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Comparative physiology and efficacy of and in 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

Volume 396 114994

Date of Publication from Ext Publisher: April 2020

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CAN UNCLASSIFIED Toxicology and Applied Pharmacology 396 (2020) 114994

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Toxicology and Applied Pharmacology

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Comparative physiology and efficacy of atropine and scopolamine in sarin T nerve agent poisoning ⁎ Alex S. Cornelissena, , Steven D. Klaassena, Tomas van Groningena, Sara Bohnertb, Marloes J.A. 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: treatment is key for effective medical treatment of nerve agent exposure. Atropine is included at Scopolamine a 2 mg intramuscular dose in so-called 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 is based on efficacy achieved in studies in a variety of species. The doseof 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 , atropine and scopolamine. The aim was to find a predictive parameter for therapeutic efficacy. Scopolamine and atropine showed a similar , 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 overactivation, an Even though the structural variation across these compounds is large, a such as or to reactivate inactivated cholines- common denominator is that they inhibit the acet- terase, and an anticonvulsant such as (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 anticholinergic (atropine) and an oxime, sometimes combined as a consequence of central overstimulation of the cholinergic system, with an anticonvulsant such as diazepam or . 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 (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 ( 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. any inter-species differences that affect the , suchas affinity or distribution. It is important to note that this method Animals were anesthetized with in (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 were fixed subcutaneously to the Department of Health and Human Services Food and Drug pectoralis major and external oblique muscles. EEG/ECG electrodes Administration Center for Drug Evaluation and Research (CDER), were attached to a plug, which was fixed to the skull of the animal using 2005). dental cement. The jugular vein was catheterized and the cannula was Establishment of pharmacokinetic-pharmacodynamic (PKPD) pro- filled with heparinized glycerol (500 IU/mL) and capped. Animals were files and linking these to observed therapeutic efficacy inanimal given pre-operative analgesia (carprofen, 5 mg/kg) and antibiotics models may provide input for improvements in dose translation to (trimethoprim 4 mg/kg and sulfadoxine 20 mg/kg) as well as post- humans. Both (ECG) and electroencephalography operative analgesia (Carprofen, 5 mg/kg) at 24 h. Additionally, local (EEG) are attractive techniques for use in translational studies. These analgesia was provided during surgery using 2% hydro- techniques are minimally-invasive and thus potentially provide a va- chloride. After the surgery, animals were housed individually and were luable link to clinical data, because clinical studies looking into such allowed to recover for 3–5 days until experiments started. physiological parameters in healthy volunteers are available (Ebert et al., 2001; Kikuchi et al., 1999). Here, we aimed to provide an initial assessment of such a transla- 2.4. Experiment design and procedure tional approach to the assessment of therapeutic efficacy of antic- holinergics by combining physiology in naïve animals with antic- The experiment consisted of two animal cohorts: the first for phy- holinergic efficacy of atropine and scopolamine in nerve agent exposed siology in naïve animals and the second group for the evaluation of the rats. Pharmacokinetic data were obtained by continuous blood sam- efficacy of selected treatments in sarin challenged animals (Table 1). pling and analysis. Physiological data were obtained using cortical For the physiology experiments in naïve animals, a balanced cross- electroencephalography (EEG) and electrocardiography (ECG). over design was used with three treatments per subject with a 48-hour washout period. The treatment consisted of either atropine (0.3, 1, 3 mg/kg), scopolamine (0.15, 0.5, 1.5 mg/kg), or phosphate-buffered 2. Materials and methods saline (PBS) which was administered intramuscularly in the hindleg (i.m., 0.3 mL/kg). Before each experiment, baseline EEG/ECG values 2.1. Animals were obtained for at least 30 minutes and baseline blood samples were collected Male Wistar WU rats (weight on arrival was 220–240 g) were ob- For the efficacy experiments in sarin-exposed animals, an up-and- tained from Charles River Laboratories (Sulzfeld, ). Animals down (UDP) approach was used for the challenge dose determination as were housed in pairs or triplets in Makrolon Type IV cages and were described by (Abal et al., 2017). A dose of 97.5 μg/kg sarin was selected allowed to acclimatize to the animal facilities for at least one week. A to be used in the experiment. For the efficacy, animals were exposed to 12-h light-dark (lights on at 7:00 a.m.) cycle was maintained; tem- sarin s.c. and received an i.m. treatment at one minute following ex- perature was kept at 19–22 °C and relative humidity at 55–65%. posure. The treatment consisted of either atropine (0.3, 3 mg/kg), Animals had ad libitum access to water and standard rodent chow scopolamine (0.15, 1.5 mg/kg), or PBS. A 10-fold range was assumed to (Teklad Global Diet, Harlan, Horst, The Netherlands). Before each be sufficiently large to detect dose responsiveness. The dose-range of

Table 1 Schematic overview of the study design.

Physiology (naïve) Efficacy (sarin challenge)

Design Balanced cross-over design with three treatments Initial dose-finding followed by single day experiment Exposure (t =0) NA 97.5 μg/kg sarin s.c. Treatment 0.3, 1, 3 mg/kg atropine 0.3, 3 mg/kg atropine 0.15, 0.5, 1.5 mg/ scopolamine 0.15, 1.5 mg/kg scopolamine i.m. at 1-minute i.m. at 1-minute

Monitoring 150 minutes on days 1 and 2. 60 minutes at day 3. 60 minutes Sampling 60 minutes following treatment, 6 samples 60 minutes following exposure, 6 samples

2 A.S. Cornelissen, et al. Toxicology and Applied Pharmacology 396 (2020) 114994 atropine was chosen to be similar to reported in the literature and was reaction gas respectively. The desolvation temperature was 350 °C at a based on BSA-corrected human doses, where 0.3 mg/kg represents 1‐2 flow of 800 L/hr. The ions were created by positive Electrospray quantities. For scopolamine, lower doses were adminis- Ionization (ESI) mode using 3.5 kV. Transitions for atropine were m/z tered, to correct for its higher reported potency, while maintaining a 290.1 to 93 and 124*, D3-Atropine m/z 293.1 to 93 an 127*, similar pharmacokinetic range (Koplovitz and Schulz, 2010). Before Scopolamine m/z 304 to 121, 138* and 156, D3-Scopolamine m/z 307 each experiment, baseline EEG/ECG values were obtained for at least to 121, 141* and 159 (*quantifier ion). Assay validation was previously 30 minutes and baseline blood samples were collected. carried out for atropine in guinea pig and human plasma (Joosen et al., 2018). Accuracy and precision were within the limit of 15% (and 20% 2.5. Telemetry data acquisition and analysis at the LLOQ). In the current study, the analytical method was adopted for scopolamine and validated in rat plasma and assay performance was Cortical EEG and ECG data were obtained using a Physiotel™ comparable. hardware system (Data Sciences International, ‘s-Hertogenbosch, The Netherlands) and TL11M2-F40-EET transmitters (naïve animals) or HD- 2.8. Data analysis S02 transmitters (sarin-exposed animals). The transmitter was attached to the plug and data was sent to a PC via a Data Exchange Matrix (or MX Plasma levels were interpolated from a calibration curve using 2.0) at 1000Hz sampling frequency. Ponemah software (v5.2 for the Graphpad Prism (version 7.00 for Windows, Graphpad Software, La F40-EET transmitters, v6.33 for the HD-S02 transmitters) was used for Jolla CA, USA). Pharmacokinetic parameters were determined using data acquisition. The Ponemah ECG module was used to analyze the Phoenix WinNonLin version 8.1 (Certara, Princeton NJ, USA). All data rate (10-second intervals) and standard deviation of normal beats was fit to a one-compartment model, 1st order elimination. Datawas (SDNN, 30-second intervals), a time-domain measure of heart varia- weighted 1/y2 and Nelder Mead minimization was applied. bility (HRV). Noise detection was enabled using the default settings. Parametric data are shown as mean ± SEM. Statistical analyses Additionally, data were manually checked for data exclusion due to were performed using one-way or two-way ANOVA followed by an signal artifacts. Cortical EEG data was analyzed using Neuroscore appropriate post-hoc test: Dunnett’s, Sidak’s or Tukey’s test. Survival software (v3.3.1). Spectral analysis was performed via Fast Fourier analysis was performed using a Log-Rank analysis. Multiple comparison Transformation (FFT). Epoch duration was set at 10 second intervals. corrections were done by p-values adjustment using the Holm-Sidak Total power was defined as the total output in the range of 0.3-50Hz. method. Type-I errors were controlled for by adjusting p-values ob- Power spectra were divided into Delta (0.5-4Hz), Theta (4-8Hz), Alpha tained in a single analysis for a family-wise error rate of < 0.05. (8-14Hz), Sigma (12-16Hz), Beta (16-24Hz), and Gamma (24-100Hz) Graphpad Prism (version 7.00 for Windows, Graphpad Software, La bands. The relative powers were calculated automatically by the di- Jolla CA, USA) was used as statistical software. viding the absolute powers of each band by the total power of all bands and normalizing versus the baseline. Epochs containing artifacts were 3. Results removed semi-automatically by using an amplitude detector and manual exclusion. 3.1. Pharmacokinetics

2.6. Sample preparation The unbound brain levels of i.m. administered atropine and sco- polamine are shown in Fig. 1 for naïve (physiology) and sarin-exposed Blood samples were immediately centrifuged and plasma was snap- (efficacy) animals. Measured levels were comparable in both naïveand frozen and stored at ‐20°C until further analysis. Internal standard, sarin-exposed animals, indicating the distribution to the brain was si- consisting of D3-atropine and D3-scopolamine, was added to each milar between studies. Plasma pharmacokinetic data are shown for plasma sample and allowed to reach equilibrium for 15 minutes. Four sarin-exposed animals only, due to the loss of reliable samples during equivalents of acetonitrile were added and samples were centrifuged for processing of naïve animal plasma. Drug plasma levels peaked around 10 minutes at 14k rpm. 200 μL of supernatant was transferred to an LC- 10 and 2 minutes post- for atropine and scopolamine respec- MS vial. tively. Scopolamine exhibited higher peak plasma levels compared to Brains were harvested immediately at the end of the final experi- atropine, given lower doses. The pharmacokinetics of atropine and ments and brain tissue samples were collected from each of three areas scopolamine were approximated by a one-compartment model and of the right hemisphere (hippocampus, caudate nucleus, somatosen- first-order elimination. A summary of the results is shownin Table 2. sory/somatomotor cortex). Samples were stored at ‐20°C. On the day of The total bioavailability of atropine was comparable to that of scopo- analysis, samples were thawed and internal standard was added ac- lamine, though the uptake and elimination was considerably faster for cording to weight. Samples were homogenized using a Bead Ruptor 24 scopolamine compared to atropine. elite (OMNI, Labconsult, Brussels, Belgium). Samples were centrifuged Brain concentrations were determined at 60 minutes after admin- for 10 minutes at 14k rpm and supernatants were subsequently filtered istration in three brain areas (hippocampus, caudate nucleus, somato- using 10kDa spin filters. The remaining eluent was transferred to anLC- sensory/somatomotor cortex). Brain region averages are shown in MS vial. Fig. 1A. Scopolamine reached much higher brain concentrations than atropine when given at lower doses. Brain concentrations were similar 2.7. Analytical assay and sample quantitation across brain regions (supplementary Figure S1).

LC-MS/MS was used for the analytical determination of plasma le- 3.2. ECG analysis vels of atropine and scopolamine. Samples were analyzed on a Waters Xevo-TQS mass spectrometer equipped with an Acquity M-Class Liquid The heart rate following administration of treatment in naïve ani- Chromatograph. 5 μL of the sample was injected and separated on a mals is shown in Fig. 2 (A,B). All animals showed statistically sig- Acquity HSS T3, 2.1 x 100 mm, 1.8 μm particles column at a flow rate of nificant after i.m. injection with atropine or scopolamine 100 μl/min. Mobile phase A was a solution of water containing 0.01% compared to vehicle treatment. The stimulatory effect on heart rate was heptafluorobutyric acid (HFBA) and mobile phase B was acetonitrile rapid, peaked around five minutes after treatment and returned to containing 0.01% HFBA. The column was maintained at room tem- baseline at 60‐90 minutes after treatment. At the doses used, no dose perature (21 °C). The MS/MS operated in Multiple Reaction Monitoring dependency was observed, except for a slightly smaller effect following (MRM) mode and and argon were used as nebulizer and scopolamine at the lowest dose of 0.15 mg/kg. This suggests a plateau

3 A.S. Cornelissen, et al. Toxicology and Applied Pharmacology 396 (2020) 114994

Figure 1. (A) Average brain levels (hippocampus, caudate nucleus, somatosensory/somatomotor cortex) of atropine and scopolamine in naïve animals (solid, n = 3) and sarin-exposed (hatch pattern, n = 8) animals at 60-minutes following administration. No differences were observed in the levels of atropine and scopolamine between studies, except for atropine 3 mg/kg, which was significantly increased sarin-exposed animals compared to naïve animals (p = 0.047). (B) Unbound plasma levels of atropine (n=5/6) and scopolamine (n=6/7) were measured at various timepoints in sarin-exposed animals up to t=60 minutes. Multiple t-tests followed by Holm-Sidak p-value adjustment were performed on brain levels. Data are shown as mean ± SEM. *: p-value < 0.05 versus naïve animals. effect induced by all atropine doses and scopolamine medium and power decrease showed a dose-dependency for scopolamine that sig- scopolamine 1.5 mg/kg. Fig. 2 (C) shows the SDNN (30-second inter- nificantly deviated from zero (p = 0.088). For theta power, nosig- vals). As expected, due to the activity of the antic- nificant correlations were found (p = 0.33, 0.27 respectively). A holinergics, SDNN decreased following treatment of atropine or sco- summary is provided in Table 3. polamine. Similar to the effect on heart rate, the effect on SDNN showed a plateau. The reduction of SDNN was longer-lasting for atropine compared to scopolamine. 3.5. Treatment efficacy

Treatment of animals with either atropine or scopolamine had a 3.3. EEG analysis beneficial, but not significant, effect on 60-minute survival compared to untreated animals. Survival was inversely correlated with oc- Results for cortical EEG analysis are shown in Fig. 3. Atropine did currence. Anticonvulsant efficacy of atropine or scopolamine was as- not induce any effect on the EEG. Animals treated with scopolamine sessed using EEG analysis. Fig. 5 shows seizure-free progression for the exhibited EEG-slowing, with an increase mostly in theta power. The various treatment groups. Animals were considered seizure-free if they activity returned to baseline at approximately 60‐90 minutes after did not exhibit uninterrupted seizure activity for at least 30 seconds. At treatment. The increased theta power coincided with a decreased beta both 0.15 mg/kg and 1.5 mg/kg, scopolamine led to a significant im- power. Both effects exhibited dose dependency, which was most pro- provement in seizure-free progression compared to untreated animals. nounced for beta power. After 60‐90 minutes after injection, animals Atropine did not elicit a significant improvement compared to un- were often observed being in a resting state, which coincided with a treated animals. One animal treated with scopolamine (1.5 mg/kg) more irregular EEG pattern. exhibited seizure activity, which subsided after two minutes. Atropine showed a minor improvement in median seizure onset time compared 3.4. Pharmacokinetic-physiology relationships to the untreated group. Results are summarized in Table 4. Acet- ylcholinesterase (AChE) activity was measured at various timepoints The relationship between the various physiological parameters and after exposure. Maximum inhibition was > 90% and no differences brain levels are shown in Fig. 4. The area under the curve (AUC) of the between groups were observed (supplementary figure S2). various physiological responses was calculated over 60 minutes fol- The tachycardia following administration of atropine or scopola- lowing treatment administration. Linear regression was performed to mine in sarin-exposed animals was similar to that observed in naïve assess the relationships between these parameters and brain drug levels. animals. The heart rate of animals treated with scopolamine showed a Whereas both atropine and scopolamine induced an elevation in heart rapid return to baseline after approximately 30 minutes, whereas the rate, there was no dose-dependent relationship for atropine or scopo- heart rate of animals treated with atropine or untreated animals showed lamine (p = 0.36, 0.14 respectively). Scopolamine showed a dose-de- a persistent increase in heart rate, which correlated with (convulsive) pendent decrease in HRV (p = 0.03), whereas atropine did not. Beta seizure activity (figure S3).

Table 2 Pharmacokinetic parameters for i.m. administered atropine sulfate and scopolamine hydrochloride in naïve rats. For each parameter, values (mean ± SD) are shown for 0.3 and 3 mg/kg for atropine and 0.15 and 1.5 mg/kg for scopolamine.

Parameter Unit Atropine Scopolamine

Dose mg/kg (i.m.) 0.3 3 0.15 1.5 n 6 6 7 6 AUC ng/mL*h 53.6 ± 4.2 550.7 ± 100.7 20.1 ± 2.9 208.5 ± 22.2

Cmax ng/mL 95.0 ± 11.6 363.3 ± 42.0 53.1 ± 14.1 604.9 ± 161.0

Tmax min 9.8 ± 1.8 7.8 ± 2.1 1.7 ± 0.4 3.4 ± 2.1 CL mL/min/kg 96.3 ± 7.8 103.2 ± 14.7 126.5 ± 16.6 121.1 ± 13.9

4 A.S. Cornelissen, et al. Toxicology and Applied Pharmacology 396 (2020) 114994

Figure 2. Baseline-corrected heart rate of naïve animals treated with either (A) atropine (n=8) or (B) scopolamine (n=8). Animals treated with either atropine or scopolamine show mild tachycardia immediately following injection. The heart rate normalizes around 90 minutes following treatment. The 60-minutes AUC of heart rate was significantly increased for atropine and scopolamine compared to the control group at all doses given in naïve animals. (C) Baseline-corrected SDNNof animals treated with atropine or scopolamine. Both substances significantly decreased this measure of heart-rate variability (HRV). Animals treated with scopolamine appeared to return to baseline faster than those treated with atropine. Data are shown as mean ± SEM. One-way ANOVA with Dunnett’s test was performed on the 60-minute AUC data. *: p-value < 0.05 versus the control (saline).

4. Discussion methods employ a means to predict pharmacokinetic parameters ex- pected in humans based on those found in one or more animal species: Anticholinergic treatment by administration of a muscarinic an- Methods based on allometric scaling consider the relationship between tagonist, such as atropine or scopolamine, is a key component of any body surface area and pharmacokinetic parameters (Mahmood, 2007; treatment regimen aimed at the mitigation of clinical signs following Reagan-Shaw et al., 2008) whereas other methods normalize the PK of a OP exposure. Therapeutic efficacy of these drugs has been studied in drug based on other parameters such as clearance and volume of dis- animals in a wide variety of exposure and treatment scenarios, with a tribution (Wajima et al., 2004). The various methods of dose scaling wide range of doses. The variety of scenarios and doses complicates the and the assumptions they rely on are extensively reviewed elsewhere translation of these results to effective and safe human treatment re- (Fan and de Lannoy, 2014; Mak et al., 2014; Blanchard and Smoliga, gimens. Translational preclinical research is essential for the improve- 2015). It is generally accepted that scaling of drug dosing based on ment of the treatment of OP poisoning, because clinical efficacy studies body mass has shortcomings, but there is no clear guidance as to what are neither ethical nor feasible (Buckley et al., 2005). Especially for method is appropriate for a given drug; often a combination of methods drugs that have a narrow therapeutic window in view of detrimental is employed to make predictions (Tuntland et al., 2014). Another ap- side effects, such as atropine, dose optimization is beneficial: Atropine proach to dose scaling is to establish PKPD models by studying phy- is associated with serious side-effects such as and tachycardia, siology in parallel to the pharmacokinetic parameters. These models caused by cholinergic deficiency (Hshieh et al., 2008; Dawson and can provide quantitative links between the physiological effect of drugs Buckley, 2016). Similarly, scopolamine is associated with various ad- and the PK profiles and can subsequently be used to convert ther- verse side-effects also including strong central (, and amnestic) apeutically efficacious doses across species. Such models have been effects (Falsafi et al., 2012). established for atropine both in healthy volunteers (Scheinin et al., A variety of dose scaling methods can be used to estimate the 1999; Picard et al., 2009) and rodent models (Perlstein et al., 2001). human equivalent dose to a safe dose found in preclinical studies. Most In the present study, two anticholinergic drugs, atropine and

5 A.S. Cornelissen, et al. Toxicology and Applied Pharmacology 396 (2020) 114994

Figure 3. Cortical EEG Power band spectra of naïve animals treated with either (A,C) atropine (n=8) or (B,D) scopolamine (n=8). Atropine did not affect theta or beta power, whereas scopolamine at 0.5 and 1.5 mg/kg i.m., induced a significant increase in theta (4-8Hz) power (p < 0.001) and decrease in beta (16-24Hz) power (p = 0.004 and 0.029 respectively). 60-minute AUC values were compared using a one-way ANOVA followed by Dunnett’s test. *: p-value < 0.05 versus the control (saline). scopolamine, were studied at multiple doses in naïve rats and in rats concentrations in the brain compared to atropine and relative to plasma exposed to sarin. The dose range of atropine and scopolamine was levels, possibly due to the differential involvement of the P-glycopro- based on the atropine BSA-corrected human equivalent of the auto- tein efflux transporter for both substances (Joosen et al., 2016). This injector used by the military. Scopolamine was given at half the dose, to tendency towards a more profound central distribution found for sco- provide a compromise between similar PK and higher potency. polamine is consistent with the stronger central effect and antic- Pharmacokinetic models were established and the physiological re- onvulsant activity observed in this study, and which has been shown in sponse in naïve animals was evaluated against the therapeutic efficacy other studies (McDonough Jr et al., 2000; Koplovitz and Schulz, 2010). at the same dose of drug administered. The therapeutic efficacy of both The effects of atropine and scopolamine on various physiological drugs was assessed as standalone treatment. Even though this treatment parameters were studied in naïve animals. Both atropine and scopola- does not represent the full treatment normally provided to poisoned mine induced tachycardia with similar dynamics. Both substances in- individuals, this approach was chosen to avoid any confounding effects duced significant reductions in HRV, which lasted longer than theef- caused by coadministered drugs and thus make it easier to compare the fects on the heart rate and were longer-lasting for atropine compared to physiological effects of atropine and scopolamine. scopolamine. This longer effect on HRV may be related to the different Both atropine and scopolamine were fit using a one-compartment PK observed for atropine and scopolamine. The parasympatholytic ac- model to estimate the pharmacokinetic parameters; atropine showed a tions on heart rate and HRV showed different dynamics, indicating delay in uptake compared to scopolamine, with about a three-fold in- potentially different sites or mechanisms of action. EEG recordings were crease in Tmax. The AUC of plasma concentration relative to dose given made to study central effects. EEG-slowing caused by atropine and was directly proportional for both atropine and scopolamine and scopolamine is a relatively well-known phenomenon and similar ob- showed similar. Total brain levels were measured in three areas of the servations have been made in healthy volunteers in a resting state brain (hippocampus, caudate nucleus, cortex), that are involved in the (Kikuchi et al., 1999; Pickworth et al., 1990; Ostfeld et al., 1960) and neuropathology of OP-induced seizures (Shih et al., 2003; Kuruba et al., animal studies (Santucci et al., 1981; Sambeth et al., 2007). Slowing 2018; Hobson et al., 2018; Kadar et al., 1995; Lemercier et al., 1983). effects on the EEG are correlated with impaired learning capacity. Even Measurements of total concentrations of scopolamine and atropine did though scopolamine is structurally similar to atropine, its central effect not reveal a differential distribution. However, the present results did is markedly different. Administration of scopolamine results in an im- not take into account any potential differences at the microenvironment pairment of cognition, correlated with its prominent EEG effects, which level, such as intra- and extracellular distribution. Moreover, the are likely mediated mainly by muscarinic M1 receptors (Reches et al., plasma levels of atropine and scopolamine may have interfered with the 2014; Snyder et al., 2005). Activation of this receptor may subsequently estimation of the brain concentrations, because correction for blood lead to involvement of the nicotinic and NMDA systems (Falsafi et al., content was not performed. Scopolamine reached much higher 2012; Erskine et al., 2004), though the relevance and contribution of

6 A.S. Cornelissen, et al. Toxicology and Applied Pharmacology 396 (2020) 114994

Figure 4. Relationships observed between brain concentration at 60 minutes after injection and physiological output for atropine and scopolamine in naïve rats. Data were baseline-corrected and the 60-minute area-under-the-curve was calculated. Data were fit using linear regression. Correlations are shown for brain concentration versus (A) heart rate, (B) SDNN, and (C) relative theta (closed symbols) and beta (open symbols) power. Results are summarized in Table 3.

Table 3 these effects were not assessed in the current study. It is not clearwhy Correlations between the unbound brain concentration and the AUC of phy- atropine did not show central effects here: Dosing and electrode pla- siological output at 60 minutes following treatment. Data are represented as cement may play a role. The magnitude of the central effects of sco- mean ± SEM. A significant correlation was found for scopolamine for beta polamine on EEG parameters were correlated with 60-minute brain power and SDNN. concentrations. Scopolamine induced clear central effects, whereas Physiological response Atropine Scopolamine atropine did not elicit any effect at a similar brain concentration. As

2 muscarinic receptor affinity is similar for both compounds, this suggests Heart rate R 0.15 0.28 that differences in microenvironment distribution could be a factor in Slope 2.47 ± 2.25 1.05 ± 0.64 p-value 0.3551 0.14 the observed difference in potency (Uchida et al., 1978; Lee, 1985). Theta power R2 0.13 0.17 However, more research is needed to elucidate this. Slope 1.96 ± 1.88 1.06 ± 0.89 Scopolamine exhibited a superior anticonvulsant effect compared to p-value 0.33 0.27 atropine, given at lower doses. A convulsive state was highly predictive Beta power R2 0.02 0.65 Slope 0.83 ± 2.00 ‐1.17 ± 0.32 of mortality in animals. The efficacious doses of scopolamine likely p-value 0.69 0.01* correspond to human doses that show severe side-effects such as de- SDNN R2 0.00 0.51 lirium. Therefore, scopolamine may be better suited for second-line Slope ‐0.49 ± 2.51 ‐1.46 ± 0.54 treatment, delivered by medical care professionals as opposed to self- or p-value 0.85 0.03* buddy-aid. At the doses used, no dose-dependent degree of efficacy was observed for either drug. For future studies, a preferred approach is to select a treatment dose based on physiological response. The current each system in the case of scopolamine is not well understood. Slowing study did, however, allow for a comparison of atropine with scopola- of the EEG by scopolamine was prominent in the current study: Sco- mine. The central effects observed for scopolamine in naïve animals polamine induced an increase in (slow-wave) theta power and a dose- showed dose-responsiveness and in a sarin model these treatments ex- dependent decrease in (fast-wave) beta power, whereas atropine did hibited superior anticonvulsant action of scopolamine compared to not elicit any such effect. This effect is expected to result in amnestic atropine. and cognitive impairment effects reported for scopolamine; however,

7 A.S. Cornelissen, et al. Toxicology and Applied Pharmacology 396 (2020) 114994

Figure 5. (A) Survival curve of cumulative seizure-free progression of animals treated with atropine or scopolamine treatments. Seizure onset was manually scored using EEG power analysis. Onset was defined as at least 30 seconds of seizure activity. Seizure-free progression was significantly higher of animals treatedwith scopolamine low (p = 0.004) and scopolamine high (p = 0.048) versus untreated animals. Atropine did not yield a significant improvement over no treatment. (B) Survival analysis. Animals treated with scopolamine exhibited improved, but not significant, survival compared to untreated animals. Survival curves were compared pairwise using a Log-rank test, p-values were adjusted using a Holm-Sidak correction. *: p-value < 0.05 versus untreated.

Table 4 References Therapeutic efficacy of atropine and scopolamine given at two doses, following s.c. exposure to 97.5 μg/kg sarin. Treatments were compared for seizure rate Abal, P, Louzao, MC, Antelo, A, Alvarez, M, Cagide, E, Vilariño, N, et al., 2017. Acute oral and 60-minute survival. Toxicity of in Mice: Determination of Lethal Dose 50 (LD50) and No Observed Adverse Effect Level (NOAEL). 9 (3), 75. https://doi.org/10.3390/ Treatment Dose Seizure activity Survival toxins9030075. (Mar). Blanchard, OL, Smoliga, JM, 2015. Translating dosages from animal models to human (i.m.) (mg/kg) Median onset Proportion (%) Proportion (%) clinical trials–revisiting body surface area scaling. FASEB J. 29 (5), 1629–1634 (min) (May). Buckley, NA, Eddleston, M, Dawson, AH, 2005. The need for translational research on Untreated ‐ 4:20 80 60 for . Clin Exp Pharmacol Physiol. 32 (11), 999–1005 Atropine 0.3 7:45 62.5 75 (Nov). 3 6:40 75 75 Connors, NJ, Harnett, ZH, Hoffman, RS, 2014. 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9

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Comparative physiology and efficacy of atropine and scopolamine in sarin nerve agent poisoning

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Cornelissen, A. S.; Klaassen, S. D.; van Groningen, T.; Joosen, M. J. A.; Bohnert, S.

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06da - CBRN Medical Countermeasures

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Scopolamine; Sarin; Atropine; Pharmacokinetics; ECG; EEG

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Anticholinergic treatment is key for effective medical treatment of nerve agent exposure. Atropine is included at a 2 mg intramuscular dose in so-called autoinjectors designed for self- and buddy-aid. As patient cohorts are not available, predicting and evaluating the efficacy of medical countermeasures relies on animal models. The use of atropine as a muscarinic antagonist is based on efficacy achieved in studies in a variety of species. The dose of atropine administered varies considerably across these studies. This is a complicating factor in the prediction of efficacy in the human situation, largely because atropine dosing also influences therapeutic efficacy of and anticonvulsants generally part of the treatment administered. To improve translation of efficacy of dosing regimens, including pharmacokinetics and physiology provide a 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 to atropine These findings show that an approach including pharmacokinetics and physiology could contribute to improved dose scaling across species and assessing the therapeutic potential of similar anticholinergic and anticonvulsant drugs against nerve agent poisoning.