A liquid chromatographic-mass spectrometric (LC-MS) method for the analysis of the bis-pyridinium oxime ICD-585 in plasma: application in a guinea pig model.

In recent animal studies, several novel oxime compounds that are better than 2-PAM as antidotes against selected organophosphate (OP) nerve agents have been identified. The purpose of this study was to develop and validate a liquid chromatographic-mass spectrometric (LC-MS) method for analysis of the bis-pyridinium oxime ICD-585 (1-(2-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-propane) in plasma and to establish the utility of the method in a guinea pig model. Calibration curves were prepared using ICD-585-spiked plasma at concentrations from 0.156 to 10 microg/ml. Curves were run over a 1-month time frame and a total of 13 (n=13) were generated. The lower limit of quantification (LLOQ) was determined to be 0.216 microg/ml. Intra- and inter-day variability was assessed by studying precision and accuracy. For intra-day studies, data from the precision determinations indicated that the % CV's ranged from 4.28 to 14.98%. The % error in the accuracy assessments ranged from -8.73 to 4.61%. For inter-day studies, precision data ranged from 3.53 to 13.20%. The % error in the accuracy assessments ranged from 0.39 to 13.77%. Room temperature, freeze-thaw and autosampler stability was also examined. For all 3 stability studies, the compound remained within +/-15% of the initial analysis. Application of the method was demonstrated by analyzing samples from guinea pigs challenged with sarin (GB) or cyclosarin (GF) (1x LD(50)) followed with intramuscular ICD-585 (58 microM/kg, 21.8 mg/kg). At 55 min after oxime administration, mean (+/-SD) plasma concentrations were 15.98 (+/-4.88)microg/ml and 14.57 (+/-3.70) microg/ml in GB- and GF-exposed animals, respectively. In summary, studies have been carried out to verify the sensitivity, precision and accuracy of the assay as well as the stability of the analyte under various conditions. The method has been demonstrated to be applicable to the analysis of plasma from nerve agent-exposed guinea pigs.

Gas chromatographic-mass spectrometric determination of british anti-lewisite in plasma.

British anti-Lewisite (BAL) (2,3-dimercapto-1-propanol) is a potential therapeutic compound when used against the effects of cutaneous sulfur mustard, and a method for its determination in plasma has been developed. BAL and the internal standard (IS) ethane dithiol were isolated from plasma samples through solid-phase extraction and then reacted with 1-pentafluoropropionylimidazole, forming stable pentafluoropropionyl derivates that are sensitive to gas chromatographic-mass spectrometric analysis. Examination of concentration versus peak-area ratios of the BAL and IS derivatives demonstrated the method to be linear over a concentration range of 0.48 to 124 ng/mL in plasma when fit to a weighted (1/y2) least-squares regression. Correlation coefficients were 0.9943 to 0.9995 for six runs, and coefficients of variation (CV) were 2.5 to 8.7% over the eight concentrations tested. The intra- and interday accuracy and precision of this method was measured by examining six groups of eight unknown test samples (n = 6). Intraday accuracy, as expressed by percent error, was found to range from -15.4 to 0.21%, whereas the precision, expressed as %CV, was less than 9.8% over all sample concentrations. Interday test unknown sample results were similar in that the accuracy was shown to be -7.1 to 0.4%, and precision was 4.7 to 9.5%. BAL levels in frozen plasma (-70 degrees C) remained constant for more than 14 days with a CV of less than 10% for the eight concentrations tested. The data indicate that the method will provide accurate and precise determination of BAL at concentrations down to approximately 1 ng/mL in plasma. This procedure has been applied to determine preliminary time-concentration profile studies of BAL in the hairless guinea pig.

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.

Pharmacokinetics of intramuscularly administered biperiden in guinea pigs challenged with soman.

Biperiden is an anticholinergic compound that has demonstrated effectiveness for treating organophosphate-induced seizure/convulsions. The plasma levels of biperiden associated with this efficacy have not yet been defined. In this study, the pharmacokinetics and tissue distribution of biperiden after intramuscular administration of 0.5 mg/kg were conducted while monitoring pharmacodynamic (electroencephalographic) data in soman-exposed guinea pigs. Overall, 59% of the animals had seizures terminated within 30 min of the biperiden administration. The mean time to seizure termination was 15.9 min. The pharmacokinetics of biperiden after i.m. administration to guinea pigs were best described by a one-compartment model with first-order absorption and elimination. The maximal plasma biperiden concentration (34.4 ng/mL) in seizure-terminated animals occurred at 26.3 min. Extensive partitioning into peripheral tissues was noted supporting the relatively large volume of distribution observed. Maximal biperiden concentrations in the cortex and brain stem were found at 30 min and were 2.3 and 1.7 times greater, respectively, than that in plasma. The time for maximal plasma concentration was found to corresponded well with the mean time to seizure termination following drug administration.

The determination of biperiden in plasma using gas chromatography mass spectrometry: pharmacokinetics after intramuscular administration to guinea pigs.

A gas chromatographic-mass spectrometric (GC-MS) method has been developed for the analysis of the biperiden from plasma. The method utilizes 290 microl of plasma and a simple hexane extraction/clean-up procedure. Standard curves were linear over the range of 1.9-250 ng/mL. The range of correlation coefficients for the individual standard curves was 0.9984-0.9999; the largest coefficient of variation expressed as a percentage (% CV) was 11.5%. Precision and accuracy were examined by assessing between-day and within-day variability. For between-day precision, the % CVs ranged from 2.86 to 5.17%. Accuracy as expressed by percentage error ranging from -2.16 to 5.83%. The study for within-day precision demonstrated % CVs from 0.95 to 5.55% with accuracy from -3.37 to 2.45%. Applicability of the method was demonstrated by examining the pharmacokinetics of intramuscular (i.m.) biperiden as an anticonvulsant treatment in a guinea pig model for organophosphate (OP)-induced seizure activity. Mean pharmacokinetic parameter estimates were similar to literature values; selected mean pharmacokinetic parameter estimates were: apparent volume of distribution, 13.9 L/kg; half-life of elimination, 93 min; time to maximal plasma concentration, 27.4 min; and maximal plasma concentration, 32.22 eta g/mL. The time to maximal plasma concentration was found to be similar to the onset time for terminating OP-induced seizure activity in guinea pigs receiving biperiden as an anticonvulsant treatment. The studies indicate that the method affords the required precision, accuracy and sensitivity to assay biperiden at the doses utilized for these pharmacokinetic studies after i.m. administration to guinea pigs.

Intramuscular diazepam pharmacokinetics in soman-exposed guinea pigs.

Intramuscular (i.m.) diazepam is included by the US military as an anticonvulsant in the standard therapeutic regimen for organophosphorus nerve agent intoxication. In this study we investigated the pharmacokinetics of diazepam after i.m. administration while monitoring pharmacodynamic (electroencephalogram, EEG) data in soman-exposed guinea pigs. Prior to experiments the animals were surgically implanted with EEG leads to monitor seizure activity. For the study, animals were administered pyridostigmine (0.026 mg x kg(-1) i.m.) 30 min prior to soman (56 microg x kg(-1), 2 x LD50; subcutaneously, s.c.), which was followed in 1 min by atropine sulfate (2 mg x kg(-1) i.m.) and pralidoxime chloride (25 mg x kg(-1) i.m.). All animals receiving this regimen developed seizure activity. Diazepam (10 mg x kg(-1) i.m.) was administered 5 min after onset of seizure activity. Based on EEG data, animals were categorized as either seizure terminated or not terminated at 30 min after diazepam. Serial blood samples were obtained from each animal. Diazepam (10 mg x kg(-1) i.m.) terminated seizure activity in 52% of the animals within 30 min. The pharmacokinetics were characterized by a one-compartment model with first-order absorption and elimination. The maximum plasma concentrations (Cmax) were 991 and 839 ng x ml(-1) for seizure terminated and not terminated, respectively. Mean plasma concentrations of diazepam were significantly different (P < 0.05) for seizure terminated vs not terminated groups at 30 min. The plasma Cmax in seizure-terminated animals in this study is similar to the minimum range of plasma diazepam (200-800 ng x ml(-1)) reported to suppress seizure activity in humans. It has been reported in an earlier study that the minimum effective i.m. dose (0.1 mg x kg(-1)) required to prevent soman-induced convulsions in Rhesus monkeys produces a mean Cmax of 50 ng x ml(-1) for diazepam. The data from our current study suggest that a higher dose (and corresponding Cmax) is necessary to terminate ongoing seizure activity.

Recovery from the lethal effects of saxitoxin: a therapeutic window for 4-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-AP's 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.

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.

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.

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.

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.

A method for determining 4-aminopyridine in plasma: pharmacokinetics in anaesthetized guinea pigs after intravenous administration.

An HPLC assay has been developed to measure 4-aminopyridine (4-AP) in guinea pig plasma. For the assay, all plasma samples (50 microL) were microfiltered following the addition of an internal standard (3,4-diaminopyridine). Filtrates (10 microL) were directly injected into a spherical silica column (100 x 2.1 mm; 5 microns); detection was achieved at 266 nm. Standard curves had correlation coefficients ranging from 0.9923 to 0.9992 and coefficients of variation expressed as a percentage (% CV) of below 8%. Precision was expressed as between-day and within-day variability of five test sample concentrations. Between-day % CV ranged from 4.0 to 6.5%. Within-day % CV ranged from 3.6 to 6.9%. Accuracy was assessed by examining expected within-day test sample concentrations against calculated concentrations; per cent errors were all below 10%. Stability studies demonstrated % CV below 5% after repeated freezing. The method was employed to study the pharmacokinetics of 4-AP after intravenous administration to anaesthetized guinea pigs. Serial blood samples (150 microL) were collected at predetermined time intervals up to 4 h post-4-AP (2 mg/kg, i.v.) administration. 4-AP demonstrated a biexponential decline in the plasma-concentration curve as a function of time indicating a two compartment model for this drug. Selected mean pharmacokinetic parameter estimates were alpha-half-life, 0.37 min; beta-half-life (biological half-life) for terminal slope, 109 min; and volume of distribution at steady state, 1036.18 mL/kg. 4-AP was found to rapidly and extensively partition into a peripheral tissue compartment and demonstrated a relatively long biological half-life. The findings from the current pharmacokinetic experiments support the pharmacology of 4-AP in its role for reversing saxitoxin (STX)- and tetrodotoxin (TTX)-induced diaphragmatic failure in terms of onset of action and duration of effect in anaesthetized guinea pigs.

The effect of ondansetron on pyridostigmine-induced blood acetylcholinesterase inhibition in the guinea pig.

The purpose of this study was to assess the compatibility, in terms of red blood cell acetylcholinesterase (AChE) inhibition, of ondansetron (OND; a 5-hydroxytryptamine subtype-3 receptor antagonist) with the organophosphorus pretreatment compound pyridostigmine (PYR) after simultaneous oral (p.o.) administration to guinea pigs. The time-course of PYR-induced (0.94 mg/kg, p.o.) AChE inhibition was determined in the absence and presence of OND. Ondansetron (10, 20 and 30 mg/kg; p.o.) did not modify AChE inhibition, whereas concurrent administration of PYR with OND (10 or 20 mg/kg; p.o.) produced significantly greater decreases in AChE activity than PYR alone. The decreases in AChE activity for PYR plus OND, 10 and 20 mg/kg, (between 30 -240 min) were 12.3 +/- 2.8% and 16.1 +/- 2.3% (mean +/- SD) respectively relative to PYR alone. The slope for recovery of AChE activity (120 - 240 min) was 0.0914 for PYR alone; recovery rates (slopes) for PYR plus OND, 10 and 20 mg/kg, were 0.0796 and 0.0433 respectively. Additionally, altered PYR-induced AChE activity profiles were ameliorated when PYR and OND (20 mg/kg) were administered 150 min apart. Since the results of this study provided evidence that the oral administration of OND alone did not inhibit AChE, the changes in PYR-induced AChE activity by the simultaneous administration of OND suggest mechanisms other than a direct action on the enzyme. The significance of these findings is that the increased AChE inhibition resulting from simultaneous oral administration of both component could result in undesirable cholinergic toxicities and subsequent perform decrements.

An HPLC method for the determination of granisetron in guinea pig plasma.

A high-performance liquid chromatographic (HPLC) method for the determination of granisetron (GRN) in guinea pig plasma has been developed. Guinea pig plasma spiked with GRN was microfiltered, and the recovered filtrate was directly injected onto the column without any further cleanup procedures. Separation was achieved on a spherical silica column and GRN was detected at 305 nm. Approximately 800-900 injections were made without any evidence of column deterioration. For the standard curves, correlation coefficients ranged from 0.9978-0.9999, and the percent standard deviation (%SD) from the mean area under the curve (AUC) was calculated to be less than 10% for all concentrations, except for the lowest concentration (0.325 ng/microL, 11.3%). Between-day and within-day coefficients of variation (%CV) ranged from 4.9 to 9.5% and 3.6 to 7.6%, respectively. Percent errors for within-day test plasma samples were not greater than 8.2% of the expected concentration for all samples except for 1.125 ng/microL (-14.6%). The limit of sensitivity was found to be 0.019 ng/microL. Estimated recovery of GRN in the microfiltrate was calculated to be 58-59% and 78-81% in plasma and water, respectively. Stability studies indicated that repeated refrigeration and warming (for six days) of microfiltered GRN plasma samples produced no changes in GRN concentrations from day to day. However, microfiltered GRN plasma samples that were repeatedly frozen and thawed demonstrated erratic concentration changes from day to day. The precision, accuracy, and small sample requirements of this method indicate its utility for pharmacokinetic studies in small animals where sample volume may be restrictive.

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.

Effects of anticholinergic-antiparkinsonian drugs on striatal neurotransmitter levels of rats intoxicated with soman.

Antimuscarinic drugs possessing antiparkinson activity that were effective in preventing convulsions induced by the organophosphorus cholinesterase (ChE) inhibitor soman were studied for their effects on spinal cord ChE activity and striatal levels of acetylcholine (ACh) and catecholamines in soman-intoxicated rats. Either biperiden (BPR) or trihexyphenidyl (THP) was administered to rats at an anticonvulsant dose (0.125 mg/kg, IM) in the presence or absence of soman (100 micrograms/kg, SC). The time course (up to 2 h) for ChE activity and levels of ACh and catecholamines were measured after soman, BPR, THP, soman and BPR, or soman and THP treatment. Soman rapidly inhibited ChE activity (65-75%; 15-120 min) and increased ACh levels (35%; at 30 min). It did not affect norepinephrine or dopamine (DA), but elevated at later time points (60-120 min) levels of the DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), thus indicating increased DA turnover. BPR and THP alone reduced striatal ACh level from control, but did not affect any other neurochemical parameters studied. THP and BPR each reversed the effects of soman on DOPAC and HVA levels, but neither affected ChE activity nor ACh level induced by soman. Thus, our findings suggest that the anticonvulsant effects of BPR and THP in soman poisoning may be attributed to their earlier reported muscarinic receptor blocking properties.

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.

Anticonvulsant actions of anticholinergic drugs in soman poisoning.

The acute effects of the organophosphorus cholinesterase inhibitor soman include hypersecretions, convulsions, and death. The purpose of this study was to evaluate the anticholinergic compounds aprophen, atropine sulfate, azaprophen, benactyzine, benztropine, biperiden, scopolamine HBr, and trihexyphenidyl for their efficacy in preventing soman-induced hypersecretions and convulsions. Male rats were injected with the oxime HI-6 (125 mg/kg, i.p.), to increase survival time, along with various intramuscular doses of the anticholinergics 30 min prior to a dose of soman (180 micrograms/kg, s.c.; equivalent to 1.6 x the median lethal dose) that produced 100% convulsions. Signs of intoxication as well as the time-to-onset of convulsions were observed. The calculated anticonvulsant median effective dose values were 0.18, 0.33, 0.36, 0.55, 2.17, 2.30, 2.45, and 31.09 mumol/kg for scopolamine HBr, biperiden, trihexyphenidyl, benactyzine, benztropine, azaprophen, aprophen, and atropine sulfate, respectively. The same rank order of potency for inhibition of hypersecretions among these compounds was observed. Parallel studies with quaternary analogs of atropine sulfate and scopolamine HBr demonstrated, however, that these charged compounds afford no protection against soman-induced hypersecretions and convulsions. The results indicate that tertiary anticholinergic compounds afford protection against soman-induced convulsions and hypersecretions and that the beneficial anticonvulsant effects are mediated through the central cholinergic system. Excitatory amino acid neurotransmitter systems may be involved in the effectiveness of these compounds.

Anticonvulsants for poisoning by the organophosphorus compound soman: pharmacological mechanisms.

Exposure to high doses of organophosphorus nerve agents such as soman, even with carbamate pretreatment, produces a variety of toxic cholinergic signs, including secretions, convulsions and death. Evidence suggests that soman-induced convulsions may be associated with postexposure brain neuropathology. The purpose of this study was to investigate the pharmacologic mechanism of action of soman-induced convulsions and of anticonvulsant drugs. Various classes of compounds were evaluated for their efficacy in preventing soman-induced convulsions in rats pretreated with the oxime HI-6 to increase survival time, along with various doses of the test compounds (IM) either in the absence or presence of atropine sulfate (16 mg/kg, IM) 30 minutes prior to a soman challenge dose (180 micrograms/kg, SC; equivalent to 1.6 x LD50) that produced 100% convulsions. Without atropine sulfate, only tertiary anticholinergics (scopolamine, trihexyphenidyl, biperiden, benactyzine, benztropine, azaprophen and aprophen), caramiphen, carbetapentane and MK-801 were effective anticonvulsants. In the presence of atropine sulfate, the benzodiazepines (diazepam, midazolam, clonazepam, loprazolam and alprazolam), mecamylamine, flunarizine, diphenylhydantoin, clonidine, CGS 19755 and Organon 6370 studied were effective. We have examined the possibility that diazepam may exert some of its anticonvulsant effects through cholinergic mechanisms and found that a reduced release of ACh into synapses after diazepam and atropine treatment may account for diazepam's anticonvulsant activity against soman. We also found that at anticonvulsant doses biperiden and trihexyphenidyl each significantly reversed the effects of soman on striatal levels of DOPAC and HVA, the metabolites of dopamine, and have concluded that in addition to actions on muscarinic receptors, the anticonvulsant effects of these anticholinergics in soman poisoning may be partially related to their actions on the striatal dopaminergic system. These findings allow us to postulate that central muscarinic cholinergic mechanisms are primarily involved in eliciting the convulsions following exposure to soman and that subsequent recruitment of other excitatory neurotransmitter systems and loss of inhibitory control may be responsible for sustaining the convulsions and for producing the subsequent brain damage. Future studies to confirm these neuropharmacological mechanisms are proposed.