratory distress and death as result. The present study evaluated the efficacy and pharmacokinetics (PK) of ATR/OBI following exposure to two different VX dose levels. The PK of ATR and OBI administered either as a single drug, combined treatment but separately injected, or administered as the ATR/OBI co-formulation, was determined in plasma of naïve guinea pigs and found to be similar for all formulations. Following subcutaneous VX exposure, ATR/OBI-treated animals showed significant improvement in survival rate and progression of clinical signs compared to untreated animals. Moreover, AChE activity after VX exposure in both blood and brain tissue was significantly higher in ATR/OBI-treated animals compared to vehicle-treated control. In conclusion, ATR/OBI has been proven to be efficacious against exposure to VX and there were no PK interactions between ATR and OBI when administered as a co-formulation.
Pharmacokinetics of Three Oximes in a Guinea Pig Model and Efficacy of Combined Oxime Therapy
2020, Toxicology Letters
Citation Excerpt :
Whole blood was spun at 4400xg for 10 min at 4 °C, plasma was aliquoted into cryovials, frozen on dry ice and stored at −70 °C until plasma analysis was completed. Quantification of oximes was determined using capillary electrophoresis (P/ACE mDQ from BeckMan Coulter (Fullerton, CA, USA)) with UV detection as previously described (Joosen et al., 2008, 2010). Briefly, imidazole as internal standard (IS), was added to each plasma sample and plasma concentrations were compared to standard curves.
Organophosphorus nerve agents (NA) inhibit acetylcholinesterase (AChE) which results in the over-stimulation of both the central and peripheral nervous systems, creating a toxic syndrome that can be lethal if left untreated (Cannard, 2006). It is standard practice to treat Sarin (GB) intoxication with an oxime, an antimuscarinic such as atropine and an anticonvulsant. Three common oximes are available: HI-6, Pralidoxime (2-PAM) and Obidoxime (Obi), all possess a nucleophile that can break the NA-AChE covalent bond. However, each oxime’s efficacy profile against various agents is different (Thiermann and Worek, 2018). In an effort to broaden therapeutic efficacy against a range of possible NA’s, consideration should be given to the use of two oximes in combination.
Using a guinea pig model, the first arm of this study was to determine the pharmacokinetics (PK) of HI-6 DMS, 2-PAM chloride and Obi chloride (at autoinjector equivalent doses) following intramuscular (i.m.) co-administration along with atropine to replicate either a single isometrically scaled dose (referred to in this study as a single autoinjector equivalent) of 2-PAM (and equimolar doses of Obi and HI-6) or double doses (referred to in this study as two autoinjector equivalents). The second arm of the study evaluated the efficacy of Obi and 2-PAM individually at a single or double autoinjector dose and also in combination against GB exposure. Pharmacokinetic profiles of each oxime were evaluated for both arms of the study and no significant change in parameters were reported. Improved cholinesterase reactivation was observed in a dose dependent manner with combined therapy showing similar reactivation to individual oximes alone at a two autoinjector equivalent dose. Seizure activity was reduced when combined oxime therapy was administered. This improvement was also reflected in the Racine seizure index score assigned at the end of the experimental period.
To the best of our knowledge, this study is the first to evaluate and compare the pharmacokinetics of three oximes and the combination of two oximes (2-PAM and Obi) administered in naïve animals or those exposed to GB. Combined oxime therapy (Obi and 2-PAM) resulted in improved seizure control, increased cholinesterase reactivation peripherally and centrally and improved behavioral signs (Racine score). This study provides evidence that combination of oximes is effective, does not result in adverse events and that the pharmacokinetics of each oxime are not affected when administered in combination.
Efficacy of atropine sulfate/obidoxime chloride co-formulation against sarin exposure in guinea pigs
2018, Chemico-Biological Interactions
The efficacy and pharmacokinetics of the aqueous co-formulation contents of the Trobigard™ (atropine sulfate, obidoxime chloride) auto-injector were evaluated in a sarin exposed guinea pig model. Two subcutaneous (sc) sarin challenge doses were evaluated in guinea pigs instrumented with brain and heart electrodes for electroencephalogram (EEG) and electrocardiogram (ECG). Sarin challenge doses were chosen to reflect exposure subclasses with sublethal (moderate to severe clinical signs) and lethal consequences. The level of protection of intramuscular human equivalent doses of the co-formulation was defined by (1) the mitigation of signs and symptoms at a sublethal level and (2) the increase of survival time at the supralethal sarin dose levels.
Pharmacokinetics of both atropine sulfate and obidoxime were proportional at 1 and 3 human equivalent doses, and only a small increase in heart rate was observed briefly as a side effect.
At both sarin challenge doses, 54 μg/kg and 84 μg/kg, the co-formulation treatment was effective against sarin-induced effects. Survival rates were improved at both sarin challenge levels, whereas clinical signs and changes in EEG activity could not in all cases be effectively mitigated, in particular at the supralethal sarin challenge dose level. Reactivation of sarin inhibited cholinesterase was observed in blood, and higher brain cholinesterase activity levels were associated with a better clinical condition of the co-formulation treated animals.
Although the results cannot be directly extrapolated to the human situation, pharmacokinetics and the effects over time related to plasma levels of therapeutics in a freely moving guinea pig could aid translational models and possibly improve prediction of efficacy in humans.
Bioscavenger is effective as a delayed therapeutic intervention following percutaneous VX poisoning in the guinea-pig
2018, Toxicology Letters
Citation Excerpt :
Similar studies using multiple i.m. administrations (Joosen et al., 2013) or i.v. infusions (Whitmore et al., 2017) have shown that pharmacological treatment needs to be extensive and continuous for it to be successful against this type of persistent poisoning. This requirement is due to the slow absorption of VX across the skin into the circulation being a lot longer than the half-lives of the therapeutic drugs (Joosen et al., 2010; Mumford et al., 2013). The reliance on MedCM treatment in percutaneous nerve agent poisoning can be reduced through the use of decontamination procedures (e.g. removing clothing, showering, adsorbent/reactive powders) to lower the overall external exposure levels, in turn protecting care-givers against secondary contamination.
The prolonged systemic exposure that follows skin contamination with low volatility nerve agents, such as VX, requires treatment to be given over a long time due to the relatively short half-lives of the therapeutic compounds used. Bioscavengers, such as butyrylcholinesterase (BChE), have been shown to provide effective post-exposure protection against percutaneous nerve agent when given immediately on signs of poisoning and to reduce reliance on additional treatments. In order to assess the benefits of administration of bioscavenger at later times, its effectiveness was assessed when administration was delayed for 2 h after the appearance of signs of poisoning in guinea-pigs challenged with VX (4 × LD₅₀). VX-challenged animals received atropine, HI-6 and avizafone on signs of poisoning and 2 h later the same combination with or without bioscavenger. Five out of 6 animals which received BChE 2 h after the appearance of signs of poisoning survived to the end of the study at 48 h, compared with 6 out of 6 which received BChE immediately on signs. All the animals (n = 6 + 6) that received only MedCM, without the addition of BChE, died within 10 h of poisoning. The toxicokinetics of a sub-lethal challenge of percutaneous VX were determined in untreated animals. Blood VX concentration peaked at approximately 4 h after percutaneous dosing with 0.4 × LD₅₀; VX was still detectable at 36 h and had declined to levels below the lower limit of quantification (10 pg/mL) by 48 h in 7 of 8 animals, with the remaining animal having a concentration of 12 pg/mL. These studies confirm the persistent systemic exposure to nerve agent following percutaneous poisoning and demonstrate that bioscavenger can be an effective component of treatment even if its administration is delayed.
The efficacy of HI-6 DMS in a sustained infusion against percutaneous VX poisoning in the guinea-pig
2018, Toxicology Letters
Post-exposure nerve agent treatment usually includes administration of an oxime, which acts to restore function of the enzyme acetylcholinesterase (AChE). For immediate treatment of military personnel, this is usually administered with an autoinjector device, or devices containing the oxime such as pralidoxime, atropine and diazepam. In addition to the autoinjector, it is likely that personnel exposed to nerve agents, particularly by the percutaneous route, will require further treatment at medical facilities. As such, there is a need to understand the relationship between dose rate, plasma concentration, reactivation of AChE activity and efficacy, to provide supporting evidence for oxime infusions in nerve agent poisoning.
Here, it has been demonstrated that intravenous infusion of HI-6, in combination with atropine, is efficacious against a percutaneous VX challenge in the conscious male Dunkin-Hartley guinea-pig. Inclusion of HI-6, in addition to atropine in the treatment, improved survival when compared to atropine alone. Additionally, erythrocyte AChE activity following poisoning was found to be dose dependent, with an increased dose rate of HI-6 (0.48 mg/kg/min) resulting in increased AChE activity. As far as we are aware, this is the first study to correlate the pharmacokinetic profile of HI-6 with both its pharmacodynamic action of reactivating nerve agent inhibited AChE and with its efficacy against a persistent nerve agent exposure challenge in the same conscious animal.

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Percutaneous exposure to the nerve agent VX: Efficacy of combined atropine, obidoxime and diazepam treatment

Abstract

Introduction

Section snippets

Experimental design

Toxicokinetics and clinical signs

Discussion

Conflict of interest statement

Toxicol. Appl. Pharmacol.

Toxicology

Toxicol. In Vitro

Organophosphorous compounds as chemical warfare agents

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Neurotoxicology

Percutaneous exposure to VX: clinical signs, effects on brain acetylcholine levels and EEG

Neurochem. Res.

Clinical aspects of percutaneous poisoning by the chemical warfare agent VX: effects of application site and decontamination

Mil. Med.