Benedict R. Capacio

The application of the fluoride reactivation process to the detection of sarin and soman nerve agent exposures in biological samples

The fluoride reactivation process was evaluated for measuring the level of sarin or soman nerve agents reactivated from substrates in plasma and tissue from in vivo exposed guinea pigs (Cava porcellus), in blood from in vivo exposed rhesus monkeys (Macaca mulatta), and in spiked human plasma and purified human albumin. Guinea pig exposures ranged from 0.05 to 44 LD50, and reactivated nerve agent levels ranged from 1.0 ng/mL in plasma obtained from 0.05 LD50 sarin‐exposed guinea pigs to an average of 147 ng/g in kidney tissue obtained from two 2.0 LD50 soman‐exposed guinea pigs. Positive dose–response relationships were observed in all low‐level, 0.05 to 0.4 LD50, exposure studies. An average value of 2.4 ng/mL for reactivated soman was determined in plasma obtained from two rhesus monkeys three days after a 2 LD50 exposure. Of the five types of guinea pig tissue studied, plasma, heart, liver, kidney and lung, the lung and kidney tissue yielded the highest amounts of reactivated agent. In similar tissue and with similar exposure procedures, reactivated soman levels were greater than reactivated sarin levels. Levels of reactivated agents decreased rapidly with time while the guinea pig was alive, but decreased much more slowly after death. This latter chemical stability should facilitate forensic retrospective identification. The high level of reactivated agents in guinea pig samples led to the hypothesis that the principal source of reactivated agent came from the agent‐carboxylesterase adduct. However, there could be contributions from adducts of the cholinesterases, albumin and fibrous tissue, as well. Quantitative analysis was performed with a GC‐MS system using selected ion monitoring of the 99 and 125 ions for sarin and the 99 and 126 ions for soman. Detection levels were as low as 0.5 ng/mL. The assay was precise and easy to perform, and has potential for exposure analysis from organophosphate nerve agents and pesticides in other animal species.

Antagonism of soman-induced convulsions by midazolam, diazepam and scopolamine

The effects of midazolam (MDZ), diazepam (DZ) and scopolamine (SCP) therapies on soman-induced electrocorticogram (ECoG) and biceps femoris electromyogram (EMG) activities and brain lesions were assessed in male rats. Animals received pyridostigmine (26 micrograms/kg, im) 30 min before soman (87.1 micrograms/kg, im) followed by therapy consisting of atropine (1.5 mg/kg) admixed with 2-PAM (25 mg/kg, im) 1 min later; MDZ (0.5 mg/kg), DZ (1.77 mg/kg) or SCP (0.43 mg/kg) was administered im at 1 min after the onset of convulsions (CVs). Typically, within 5 min after soman the ECoG profile changed to a full-blown, spike-and-dome epileptiform (SDE) pattern followed by CVs and increased amplitude of EMG activity. Treatment with SCP restored ECoG and EMG profiles by 30 min. At 2 hr after exposure only 1 animal demonstrated a slight abnormality in ECoG activity which was normal at 24 hr. Similarly, DZ and MDZ restored EcoG and EMG profiles by 30 min; however, in contrast to SCP, 83% of the animals demonstrated reappearance of SDE 2 hrs after soman. SCP therapy also enabled rats to move about in their cages by 30 min post treatment. In contrast, DZ- and MDZ-treated rats remained incapacitated as late as 2 hr post-exposure. Animals were euthanized at 24 hr, and the extent of soman-induced brain lesions was determined by light microscopic analysis. When present, brain lesions were minimal in SCP-treated rats. The mean brain lesion scores across all experimental conditions ranked as follows: soman control > MDZ > DZ > or = SCP = saline control. These observations suggest that SCP may be highly effective in severe soman intoxication.

Antagonism of botulinum toxin A-mediated muscle paralysis by 3, 4-diaminopyridine delivered via osmotic minipumps.

The ability of 3,4-diaminopyridine (3,4-DAP) to antagonize muscle paralysis following local injection of botulinum neurotoxin A (BoNT/A) complex was evaluated in the in situ rat extensor digitorum longus (EDL) preparation. The minipumps were implanted 6 h prior to BoNT/A administration and delivered their contents over a 7-day period producing a steady plasma 3,4-DAP concentration of 27-29 microM. In the absence of 3,4-DAP, a local injection of five mouse LD(50) units of BoNT/A led to total paralysis of EDL muscles within 24 h of application. Recovery from paralysis was slow, remaining at <30% of control 14 days after toxin injection. 3,4-DAP delivery by osmotic minipumps antagonized the actions of BoNT/A on neuromuscular transmission. Seven days after the onset of 3,4-DAP infusion, indirectly elicited twitch and tetanic tensions in BoNT/A-injected EDL muscles were 72.4 and 46.9% of control, respectively. In the absence of 3,4-DAP, twitch and tetanic tensions were only 5.4 and 15. 1% of control. The benefits conferred by 3,4-DAP treatment were not maintained after minipumps were removed. Seven days after cessation of 3,4-DAP infusion, twitch and tetanic tensions were not significantly different from those observed in muscles receiving BoNT/A alone. It is concluded that 3,4-DAP may be useful for treatment of BoNT/A-induced muscle paralysis, but sustained delivery of the drug would be required for the entire period of BoNT intoxication to maintain muscle function.

4-Aminopyridine reverses saxitoxin (STX)- and tetrodotoxin (TTX)-induced cardiorespiratory depression in chronically instrumented guinea pigs

The extent to which cardiorespiratory infirmity and other sublethal effects of saxitoxin (STX) and tetrodotoxin (TTX) can be reversed by 4-aminopyridine (4-AP) was investigated in guinea pigs chronically instrumented for the concurrent electrophysiological recordings of electrocorticogram (ECoG), diaphragmatic electromyogram (DEMG), Lead II electrocardiogram, and neck skeletal muscle electromyogram. Animals were intoxicated with either STX or TTX (2 and 3 microg/kg, im) to produce a state of progressive cardiorespiratory depression (depicted by decreasing DEMG amplitude, bradypnea, and bradycardia). At the point where cardiorespiratory performance was most seriously compromised (approximately 30 min posttoxin), 4-AP (1 or 2 mg/kg, im) was administered. The therapeutic effect of 4-AP was striking in that, within minutes, the toxin-induced diaphragmatic blockade, bradypnea, bradycardia, and depressed cortical activity were all restored to a level either comparable to, or surpassing, that of control. The optimal 4-AP dose level was determined to be 2 mg/kg (im) based on analyses of cardiorespiratory activity profiles throughout the course of intoxication and 4-AP treatment. At the dose levels (either 1 or 2 mg/kg) used to restore ventilatory function and cardiovascular performance, 4-AP produced no sign of seizures and convulsions. Although less serious secondary effects such as cortical excitant/arousal effect (indicated by ECoG power spectral analysis) and transient periods of skeletal muscle fasciculation were observed, these events were of minor concern particularly in view of the remarkable therapeutic effects of 4-AP.

Central and peripheral cardio-respiratory effects of saxitoxin (STX) in urethane-anesthetized guinea-pigs

Effects of saxitoxin (STX; 10 micrograms/kg; i.p.) on cardio-respiratory activities were evaluated in urethane-anesthetized guinea-pigs. Concurrent recordings were made of electrocorticogram (ECoG), bulbar respiratory-related unit activities, diaphragmatic electromyogram (DEMG), electrocardiogram (Lead II ECG), blood pressure, heart rate, end-tidal CO2, arterial O2/CO2 tensions, and arterial pH. The average time to STX-induced respiratory failure was about 10 min. The most striking effect prior to apnea was a state of progressive bradypnea which emerged 5-7 min after the toxin administration. Other noteworthy responses included (i) a time-dependent decrease in ECoG amplitudes which typically began before the development of a bradypneic profile; (ii) an increasing degree of diaphragm neuromuscular blockade; (iii) a state of combined hypercapnia and uncompensated acidemia; (iv) a declining blood pressure; (v) an incrementally dysfunctional myocardial performance; and (vi) an increasingly degenerative central respiratory activity profile which ultimately culminated in a complete loss of central respiratory drive. The therapeutic effect of intratracheally administered oxygen was equivocal in that the cardio-respiratory activities, be they of central of peripheral nature, remained conspicuously dysfunctional and precarious despite 100% oxygen ventilation. What can be inferred from this study is two-fold. First, STX-induced ventilatory insufficiency can be attributed to a loss of functional integrity of both central and peripheral respiratory system components. That is, although diaphragm blockade contributes significantly to STX-induced respiratory failure, analyses of single respiratory unit activity data revealed that the central respiratory rhythmogenic mechanism also appeared to play a pivotal role in the development of a bradypneic profile which promotes, and directly causes, a complete loss of respiratory drive. Second, a state of unabating depression of central respiratory activities, which seemed to be refractory to the effect of O2, suggests STX has a direct and persistent action on medullary rhythmogenic mechanisms. In conclusion, these findings indicate that both central and peripheral cardio-respiratory components are critically involved in STX-induced apnea, dysfunctional cardiovascular performance, and lethality.

Pharmacokinetic Studies of Intramuscular Midazolam in Guinea Pigs Challenged with Soman

Studies have demonstrated that benzodiazepine compounds are effective at antagonizing seizure activity produced by the organophosphate (OP) cholinesterase inhibitor soman. In this present study we have investigated the pharmacokinetics of midazolam and its associated effects on electroencephalographic (EEG) activity following intramuscular (im) injection to soman‐exposed guinea pigs (Crl:(HA)BR). Prior to experiments, the animals were surgically implanted with EEG leads to monitor seizure activity. For the study, animals were administered the following pretreatment/OP/treatment regimen. Pyridostigmine bromide (0.026 mg/kg, im) was given 30 min prior to soman (56 µg/kg, 2 × LD50; subcutaneously, sc), followed in one minute by atropine sulfate (2 mg/kg, im) and pralidoxime chloride (25 mg/kg, im). All animals receiving this regimen developed seizure activity. Midazolam 0.8 mg/kg, im, was administered 5 min after onset of seizure activity. Based on EEG data, animals were categorized as either seizure‐terminated or seizure not‐terminated at 30 min following anticonvulsant administration. Serial blood samples were collected for the plasma midazolam analysis; the assay was accomplished with a gas chromatograph/mass spectrometer. The mean time to seizure termination was 8.8 ± 1.6 min. The mean time‐plasma concentration data were fit to standard pharmacokinetic models. The following parameter estimates were determined from the model‐fit for seizure terminated and not‐terminated animals respectively: apparent volumes of distribution (Vd) were 1.4 and 1.7 l/kg; area under the time‐concentration curves (AUC), 15,990 and 15,120 ng · min/ml; times to maximal plasma concentration (Tmax), 1.66 and 2.91 min and maximal plasma concentrations (Cmax) 535.1 and 436.6 ng/ml. These data indicate that im injection of midazolam is effective at terminating ongoing soman‐induced seizure activity. Additionally, the relatively short Tmax and latency to seizure termination demonstrate the rapidity of drug absorption and action respectively.

4-Aminopyridine antagonizes saxitoxin- and tetrodotoxin-induced cardiorespiratory depression

Antagonism of saxitoxin-and tetrodotoxin-induced lethality by 4-aminopyridine was studied in urethane-anesthetized guinea pigs instrumented for the concurrent recordings of medullary respiratory-related unit activities (Bötzinger complex and Nu. para-Ambiguus), diaphragmatic electromyogram, electrocorticogram, Lead II electrocardiogram, blood pressure, end-tidal CO2 and arterial O2/CO2/pH. The toxin (either saxitoxin or tetrodotoxin) was infused at a dose rate of 0.3 microgram/kg/min (i.v.) to produce a state of progressive cardiorespiratory depression. The animals were artificially ventilated when the magnitude of integrated diaphragm activities was reduced to 50% of control. Immediately after the disappearance of the diaphragm electromyogram, the toxin infusion was terminated, and 4-aminopyridine (2 mg/kg, i.v.) was administered. The therapeutic effect of 4-aminopyridine was striking in that the toxin-induced blockade of diaphragmatic neurotransmission, vascular hypotension, myocardial anomalies, bradycardia and aberrant discharge patterns of medullary respiratory-related neurons could all be promptly restored to a level comparable to that of control condition. The animals were typically able to breathe spontaneously within minutes after 4-aminopyridine. At the dose level used to achieve the desired therapeutic responses, 4-aminopyridine produced no sign of seizure and convulsion. Although less serious side-effects such as cortical excitant/arousal and transient periods of fascicular twitch could be observed, these events were of minor concern, in our opinion, particularly in view of the remarkable therapeutic effects of 4-aminopyridine.

Use of the accelerating rotarod for assessment of motor performance decrement induced by potential anticonvulsant compounds in nerve agent poisoning

The accelerating rotarod was used to assess motor performance decrement in rats after administration of candidate anticonvulsant compounds (acetazolamide, amitriptyline, chlordiazepoxide, diazepam, diazepam-lysine, lorazepam, loprazolam, midazolam, phenobarbital and scopolamine) against nerve agent poisoning. All compounds were tested as the commercially available injectable preparation except for diazepam-lysine and loprazolam, which are not FDA approved. A peak effect time, as well as a dose to decrease performance time by 50% from control (PDD50), was determined. The calculated PDD50 (mumol/kg) values and peak effect times were midazolam, 1.16 at 15 min; loprazolam, 1.17 at 15 min; diazepam-lysine, 4.17 at 30 min; lorazepam, 4.98 at 15 min; diazepam, 5.27 at 15 min; phenobarbital, 101.49 at 45 min; chlordiazepoxide, 159.21 at 30 min; scopolamine, amitriptyline and acetazolamide did not demonstrate a performance decrement at any of the doses tested. The PDD50 values were compared with doses which have been utilized against nerve agent-induced convulsions or published ED50 values from standard anticonvulsant screening tests (maximal electroshock [MES] and subcutaneous pentylenetetrazol [scMET]). The results suggest that at anticonvulsant doses against nerve agents, all the benzodiazepines and phenobarbital have the potential to cause a performance decrement, whereas candidate anticonvulsants of the non-benzodiazepine or non-barbiturate type would not be expected to demonstrate this effect on motor performance. It is concluded that compounds such as acetazolamide, amitriptyline and scopolamine offer alternatives to the highly decrementing benzodiazepines and phenobarbital and should be further tested as anticonvulsant candidates against nerve agent intoxication.

Pharmacokinetics and Pharmacodynamics of 4-Aminopyridine in Awake Guinea Pigs

The selective blockade of potassium channels on excitable membranes by 4-aminopyridine (4-AP) leads to facilitation of neurotransmitter release at a wide variety of synapses. This compound has been shown to be efficacious against lethality induced by saxitoxin (STX) and tetrodotoxin (TTX) in guinea pigs. To characterize the actions of 4-AP in guinea pigs we have investigated its pharmacokinetics (PK) and pharmacodynamics following a 2 mg/kg, intramuscular (im) dose in awake chronically instrumented (IN) animals. Animals were chronically instrumented for electrophysiologic recordings of diaphragmatic electromyogram (DEMG), lead II electrocardiogram (ECGII) and electrocorticogram (ECoG). Also, PK studies were carried out in uninstrumented (UN) guinea pigs. Blood and electrophysiologic data were collected at predetermined time intervals up to 4 hours post 4-AP administration. High performance liquid chromatography was used to determine plasma 4-AP concentrations. For IN and UN animals, plasma concentration-time data best fit a one-compartment model, and PK parameter estimates were similar for both groups. Peak plasma levels were found to occur between 16 and 17 min, and the half-lives of elimination were 65 and 71 min for IN and UN animals respectively. Heart and respiratory rates were elevated as early as 5 and 15 min respectively in response to 4-AP administration. The duration of action was approximately 1-1.5 half-lives of elimination beyond peak plasma levels. Maximum ECoG responses were observed between 12-15 min after 4-AP injection; some residual drug effects were still apparent at 240 min. The difference between the heart and respiratory rates and ECoG profiles suggests that these different physiological systems respond with varying degrees of sensitivity to plasma 4-AP concentrations. The stimulation of these systems is consistent with the action of 4-AP in reversing STX- and TTX-induced cardiorespiratory depression and decreased ECoG power in guinea pigs.

Recovery from the lethal effects of saxitoxin: a therapeutic window for4-aminopyridine (4-AP)

We have shown that saxitoxin (STX) induced lethality can be reversed by 4-AP when it is administered at the time of respiratory arrest [Benton, B. J., Spriggs, D. L., Capacio, B. R. and Chang, F.-C. T. (1995) 4-Aminopyridine antagonizes the lethal effects of saxitoxin (STX) and tetrodotoxin (TTX). International Society of Toxicology, 5th Pan American Symposium on Animal, Plant and Microbial Toxins, Frederick, MD. July/August 1995, p. 217]. The purpose of this study was to determine whether 4-APs efficacy could be enhanced further when administered at different times relative to STX intoxication. The animals used in this study were chronically instrumented for concurrent recordings of diaphragm electromyogram (DEMG), neck skeletal muscle electromyogram, Lead II electrocardiogram, and electrocorticogram (ECoG). There were five groups of unanesthetized guinea pigs. The first group served as 4-AP controls and received a 2 mg/kg i.m. dose of 4-AP. The four remaining groups were given a lethal dose of STX (5 microg/kg i.m.); the second group, STX controls, received no 4-AP; the third group, the 4-AP treatment group, received 4-AP immediately following cardiorespiratory collapse; the fourth group was the 4-AP/STX co-administration group and 4-AP was given concurrently with STX; and the fifth group was the 4-AP pretreatment group in which 4-AP was given 10 min before STX. At the point of STX-induced cardiorespiratory collapse, the guinea pigs were ventilated and given an i.p. injection of sodium bicarbonate. Results showed that 4-AP prevented cardiorespiratory collapse in 3/7 animals in the 4-AP pretreatment group. Also, 4-AP in conjunction with artificial ventilation and sodium bicarbonate accelerated recovery from STX-induced cardiorespiratory collapse in all the treatment groups compared to the STX controls.