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.

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.

CART peptide inhibits locomotor activity induced by simultaneous stimulation of D1 and D2 receptors, but not by stimulation of individual dopamine receptors.

CART (Cocaine- and amphetamine-regulated transcript) peptide has been implicated in playing a modulatory role in reward and reinforcement. Previously, our laboratory demonstrated that injections of CART peptide (CART 55-102) into the nucleus accumbens (NAc) attenuated both cocaine- and dopamine-induced increases in locomotor activity (LMA), and attenuated cocaine reward as well. In this study, the effects of CART peptide on LMA induced by dopamine receptor agonists were evaluated after intraaccumbal injections in male, Sprague-Dawley rats. Effects of the D1 receptor agonist SKF-81,297, saline, CART 55-102, or CART 55-102 and SKF-81,297 together were compared. The SKF-81,297-induced increase in LMA was potentiated by coadministration of CART, while injection of CART alone had no significant effect. Injection of the D2 agonist 7-OH-DPAT had no effect on LMA, and the combination of both 7-OH-DPAT and CART peptide also had no effect. Quinelorane, a D3 receptor agonist, did not alter LMA, nor did the combination of CART peptide and quinelorane. The next experiment examined the effects of CART peptide on LMA induced by coinjection of both the D1 agonist SKF-81,297 and the D2 agonist 7-OH-DPAT. The combination of SKF-81,297 and 7-OH-DPAT induced greater LMA than SKF-81,297 alone. Coadministration of CART peptide along with the D1 and D2 agonists reduced LMA. These results strongly suggest that CART peptide reduces the effects of psychostimulants by modulating the simultaneous activation of both D1 and D2 dopamine receptors rather than by affecting the action of any individual dopamine receptor.

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.

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.

CART peptides as modulators of dopamine and psychostimulants and interactions with the mesolimbic dopaminergic system.

Cocaine- and amphetamine-regulated transcript (CART) peptides (CART 55-102 and CART 62-102) are peptidergic neurotransmitters that are widely but specifically distributed throughout the brain, gut and other parts of the body. They are found in many brain regions associated with drug addiction including the nucleus accumbens, ventral tegmental area and ventral pallidum. Injections of CART 55-102 into the nucleus accumbens have no effect on basal locomotor activity. However, an injection of CART just before an i.p. injection of cocaine reduces the locomotor activating effects of cocaine. These and other data suggest that CART in the accumbens blunts the effects of cocaine. A hypothesis is that CART is homeostatic in the accumbens and tends to oppose large increases in dopamine signaling. These actions would therefore be able to regulate the effects of some abused drugs such as the psychostimulants.

Age-Related Susceptibility to Epileptogenesis and Neuronal Loss in Male Fischer Rats Exposed to Soman and Treated With Medical Countermeasures.

Elderly individuals compose a large percentage of the world population; however, few studies have addressed the efficacy of current medical countermeasures (MCMs) against the effects of chemical warfare nerve agent exposure in aged populations. We evaluated the efficacy of the anticonvulsant diazepam in an old adult rat model of soman (GD) poisoning and compared the toxic effects to those observed in young adult rats when anticonvulsant treatment is delayed. After determining their respective median lethal dose (LD50) of GD, we exposed young adult and old adult rats to an equitoxic 1.2 LD50 dose of GD followed by treatment with atropine sulfate and the oxime HI-6 at 1 min after exposure, and diazepam at 30 min after seizure onset. Old adult rats that presented with status epilepticus were more susceptible to developing spontaneous recurrent seizures (SRSs). Neuropathological analysis revealed that in rats of both age groups that developed SRS, there was a significant reduction in the density of mature neurons in the piriform cortex, thalamus, and amygdala, with more pronounced neuronal loss in the thalamus of old adult rats compared with young adult rats. Furthermore, old adult rats displayed a reduced density of cells expressing glutamic acid decarboxylase 67, a marker of GABAergic interneurons, in the basolateral amygdala and piriform cortex, and a reduction of astrocyte activation in the piriform cortex. Our observations demonstrate the reduced effectiveness of current MCM in an old adult animal model of GD exposure and strongly suggest the need for countermeasures that are more tailored to the vulnerabilities of an aging population.

Impaired auditory and contextual fear conditioning in soman-exposed rats.

Exposure to soman (GD) can result in prolonged seizures and subsequent neuropathology in a variety of brain regions including the amygdala and hippocampus. Both regions are believed to play important roles in the development and expression of fear conditioning. The purpose of this experiment was to test these conditioning tasks as a possible behavioral correlate of the observed neuropathology. Male rats were exposed to GD (1.0 or 1.2×LD50) or saline followed with injections of atropine sulfate, the oxime HI-6 and diazepam. Fear conditioning was conducted on post-exposure day (PED) 8 followed by measuring freezing to contextual and auditory conditioned stimuli on PED 9 and 10 respectively. Contextual and auditory fear conditioning was severely impaired in both the 1.0×LD50 and 1.2×LD50 GD groups. Both GD groups spent less time freezing than controls when returned to the context in which conditioning occurred. The 1.0×LD50 and 1.2×LD50 groups had very low levels of freezing following presentation of the auditory conditioned stimulus. Neuronal fiber degeneration was present in the piriform cortex, thalamus, and amygdala in GD-exposed animals regardless of dose. The present study suggests that contextual and auditory fear conditioning is impaired in GD-exposed rats possibly due to neuropathology observed in the hippocampus, amygdala and thalamus.

Maternal separation and handling affects cocaine self-administration in both the treated pups as adults and the dams.

Repeated maternal separation of pups from dams is often used as an early life stressor that causes profound neurochemical and behavioral changes in the pups that persist into adulthood. The effects of maternal separation on both the dams and the treated pups as adults on cocaine self-administration were examined using four separation conditions: 15- or 180-min separation (MS15 and MS180), brief handling without separation (MS0), and a nonhandled group (NH). The separations and handling occurred daily on postnatal days 2 to 15. The acquisition of cocaine self-administration (0.0625-1.0 mg/kg/infusion) was evaluated in the treated pups as adults. The MS180 group acquired cocaine self-administration at the lowest dose tested (0.0625 mg/kg/infusion), whereas the MS15s did not respond for cocaine at rates greater than that seen with saline administration. The NH group received the greatest number of infusions and intake at the highest doses. After self-administration, no differences were observed between groups in activity of two liver carboxylesterases involved in the inactivation of cocaine, ES10 and ES4. Maternal separation affected cocaine self-administration in the dams as well. Although there was an overall significant affect of treatment on cocaine self-administration, the length of separation (15 or 180 min) did not affect cocaine self-administration on the dams. The MS0 dams averaged a greater number of infusions per session than NH group during the 1st week of acquisition. These data suggest that in addition to the profound changes that occur in pups as result of maternal separation, the dams are also susceptible to alterations in behaviors.