Pharmacokinetics and pharmacodynamics of standard nerve agent medical countermeasures in Göttingen Minipigs.

Animal research continues to serve a critical role in the testing and development of medical countermeasures. The Göttingen minipig, developed for laboratory research, may provide many benefits for addressing research questions within chemical defense. Targeted development of the Göttingen minipig model could reduce reliance upon non-human primates, and improve study design, statistical power, and throughput to advance medical countermeasures for regulatory approval and fielding. In this vein, we completed foundational pharmacokinetics and physiological safety studies of intramuscularly administered atropine sulfate, pralidoxime chloride (2-PAM), and diazepam across a broad range of doses (1-6 autoinjector equivalent) using adult male Göttingen minipigs (n=11; n=4-8/study) surgically implanted with vascular access ports and telemetric devices to monitor cardiovascular, respiratory, arterial pressure, and temperature signals. Pharmacokinetic data were orderly and the concentration maximum mirrored available human data at comparably scaled doses clearly for atropine, moderately for 2-PAM, and poorly for diazepam. Time to peak concentration approximated 2, 7, and 20 min for atropine, 2-PAM, and diazepam, respectively, and the elimination half-life of these drugs approximated 2 hr (atropine), 3 hr (2-PAM), and 8 hr (diazepam). Atropine sulfate dose-dependently increased the magnitude and duration of tachycardia and decreased the PR and ST intervals (consistent with findings obtained from other species). Mild hypothermia was observed at the highest diazepam dose. Göttingen minipigs appear to provide a ready and appropriate large animal alternative to non-human primates, and further development and evaluation of novel nerve agent medical countermeasures and treatment strategies in this model are justified.

Development and characterization of an automated behavioral assessment platform for the Göttingen minipig.

The Göttingen minipig is fast becoming the standard for assessing dermal chemical hazards because, like most swine, its skin is predictive of human skin response and because this strain's smaller size makes laboratory manipulations and husbandry easier. Unfortunately, standard behavioral tests and apparatus have not been developed for behavioral assessments of this swine strain. Indeed, computer-controlled automated behavioral testing procedures are much needed. The present research advanced this goal by producing a home-cage behavioral testing system that could accommodate minipigs of various sizes (ages). An aluminum frame housed three levers for recording operant responses, and LEDs above and below each lever served as discriminative stimuli. A commercially available food pellet dispenser was attached to a specialized pellet receptacle capable of measuring pellet retrieval. Two behavioral tests were selected and adapted from our commonly used non-human primate behavioral assessments: delayed match-to-sample (a memory test) and temporal response differentiation (a time-estimation test). Minipigs were capable of learning both tests and attaining stable performance. Next, scopolamine was used to validate the sensitivity of the behavioral tests for gauging behavioral perturbations in this swine strain. Scopolamine dose-effect functions were comparable to those observed in other species, including non-human primates, wherein 37.5 µg/kg of scopolamine (administered intramuscularly) reduced responding approximately 50%. Thus, we were successful in developing the apparatus and automated operant behavioral tests necessary to characterize drug safety in this swine strain. This capability will be valuable for characterizing chemical agent toxicity as well as the safety and efficacy of medical countermeasures.

Evaluation of adenosine A1 receptor agonists as neuroprotective countermeasures against Soman intoxication in rats.

Soman, an organophosphorus (OP) compound, disrupts nervous system function through inactivation of acetylcholinesterase (AChE), the enzyme that breaks down acetylcholine at synapses. Left untreated, a state of prolonged seizure activity (status epilepticus, SE) is induced, causing widespread neuronal damage and associated cognitive and behavioral impairments. Previous research demonstrated that therapeutic stimulation of A1 adenosine receptors (A1ARs) can prevent or terminate soman-induced seizure. This study examined the ability of three potent A1AR agonists to provide neuroprotection and, ultimately, prevent observable cognitive and behavioral deficits following exposure to soman. Sprague Dawley rats were challenged with a seizure-inducing dose of soman (1.2 x LD50) and treated 1 min later with one of the following A1AR agonists: (6)-Cyclopentyladenosine (CPA), 2-Chloro-N6-cyclopentyladenosine (CCPA) or N-bicyclo(2.2.1)hept-2-yl-5'-chloro-5'-deoxyadenosine (cdENBA). An active avoidance shuttle box task was used to evaluate locomotor responses to aversive stimuli at 3, 7 and 14 days post-exposure. Animals treated with CPA, CCPA or cdENBA demonstrated a higher number of avoidance responses and a faster reaction to the aversive stimulus than the soman/saline control group across all three sessions. Findings suggest that A1AR agonism is a promising neuroprotective countermeasure, capable of preventing the long-term deficits in learning and memory that are characteristic of soman intoxication.

Carfentanil toxicity in the African green monkey: Therapeutic efficacy of naloxone.

Carfentanil is an ultra-potent opioid with an analgesic potency 10,000 times that of morphine but has received little scientific investigation. Three experiments were conducted to evaluate the toxicity of carfentanil and the efficacy of naloxone in adult male African green monkeys. The first experiment determined the ED50 (found to be 0.71 µg/kg) of subcutaneous carfentanil for inducing bradypnea and/or loss of posture. Experiment 2 attempted to establish the ED50 of naloxone for rapidly reversing the bradypnea/loss of posture induced by carfentanil (1.15 µg/kg). Experiment 3 evaluated the effects of carfentanil (0.575 µg/kg) alone, the safety of naloxone (71-2841 µg/kg), and the efficacy of naloxone (71-710 µg/kg) administration at two time points following carfentanil (1.15 µg/kg) on operant choice reaction time. Collectively, these experiments characterized the temporal progression of carfentanil-induced toxic signs, determined the range of naloxone doses that restored respiratory and gross behavioral function, and determined the time course and range of naloxone doses that partially or completely reversed the effects of carfentanil on operant choice reaction time performance in African green monkeys. These results have practical relevance for the selection of opioid antagonists, initial doses, and expected functional outcomes following treatment of synthetic opioid overdose in a variety of operational/emergency response contexts.

Behavioral toxicity of sodium cyanide following oral ingestion in rats: Dose-dependent onset, severity, survival, and recovery.

Sodium cyanide (NaCN) is a commonly and widely used industrial and laboratory chemical reagent that is highly toxic. Its availability and rapid harmful/lethal effects combine to make cyanide a potential foodborne/waterborne intentional-poisoning hazard. Thus, laboratory studies are needed to understand the dose-dependent progression of toxicity/lethality following ingestion of cyanide-poisoned foods/liquids. We developed an oral-dosing method in which a standard pipette was used to dispense a sodium cyanide solution into the cheek, and the rat then swallowed the solution. Following poisoning (4-128 mg/kg), overt toxic signs were recorded and survival was evaluated periodically up to 30 hours thereafter. Toxic signs for NaCN doses higher than 16 mg/kg progressed quickly from head burial and mastication, to lethargy, convulsions, gasping/respiratory distress, and death. In a follow-on study, trained operant-behavioral performance was assessed immediately following cyanide exposure (4-64 mg/kg) continuously for 5 h and again the following day. Onset of behavioral intoxication (i.e., behavioral suppression) occurred more rapidly and lasted longer as the NaCN dose increased. This oral-consumption method with concomitant operantbehavioral assessment allowed for accurate dosing and quantification of intoxication onset, severity, and recovery, and will also be valuable in characterizing similar outcomes following varying medical countermeasure drugs and doses.

Behavioral intoxication following voluntary oral ingestion of tetramethylenedisulfotetramine: Dose-dependent onset, severity, survival, and recovery.

Tetramethylenedisulfotetramine (tetramine, or TETS) is a highly toxic rodenticide that has been responsible for over 14,000 accidental and intentional poisonings worldwide. Although the vast majority of TETS poisonings involved tainted food or drink, the laboratory in vivo studies of TETS intoxication used intraperitoneal injection or gavage for TETS exposure. Seeking to develop and characterize a more realistic model of TETS intoxication in the present study, rats were trained to rapidly and voluntarily consume a poisoned food morsel. Initially, the overt toxic effects of TETS consumption across a large range of doses were characterized, then a focused range of doses was selected for more intensive behavioral evaluation (in operant test chambers providing a variable-interval schedule of food reinforcement). The onset of intoxication following voluntary oral consumption of TETS was rapid, and clear dose-dependent response-rate suppression was observed across multiple performance measures within the operant-chamber environment. At most doses, recovery of operant performance did not occur within 30h. Food consumption and body weight changes were also dose dependent and corroborated the behavioral measures of intoxication. This voluntary oral-poisoning method with concomitant operant-behavioral assessment shows promise for future studies of TETS (and other toxic chemicals of interest) and may be extremely valuable in characterizing treatment outcomes.

VX toxicity in the Göttingen minipig.

The present experiments determined the intramuscular LD50 of VX in male Göttingen minipigs at two stages of development. In pubertal animals (115 days old), the LD50 of VX was indeterminate, but approximated 33.3µg/kg. However, in sexually mature animals (152 days old), the LD50 was estimated to be only 17.4µg/kg. Signs of nerve agent toxicity in the Göttingen minipig were similar to those described for other species, with some notable exceptions (such as urticaria and ejaculation). Latencies to the onset of sustained convulsions were inversely related to the administered dose of VX in both ages of minipigs. Additionally, actigraphy was used to quantify the presence of tremor and convulsions and, in some cases, was useful for precisely estimating time of death. The main finding indicates that in minipigs, as in other species, even relatively small differences in age can substantially alter the toxicity of nerve agents. Additionally, actigraphy can serve as a non-invasive method of characterizing the tremors and convulsions that often accompany nerve agent intoxication.

Characterizing the behavioral effects of nerve agent-induced seizure activity in rats: increased startle reactivity and perseverative behavior.

The development and deployment of next-generation therapeutics to protect military and civilian personnel against chemical warfare nerve agent threats require the establishment and validation of animal models. The purpose of the present investigation was to characterize the behavioral consequences of soman (GD)-induced seizure activity using a series of behavioral assessments. Male Sprague-Dawley rats (n=24), implanted with a transmitter for telemetric recording of encephalographic signals, were administered either saline or 1.0 LD50 GD (110 µg/kg, sc) followed by treatment with a combination of atropine sulfate (2 mg/kg, im) and the oxime HI-6 (93.6 mg/kg, im) at 1 min post-exposure. Seizure activity was allowed to continue for 30 min before administration of the anticonvulsant diazepam (10 mg/kg, sc). The animals that received GD and experienced seizure activity had elevated startle responses to both 100- and 120-dB startle stimuli compared to control animals. The GD-exposed animals that had seizure activity also exhibited diminished prepulse inhibition in response to 120-dB startle stimuli, indicating altered sensorimotor gating. The animals were subsequently evaluated for the acquisition of lever pressing using an autoshaping procedure. Animals that experienced seizure activity engaged in more goal-directed (i.e., head entries into the food trough) behavior than did control animals. There were, however, no differences between groups in the number of lever presses made during 15 sessions of autoshaping. Finally, the animals were evaluated for the development of fixed-ratio (FR) schedule performance. Animals that experienced GD-induced seizure activity engaged in perseverative food trough-directed behaviors. There were few differences between groups on other measures of FR schedule-controlled behavior. It is concluded that the GD-induced seizure activity increased startle reactivity and engendered perseverative responding and that these measures are useful for assessing the long-term effects of GD exposure in rats.

Diet composition modifies the toxicity of repeated soman exposure in rats.

It was previously demonstrated that diet potently modulates the toxic effects of an acute lethal dose of the nerve agent soman. The current investigation was undertaken to examine the influence of diet on the cumulative toxicity of repeated soman administration. Rats were fed one of four distinct diets (standard, choline-enriched, glucose-enriched, or ketogenic) for four weeks prior to and throughout a repeated soman dosing and recovery regimen. Each diet group included animals exposed to an equivalent volume of saline that served as negative controls. In exposure Week 1, animals received three consecutive daily doses of 0.4 LD(50) soman. In exposure Week 2, animals received four consecutive daily doses of 0.5 LD(50) soman. In exposure Week 3, animals received five consecutive daily doses of 0.5 LD(50) soman. Week 4 constituted a post-exposure recovery evaluation. Throughout the experiment, behavioral function was assessed by a discriminated avoidance test that required intact sensory and motor function. Survival and body weight changes were recorded daily. Differences in toxicity as a function of diet composition became apparent during the first week. Specifically, rats fed the glucose-enriched diet showed pronounced intoxication during Week 1, resulting in imperfect survival, weight loss, and deteriorated avoidance performance relative to all other groups. All rats fed the glucose-enriched diet died by the end of exposure Week 2. In contrast, only 10% of animals fed the standard diet died by the end of Week 2. Also in Week 2, weight loss and disrupted avoidance performance were apparent for all groups except for those fed the ketogenic diet. This differential effect of diet composition became even more striking in Week 3 when survival in the standard and choline diet groups approximated 50%, whereas survival equaled 90% in the ketogenic diet group. Avoidance performance and weight loss measures corroborated the differential toxicity observed across diet groups. Upon cessation of soman exposure during the final week, recovery of weight and avoidance performance in survivors was comparable across diet groups. These results systematically replicate previous findings demonstrating that diet composition exacerbates or attenuates toxicity in rodents exposed acutely to organophosphorus compounds.

Diet composition exacerbates or attenuates soman toxicity in rats: implied metabolic control of nerve agent toxicity.

To evaluate the role of diet composition on nerve agent toxicity, rats were fed four distinct diets ad libitum for 28 d prior to challenge with 110 µg/kg (1.0 LD(50), sc) soman. The four diets used were a standard rodent diet, a choline-enriched diet, a glucose-enriched diet, and a ketogenic diet. Body weight was recorded throughout the study. Toxic signs and survival were evaluated at key times for up to 72 h following soman exposure. Additionally, acquisition of discriminated shuttlebox avoidance performance was characterized beginning 24h after soman challenge and across the next 8 d (six behavioral sessions). Prior to exposure, body weight was highest in the standard diet group and lowest in the ketogenic diet group. Upon exposure, differences in soman toxicity as a function of diet became apparent within the first hour, with mortality in the glucose-enriched diet group reaching 80% and exceeding all other groups (in which mortality ranged from 0 to 6%). At 72 h after exposure, mortality was 100% in the glucose-enriched diet group, and survival approximated 50% in the standard and choline-enriched diet groups, but equaled 87% in the ketogenic diet group. Body weight loss was significantly reduced in the ketogenic and choline-enriched diet groups, relative to the standard diet group. At 1 and 4h after exposure, rats in the ketogenic diet group had significantly lower toxic sign scores than all other groups. The ketogenic diet group performed significantly better than the standard diet group on two measures of active avoidance performance. The exacerbated soman toxicity observed in the glucose-enriched diet group coupled with the attenuated soman toxicity observed in the ketogenic diet group implicates glucose availability in the toxic effects of soman. This increased glucose availability may enhance acetylcholine synthesis and/or utilization, thereby exacerbating peripheral and central soman toxicity.

Effects of sarin on the operant behavior of guinea pigs.

The present study evaluated the dose-response effects of subacute exposure to sublethal doses of the organophosphorus (OP) chemical warfare nerve agent (CWNA) sarin (GB) on the operant behavior of guinea pigs. Dietary restricted guinea pigs, trained to respond for food under a progressive ratio (PR) schedule of reinforcement, were injected five times per week (Monday-Friday) for 2 weeks with fractions (0.1, 0.2, and 0.4) of the established LD(50) of GB (42 microg/kg). Changes in body weight, whole blood (WB) acetylcholinesterase (AChE) levels, and operant performances were monitored over the 2 weeks of GB exposure and for an additional 2 weeks following the termination of exposures. There were dose-related changes in body weight and WB AChE levels throughout the exposure and post-exposure periods. Several parameters of PR performance were disrupted during exposure to 0.4 LD50 GB, however, concurrent weight loss indicated the presence of overt toxicity. PR performance recovered following the termination of exposures. Lower doses (0.1 and 0.2 LD50) of GB failed to produce reliable effects on operant performance during the exposure period. Overall responding decreased during exposure to 0.4 LD50 GB, resulting in reduced response rates and break points. The decrease in overall response rates was attributed to an increase in pausing since there was no decrease in running rate. Motor effects of 0.4 LD50 GB were evident as an increase in the proportion of lever press durations > or = 1.0 s. In the present study, doses of GB lower than 0.4 LD50 produced no marked alteration of operant performance in guinea pigs, although WB AChE levels were maximally inhibited to 20% of control.

Gestational exposure to methylmercury retards choice in transition in aging rats.

Developmental exposure to methylmercury has behavioral effects that extend into adulthood and aging. In this study, methylmercury's prolonged effects on the acquisition of choice and sensitivity to changes in reinforcement rates were studied. Pregnant female rats were exposed to drinking water containing 0, 0.5, or 6.4 ppm Hg as methylmercury, resulting in about 40 and 500 microg/kg/day of mercury intake. Maternal exposure began at least 4 weeks before mating, and continued to postnatal day 16. Then all mercury exposure ended. The behavior of 1.7- and 2.3-year-old offspring was maintained under various concurrent schedules of reinforcement. Thus, one reinforcement schedule maintained left-lever responding and a separate one maintained right-lever responding. The animal could switch ("changeover") between the two levers at any time. For the first 30 min of a 3-h session, the left and right levers each produced reinforcement at the same rate and left:right response ratios were about 1:1. After 30 min, either the left lever became richer than the right; the right lever became richer than the left, or there was no change. Terminal reinforcer ratios (left:right) used were 9:1, 4:1, 3:1, 1:1, 1:3, 1:4, and 1:9. Response rates on the two levers were tracked continuously through a session. This novel procedure for examining choice, and its acquisition, in a single session, was validated through many comparisons with the extant literature. Both response rates and changeover rates were influenced by the reinforcer ratios for the 1.7-year-olds. Changeover rates were not influenced by reinforcement rate for their 2.3-year-old littermates. For the 1.7-year-olds, there was no effect of methylmercury on changeover or response rates and there was no interaction between exposure and reinforcer ratio. In controls and most methylmercury-exposed rats, response ratios (the measure of choice) approximately matched reinforcer ratios by the end of the single session. This is commonly interpreted as reflecting sensitivity to reinforcement rates. Methylmercury exposure did not affect this measure systematically. The single-session transition from baseline (response ratios about 1:1) to terminal performance was retarded in many methylmercury-exposed rats relative to controls, especially in the older rats. The 2.3-year-old control rats required about 20 to 25 reinforcers to complete one half of the 9:1 and 4:1 transitions, respectively, and exposed rats required about twice as many. Thus, prenatal methylmercury exposure specifically retarded the acquisition of choice in older rats. Methylmercury did not interfere with the final expression of choice. Moreover, two rate measures, lever-press rates and changeover rates, were not systematically affected by methylmercury. The acquisition of choice appears to be very sensitive to subtle consequences of developmental methylmercury exposure. The specific tactics greatly reduced the time required to study behavior in transition from a month in previous reports to a single session here.