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.

Behavioral, Pharmacokinetic, and Cardiovascular Evaluation of Candidate Oximes in African Green Monkeys.

Oxime reactivators are critical antidotes after organophosphate pesticide or nerve agent poisoning, directly restoring the function of inhibited acetylcholinesterase. In the continuing search for more broad-spectrum acetylcholinesterase reactivators, this study evaluated one of the leading next-generation oxime reactivators: methoxime, (1,1'-trimethylene bis[4-(hydroxyimino)methyl]pyridinium dichloride (MMB-4). The pharmacokinetics of both salts of MMB-4 (dichloride [2Cl] and dimethanesulphonate [DMS]) were characterized across a range of relevant doses (19, 58, and 116 µmol/kg, intramuscular) in a nonhuman primate model (male African green monkeys), and only subtle differences were observed between the salts. Additionally, the behavioral and cardiovascular safety of these MMB-4 salts was compared directly to other available oximes (HI-6 2Cl, HI-6 DMS, and pyridine-2-aldoxime chloride (2-PAM Cl)) at comparable projected doses. Automated operant behavioral tests were used to examine attention, motivation, visual discrimination, concept execution, and fine motor coordination after high doses of all oxime salts, and of all oximes studied, only the highest dose of 2-PAM Cl (447 µmol/kg) disrupted behavioral performance. Likewise, the effects of a range of doses of MMB-4 2Cl or DMS, HI-6 2Cl or DMS, or 2-PAM Cl on cardiovascular parameters were measured in African green monkeys implanted with telemetry devices. Only a small transient decrease in pulse pressure was observed following administration of the highest dose of MMB-4 DMS (116 µmol/kg). Thus, MMB-4 salts, up to the 9× equivalent of a projected autoinjector dose in humans, did not produce behavioral or cardiovascular toxicity in African green monkeys in the current study, and the pharmacokinetic parameters were orderly and predictable.

Time Course, Behavioral Safety, and Protective Efficacy of Centrally Active Reversible Acetylcholinesterase Inhibitors in Cynomolgus Macaques.

Galantamine hydrobromide and (-)huperzine A, centrally active reversible acetylcholinesterase inhibitors, are potentially superior to the current standard, pyridostigmine bromide, as a pretreatment for organophosphorus chemical warfare nerve agent intoxication. Galantamine, huperzine, and pyridostigmine were compared for time course of acetylcholinesterase inhibition in 12 cynomolgus macaques. Although both galantamine and huperzine shared a similar time course profile for acetylcholinesterase inhibition, huperzine was 88 times more potent than galantamine. The dose for 50% acetylcholinesterase inhibition (ID50) was 4.1 ug/kg for huperzine, 362 ug/kg for galantamine, and 30.9 ug/kg for pyridostigmine. In a safety assessment, galantamine, huperzine, and pyridostigmine were examined using an operant time-estimation task. Huperzine and pyridostigmine were devoid of behavioral toxicity, whereas galantamine was behaviorally toxic at doses producing peak acetylcholinesterase inhibition of about 50% and higher. Following pretreatment with galantamine, huperzine or pyridostigmine, monkeys were challenged with the median lethal dose of soman at the time of peak acetylcholinesterase inhibition and evaluated for overt signs of soman toxicity (cholinergic crisis, convulsions). Both huperzine and galantamine were equally effective at preventing overt signs of soman toxicity, but neither drug was capable of preventing soman-induced neurobehavioral disruption. In contrast, three of four pyridostigmine-pretreated animals exposed to soman exhibited convulsions and required therapy. Full functional recovery required 3-16 days. The degree of acetylcholinesterase inhibition was lower for pyridostigmine, but rates of recovery of acetylcholinesterase activity following soman challenge were comparable for all drug pretreatments. Huperzine may be the more promising centrally active reversible acetylcholinesterase inhibitor due to its greater potency and superior safety profile.

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.

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.

Delayed match-to-sample performance in African green monkeys (Chlorocebus aethiops sabaeus): effects of benzodiazepine, cholinergic, and anticholinergic drugs.

Delayed match-to-sample (DMTS) procedures are among the most commonly used attention and memory tasks in behavioral pharmacology and have been utilized in a variety of species. Although macaque species such as the rhesus and cynomolgus macaque are often used for such studies, availability and disease transmission raise concerns over their use. The present study investigated whether the African green monkey might function as a suitable alternative by evaluating operant performance on a DMTS task and comparing this species' response to some commonly used drugs (0.025-0.075 mg/kg physostigmine, 0.0033-0.03 mg/kg scopolamine, 0.014-0.44 mg/kg atropine, 0.125-1.0 mg/kg midazolam, and 0.125-2.0 mg/kg diazepam) to the responses previously reported in macaques. Results demonstrated that African green monkeys are capable of learning and performing a DMTS task, and dose-effect functions for behavioral pharmacology were quite similar to those reported for rhesus macaques and other nonhuman primate species. Thus, the African green monkey may function as a suitable alternative to macaque species in behavioral pharmacology research.

The oxime pro-2-PAM provides minimal protection against the CNS effects of the nerve agents sarin, cyclosarin, and VX in guinea pigs.

This study examined whether pro-2-PAM, a pro-drug dihydropyridine derivative of the oxime 2-pralidoxime (2-PAM) that can penetrate the brain, could prevent or reverse the central toxic effects of three nerve agents; sarin, cyclosarin, and VX. The first experiment tested whether pro-2-PAM could reactivate guinea pig cholinesterase (ChE) in vivo in central and peripheral tissues inhibited by these nerve agents. Pro-2-PAM produced a dose-dependent reactivation of sarin- or VX-inhibited ChE in both peripheral and brain tissues, but with substantially greater reactivation in peripheral tissues compared to brain. Pro-2-PAM produced 9-25% reactivation of cyclosarin-inhibited ChE in blood, heart, and spinal cord, but no reactivation in brain or muscle tissues. In a second experiment, the ability of pro-2-PAM to block or terminate nerve agent-induced electroencephalographic seizure activity was evaluated. Pro-2-PAM was able to block sarin- or VX-induced seizures (16-33%) over a dose range of 24-32 mg/kg, but was ineffective against cyclosarin-induced seizures. Animals that were protected from seizures showed significantly less weight loss and greater behavioral function 24 h after exposure than those animals that were not protected. Additionally, brains were free from neuropathology when pro-2-PAM prevented seizures. In summary, pro-2-PAM provided modest reactivation of sarin- and VX-inhibited ChE in the brain and periphery, which was reflected by a limited ability to block or terminate seizures elicited by these agents. Pro-2-PAM was able to reactivate blood, heart, and spinal cord ChE inhibited by cyclosarin, but was not effective against cyclosarin-induced seizures.

Conjugates of human serum butyrylcholinesterase and nerve agents are behaviorally safe in rhesus macaques.

Exogenously administered human serum butyrylcholinesterase (Hu BChE) affords protection by binding to organophosphorus (OP) nerve agents and pesticides in circulation. The resulting Hu BChE-OP conjugate undergoes 'aging' and the conjugate circulates until cleared from the body. Thus, we evaluated the effects of Hu BChE-OP conjugates on the general health and operant behavior of macaques. Rhesus macaques trained to perform a six-item serial probe recognition (SPR) task were administered 30 mg/kg of Hu BChE-soman conjugate (n = 4) or Hu BChE-VX conjugate (n = 4) by intramuscular injection. Performance on the SPR task was evaluated at 60-90 min after conjugate administration and daily thereafter for the next 4 weeks. Diazepam (3.2 mg/kg), a positive control, was administered 5 weeks after conjugate administration and performance on the SPR task was evaluated as before. Blood collected throughout the study was analyzed for acetylcholinesterase (AChE) and BChE activities. Residual BChE activity of conjugates displayed a similar pharmacokinetic profile as free Hu BChE. Neither of the Hu BChE-OP conjugates produced clear or pronounced degradations in performance on the SPR task. In contrast, diazepam clearly impaired performance on the SPR task on the day of administration in 7 of 8 macaques (and sometimes longer). Taken together, these results suggest that Hu BChE-OP conjugates are safe and provide further support for the development of Hu BChE as a bioscavenger for use in humans.

Implementation of Manual and Automated Water Regulation for Rats (Rattus norvegicus) and Ferrets (Mustela putorius).

Water regulation is a procedure that allows animals to consume water volumes equivalent to ad libitum access, but access is limited to specific time intervals (that is, water is not available outside of the designated access periods). Despite the relatively common use of water regulation in research, the implementation method is rarely detailed, stating only that water was available in the animal's home cage at specific times. For planned toxicologic assessments, we placed rats (n = 510) and ferrets (n = 16) on water regulation using both automated and manual methods. In testing our systems, we defined "successful implementation" as maintenance of appropriate weight gain and health status. An automated system that controlled water access to an entire rat rack was successful for most rats, but several rats failed to consume enough water even after 2 wk of experience. Manual methods of water regulation were successful in rats by either moving the cage to prevent access to the drinking valve or by placing/removing water bottles. An automated system that controlled water access from water bottles was implemented for ferrets and was maintained for up to 30 wk. Retrospective comparison of body weights to standard growth curves for both species showed that all animals grew normally despite water regulation. Differences in the systems and some species considerations provide insights into the key elements necessary for successful water regulation in rats and ferrets.

Organoleptic assessment and median lethal dose determination of oral aldicarb in rats.

Aldicarb, a carbamate pesticide, is an acetylcholinesterase inhibitor, with oral median lethal dose (LD50 ) estimates in rats ranging from 0.46 to 0.93 mg/kg. A three-phase approach was used to comprehensively assess aldicarb as an oral-ingestion hazard. First, the solubility of aldicarb in popular consumer beverages (bottled water, apple juice, and 2% milk) was assessed. Lethality was then assessed by administering aldicarb in bottled water via gavage. A probit model was fit to 24-h survival data and predicted a median lethal dose of 0.83 mg/kg (95% CI: 0.54-1.45 mg/kg; slope: 4.50). Finally, organoleptic properties (e.g., taste, smell, and texture) were assessed by allowing rats to voluntarily consume 3.0 mL of the above beverages as well as liquid eggs adulterated with aldicarb at various concentrations. This organoleptic assessment determined that aldicarb was readily consumed at lethal and supralethal doses. Overt toxic signs presented within 5 min post-ingestion, and all rats died within 20 min after consuming the highest concentration (0.542 mg/mL), regardless of amount consumed. Because rats have more developed chemoreceptive capabilities than humans, these results suggest that aldicarb may be consumed in toxic or even lethal concentrations by humans in a variety of beverages or foods.

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.

Human plasma-derived butyrylcholinesterase is behaviorally safe and effective in cynomolgus macaques (Macaca fascicularis) challenged with soman.

Organophosphorus compounds (OP) pose a significant threat. Administration of human butyrylcholinesterase (HuBChE) may reduce or prevent OP toxicity. Thus, we evaluated the safety and efficacy of HuBChE in monkeys using sensitive neurobehavioral tests while concurrently characterizing absorption and elimination in the presence and absence of high-dose soman exposure to predict time course and degree of protection. Eight young adult male cynomolgus macaques were trained on two distinct automated tests of neurobehavioral functioning. HuBChE purified under current Good Manufacturing Practices (CGMP) was injected intramuscularly at 13.1 mg/kg, producing an average peak plasma value (Cmax) of over 27 Units/ml. The apparent time to maximum concentration (Tmax) approximated 7 h, the elimination half-life approximated 102 h, and plasma levels returned to pre-administration (baseline) levels by 14 days. No behavioral disruptions following HuBChE administration were observed on either neurobehavioral test, even in monkeys injected 24 h later with an otherwise lethal dose of soman. Thus, HuBChE provided complete neurobehavioral protection from soman challenge. The present data replicate and extend previous results from our laboratory that had used a different route of administration (intravenous), a different species (rhesus macaque), and a different BChE product (non-CGMP material). The addition of two sensitive neurobehavioral tests coupled with the PK/PD results convincingly demonstrates the neurobehavioral safety of plasma-derived HuBChE at therapeutic levels. Protection against an otherwise-lethal dose of soman by a pre-exposure treatment dose that is devoid of side effects establishes a foundation for additional testing using other exposure routes and treatment times, other challenge agents/routes, or other classes of organophosphate scavengers.

Survey of drug therapies against acute oral tetramethylenedisulfotetramine poisoning in a rat voluntary consumption model.

Ingestion of the noncompetitive GABAA receptor antagonist tetramethylenedisulfotetramine (TETS) results in arrhythmias, respiratory depression, and life-threatening convulsive status epilepticus. We have previously developed a realistic model of voluntary TETS consumption, in which rats promptly consumed a piece of cereal containing a dose of TETS that led to rapid progression of toxic signs (including convulsions) and profound and enduring behavioral suppression. Recently, this model was used to survey nine different drugs from distinct drug classes over a large range of doses to identify possible therapeutics. The drugs included three benzodiazepines (diazepam, midazolam, and lorazepam), two barbiturates (phenobarbital and pentobarbital), the GABAA allosteric modulator allopregnanolone, and three non-traditional therapeutics (dexmedetomidine, ketamine, and ethanol). Treatment was administered intraperitoneally 10 min after consumption of the cereal morsel containing TETS (600 µg/kg). This exposure model resulted in a survival rate of 30% in vehicle-treated rats. Diazepam (12.5 mg/kg) and midazolam (25 mg/kg), compared to vehicle, significantly increased survival (75 and 100% respectively) but at only one of the three doses tested. Lorazepam increased survival across a wide range of doses (1.56-25 mg/kg) with survival rates between 80-100%. Phenobarbital (100 mg/kg) was the only other drug and non-benzodiazepine to improve survival rates (80%). Although the four aforementioned therapeutics increased survival, TETS-induced weight loss, food wastage, and behavioral deficits remained in survivors.

Behavioural and physiological assessments of dimethyl trisulfide treatment for acute oral sodium cyanide poisoning.

Sodium cyanide (NaCN) is a commonly and widely used industrial and laboratory chemical that is highly toxic. Its availability and rapid harmful/lethal effects combine to make cyanide a potential foodborne/waterborne intentional-poisoning hazard. Effective antidotes to cyanide poisoning are currently approved only for intravenous administration. Therefore, an effective cyanide antidote that can be administered intramuscularly in pre-hospital and/or mass-casualty settings is needed. Dimethyl trisulfide (DMTS) is a naturally occurring substance used as a flavour enhancer in foods. DMTS has shown antidotal efficacy in cyanide poisoning and is thought to act as both a sulphur donor and partial methaemoglobin inducer. In this study, an intramuscular injection of DMTS (6.25-200 mg/kg) was given to rats 1 minute after an oral dose of NaCN (98.2 mg/kg; twice the median lethal dose) to test the antidotal efficacy and safety of DMTS treatment. Toxic signs and survival were examined along with behavioural function (up to 30 hour after ingestion) using a previously established operant behavioural model. A large range of DMTS doses (6.25-100 mg/kg) increased survival after oral cyanide poisoning, and the lower DMTS doses (6.25-25 mg/kg) also proved to be behaviourally and physiologically safe. Larger DMTS doses (50-200 mg/kg) produced side effects (ie, inflammation and limping) that were more severe and protracted than those observed at lower DMTS doses. The 25 mg/kg DMTS proved to be the most efficacious (increasing survival from 20% to 75%) and also produced minimal side effects (eg, inflammation) that resolved within 24-72 hour. Thus, DMTS shows promise as an intramuscularly administered cyanide antidote useful for prompt pre-hospital or mass-casualty emergency medical treatment.

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.

Sex and the stimulus-movement effect: Differences in acquisition of autoshaped responding in cynomolgus monkeys.

The stimulus-movement effect refers to the phenomenon in which stimulus discrimination or acquisition of a response is facilitated by moving stimuli as opposed to stationary stimuli. The effect has been found in monkeys, rats, and humans, but the experiments conducted did not provide adequate female representation to investigate potential sex differences. The current experiment analyzed acquisition of stimulus touching in a progressive series of classical conditioning procedures in cynomolgus monkeys (Macaca fascicularis) as a function of sex and stimulus movement. Classical conditioning tasks arrange two or more stimuli in relation to each other with different temporal and predictive relations. Autoshaping procedures overlay operant contingencies onto a classical-conditioning stimulus arrangement. In the present case, a neutral stimulus (a small gray square displayed on a touchscreen) functioned as the conditional stimulus and a food pellet functioned as the unconditional stimulus. Although touching is not required to produce food, with repeated stimulus pairings subjects eventually touch the stimulus. Across conditions of increasing stimulus correlation and temporal contiguity, male monkeys acquired the response faster with a moving stimulus. In contrast, females acquired the response faster with a stationary stimulus. These results demonstrate that the stimulus-movement effect may be differentially affected by sex and indicate that additional experiments with females are needed to determine how sex interacts with behavioral phenomena discovered and elaborated almost exclusively using males.

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.

Bioscavenger for protection from toxicity of organophosphorus compounds.

Current antidotal regimens for organophosphorus compound (OP) poisoning consist of a combination of pretreatment with a spontaneously reactivating AChE inhibitor such as pyridostigmine bromide, and postexposure therapy with anticholinergic drugs such as atropine sulfate and oximes such as 2-PAM chloride (Gray, 1984). Although these antidotal regimens are effective in preventing lethality of animals from OP poisoning, they do not prevent postexposure incapacitation, convulsions, performance deficits, or, in many cases, permanent brain damage (Dunn and Sidell, 1989). These problems stimulated the development of enzyme bioscavengers as a pretreatment to sequester highly toxic OPs before they reach their physiological targets. Several studies over the last two decades have demonstrated that exogenously administered human serum butyrylcholinesterase (Hu BChE) can be used successfully as a safe, efficacious, and single prophylactic treatment to counteract the toxicity of OPs. It also has potential use for first responders (civilians) reacting to terrorist nerve gas release, pesticide overexposure, or succinylcholine-induced apnea. A dose of 200 mg of Hu BChE in humans is envisioned as a prophylactic treatment that can protect from exposure of 2-5 x LD50 of nerve agents (Ashani, 2000).

Serial-probe recognition in rhesus macaques: effects of midazolam.

A serial-probe recognition task was used to assess the effects of midazolam on visual attention and short-term memory in three rhesus monkeys. On each trial, six unique alphanumeric sample stimuli (list items) were presented sequentially followed by a choice period. Choosing the 'probe' stimulus was correct if the probe matched one of the list items; otherwise, choosing the 'default' stimulus (a white square) was correct. Behavior was examined under a range of doses of midazolam (0.065, 0.13, 0.26, and 0.52 mg/kg IM). Midazolam did not significantly reduce choice accuracy or change the shape of the serial position function and did not significantly reduce choice responding. However, choice reaction time was significantly increased by the two highest doses of midazolam. Responding directed at the sample stimuli was reduced at the two highest doses of midazolam. Furthermore, 0.52 mg/kg midazolam significantly increased sample-stimulus reaction time at all six serial positions. Overall, these data suggest that the two highest doses of midazolam tested increase reaction time, but do not directly impair short-term visual recognition memory. This is noteworthy because such doses appear capable of protecting against nerve agent-induced seizures.

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.