CAN UNCLASSIFIED Comparative physiology and efficacy of atropine and scopolamine in sarin nerve agent poisoning Alex S. Cornelissen Steven D. Klaassen Tomas van Groningen Marloes J. A. Joosen TNO Defense Sara Bohnert DRDC – Suffield Research Centre Toxicology and Applied Pharmacology Volume 396 114994 Date of Publication from Ext Publisher: April 2020 The body of this CAN UNCLASSIFIED document does not contain the required security banners according to DND security standards. However, it must be treated as CAN UNCLASSIFIED and protected appropriately based on the terms and conditions specified on the covering page. Defence Research and Development Canada External Literature (P) DRDC-RDDC-2020-P225 December 2020 CAN UNCLASSIFIED CAN UNCLASSIFIED IMPORTANT INFORMATIVE STATEMENTS This document was reviewed for Controlled Goods by Defence Research and Development Canada using the Schedule to the Defence Production Act. 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Joosena a TNO Defense, Security and Safety, CBRN Protection, Lange Kleiweg 137, 2288, GJ, Rijswijk, the Netherlands b Defence Research and Development Canada-Suffield Research Centre, Department of National Defence, Suffield, Alberta, Canada. ARTICLE INFO ABSTRACT Keywords: Anticholinergic treatment is key for effective medical treatment of nerve agent exposure. Atropine is included at Scopolamine a 2 mg intramuscular dose in so-called autoinjectors designed for self- and buddy-aid. As patient cohorts are not Atropine available, predicting and evaluating the efficacy of medical countermeasures relies on animal models. The useof Sarin atropine as a muscarinic antagonist is based on efficacy achieved in studies in a variety of species. The doseof Pharmacokinetics atropine administered varies considerably across these studies. This is a complicating factor in the prediction of ECG efficacy in the human situation, largely because atropine dosing also influences therapeutic efficacy ofoximes EEG and anticonvulsants generally part of the treatment administered. To improve translation of efficacy of dosing regimens, including pharmacokinetics and physiology providea promising approach. In the current study, pharmacokinetics and physiological parameters obtained using EEG and ECG were assessed in naïve rats and in sarin-exposed rats for two anticholinergic drugs, atropine and scopolamine. The aim was to find a predictive parameter for therapeutic efficacy. Scopolamine and atropine showed a similar bioavailability, but brain levels reached were much higher for scopolamine. Scopolamine exhibited a dose-dependent loss of beta power in naïve animals, whereas atropine did not show any such central effect. This effect was correlated with an enhanced anticonvulsant effect of scopolamine compared toatropine. These findings show that an approach including pharmacokinetics and physiology could contribute toim- proved dose scaling across species and assessing the therapeutic potential of similar anticholinergic and antic- onvulsant drugs against nerve agent poisoning. 1. Introduction 2003; Treiman, 2007). The standard treatment for nerve agent exposure consists of a combination of a muscarinic antagonist such as atropine, Nerve agents are highly toxic organophosphorus (OP) compounds. intended to directly counteract the cholinergic overactivation, an oxime Even though the structural variation across these compounds is large, a such as pralidoxime or obidoxime to reactivate inactivated cholines- common denominator is that they inhibit the enzyme acet- terase, and an anticonvulsant such as diazepam (Eddleston et al., 2008). ylcholinesterase (AChE) by acting as false substrates. Upon exposure, Early treatment in a military setting by self-administration or these compounds can rapidly elicit severely incapacitating and life- buddy-aid consists of three autoinjectors, each containing 2 mg of an threatening symptoms. Among these symptoms are convulsive seizures anticholinergic drug (atropine) and an oxime, sometimes combined as a consequence of central overstimulation of the cholinergic system, with an anticonvulsant such as diazepam or midazolam. The dose of leading to uncontrolled firing in the brain (Shih and McDonough Jr., atropine in these autoinjectors is such as to prevent severe side-effects 1997). These seizures can become self-sustaining and progress to status following administration in case of misdiagnosis of OP poisoning, as epilepticus if left untreated. Over time, they become more resistant to described in studies in healthy volunteers (Cullumbine et al., 1955; anticholinergic treatment, likely as a result of involvement of the glu- Moylan-Jones, 1969). Multiple treatment regimens exist for civilian tamatergic system (Shih and McDonough, 2000; Lallement et al., 1991). settings, which are focused on achieving rapid atropinization (Connors Studies have shown that longer-lasting seizures (> 10 min) may induce et al., 2014). However, assessment of these regimens is difficult due toa irreversible brain injury (Shih and McDonough Jr., 1997; Shih et al., lack of controlled studies (Eddleston et al., 2004). ⁎ Corresponding author. E-mail address: [email protected] (A.S. Cornelissen). https://doi.org/10.1016/j.taap.2020.114994 Received 28 December 2019; Received in revised form 30 March 2020; Accepted 2 April 2020 Available online 03 April 2020 0041-008X/ © 2020 Elsevier Inc. All rights reserved. A.S. Cornelissen, et al. Toxicology and Applied Pharmacology 396 (2020) 114994 There is extensive evidence for the efficacy of atropine and other experiment, the animals were placed in Makrolon type III cages. All anticholinergic drugs in preclinical nerve agent poisoning studies. experiments were in agreement with a project license according to EU Atropine dosages reported in animal studies range from approximately Directive 2010/63EU for animal experiments and approved by the 0.1 mg/kg to well over 10 mg/kg, depending on the agent used, the Animal Welfare Body of TNO. challenge dose, and co-administered (pre)treatments (Lennox et al., 1985; McDonough Jr et al., 2000; McDonough Jr and Shih, 1995; Gilat 2.2. Chemicals et al., 2005; Koplovitz and Schulz, 2010; Shih et al., 2007). Scopola- mine is an alternative anticholinergic drug with a high potential for use Sarin (GB, Isopropylmethylphosphonofluoridate) was obtained from against nerve agent poisoning, showing efficacy in suppressing seizures, in-house synthetized stocks of TNO Rijswijk and purity was > 98%. either as pretreatment or as adjunct therapy (Koplovitz and Schulz, Atropine (sulfate monohydrate, > 95% purity), D3-atropine, and sco- 2010; Raveh et al., 2002). Therapeutic doses established in these pre- polamine (hydrochloride, 100% purity) were purchased from Sigma- clinical studies are difficult to translate to human equivalents. Aldrich (Zwijndrecht, The Netherlands). D3-scopolamine (hydro- For the determination of human equivalent doses of drugs used to bromide) was obtained from CDN Isotopes (Pointe-Claire, Quebec, treat nerve agent poisoning, the currently accepted method of dose Canada). scaling across species is based on allometric as published by Reagan- Shaw, et al. (2008). However, this method does not take into account 2.3. Surgery any inter-species differences that affect the pharmacodynamics, suchas receptor affinity or distribution. It is important to note that this method Animals were anesthetized with isoflurane in oxygen (4–5% for is in particular recommended for establishing No Observed Adverse induction, 2–3% for maintenance). The EEG electrodes were placed on Effect Levels (NOAEL) for drugs when there is no clinical data available the dura mater at A1.0 and P6.0 mm relative to Bregma and 1 mm from and not designed for finding Pharmacologically Active Dosages (U.S. the sagittal suture. ECG electrodes