Pharmacokinetics of diazepam intramuscularly administered to rhesus monkeys.

The purpose of this study was to define the pharmacokinetics of diazepam in monkeys following an im injection of 100 micrograms/kg (the minimum effective dose that prevents nerve agent-induced convulsions in pyridostigmine-pretreated, atropine- and 2-PAM-treated monkeys) in order to predict what im dose in humans is needed to prevent nerve agent-induced convulsions. Six rhesus monkeys were administered diazepam in the hind limb. Blood (3 mL) was collected via an indwelling saphenous catheter immediately prior to and 5, 10, 15, 25, 40, 60, 90, 120, 180, and 240 min after diazepam dosing. A contract laboratory, blind to the labeling code, analyzed diazepam serum concentrations by electron-capture gas chromatography and the percentage of unbound diazepam by equilibrium dialysis. The concentration-time data for total (unbound and bound) diazepam individually determined for each animal was best described by a one-compartment open model with first-order absorption and elimination. The average maximum serum concentration (50 ng/mL) was reached in 29 min. The volume of distribution and systemic clearance, assuming 100% bioavailability, were 1.5 L/kg and 19.4 mL/min/kg, respectively. The percentage of diazepam unbound to serum proteins was 4.6% and, therefore, the maximum concentration of free diazepam was 2.3 ng/mL. These results, when compared with human pharmacokinetic studies, allow a means of extrapolating effective monkey anticonvulsant doses to humans on a pharmacokinetic basis.

Behavioral efficacy of diazepam against nerve agent exposure in rhesus monkeys.

The possibility that nerve agents will be used on the battlefield is real. The traditional therapy against nerve agent exposure consists of pyridostigmine pretreatment and atropine-pralidoxime chloride therapy administered after nerve agent exposure. This therapy regimen is extremely effective in preventing mortality in laboratory animals exposed to multilethal concentrations of nerve agent, yet these animals often display convulsions, brain damage, and behavioral incapacitation. We report here that the addition of diazepam to the traditional therapy for nerve agent (soman) exposure not only decreases the incidence of convulsions, but also attenuates the cognitive impairments of rhesus monkeys trained on a Serial Probe Recognition (SPR) task. Monkeys which received diazepam treatment required only 6 days before their performance on the SPR task returned to presoman exposure levels, compared to nondiazepam-treated monkeys which required 15 days. Moreover, only 1 out of the 5 monkeys which received diazepam treatment suffered tonic-clonic convulsions; in contrast all 5 monkeys which did not receive diazepam treatment experienced severe convulsive episodes. These results suggest that diazepam would be an excellent adjunct to traditional nerve agent therapy to facilitate behavioral recovery from nerve agent intoxication that might be encountered by US military personnel on the battlefield or accidental organophosphate poisoning encountered in industrial or agricultural accidents.

Behavioral decrements persist in rhesus monkeys trained on a serial probe recognition task despite protection against soman lethality by butyrylcholinesterase.

Recently, it has been demonstrated that an exogenously administered enzyme such as butyrylcholinesterase (BuChE) can prevent death in rhesus monkeys exposed to multiple-lethal doses of the acetylcholinesterase inhibitor soman when the enzyme is given prior to soman exposure (3). We report that despite BuChE protecting against soman-induced lethality, behavioral effects are seen in these monkeys which last for at least 6 days as measured by performance on a serial probe recognition (SPR) task. Analyses of the serial position curves showed that performance was lower on the probe trials when the probe items were from the middle of the list than when the probe items were from the beginning or end of the list which were unaffected. BuChE given alone also produced behavioral effects, causing all animals not to respond on the probe trials until 8 h following BuChE administration. Taken together, these findings suggest that the BuChE is not completely binding all of the soman and that a concentration of soman which is capable of causing behavioral effects may be entering the CNS.

Effects of subacute pretreatment with carbamate together with acute adjunct pretreatment against nerve agent exposure.

Visual observations were made to compare the pretreatment benefits of subacute (75 micrograms/hr, sc) and acute (146 micrograms/kg, im, at 30 min) deliveries of physostigmine salicylate (Phy) against 2 or 5 LD50s (60 or 150 micrograms/kg, sc) of soman in guinea pigs; scopolamine, 80 micrograms/kg, im, was given routinely at 30 min. In a second set of studies, pretreatment with subacute carbamate [sc, Phy 36 micrograms/hr or pyridostigmine (Pyr), 50 micrograms/hr] and acute adjunct (im, scopolamine, 0.48 mg/kg, or trihexyphenidyl, 2 mg/kg) at 30 min, was used against soman (5 LD50s, sc) and VX (18.4 micrograms/kg, sc; 2 LD50s); atropine (16 mg/kg, im) and 2-PAM (25 mg/kg, im) were given at 1 min post soman. In all studies, lethality, % convulsing, convulsive/subconvulsive score, and recovery time were noted. Subacute dosing for 7 days was done via 14-day osmotic minipumps (OMPs). Results of the first set of studies indicate that subacute and acute deliveries of Phy give essentially comparable protection against 2 or 5 LD50s of soman. The second set of studies show that against soman, the adjuncts scopolamine and trihexyphenidyl when compared, and the carbamates, Phy and Pyr when compared, gave similar protective benefits as indicated by all four monitored measures of toxicity. Phy with either adjunct provided excellent protection against VX induced mortality and convulsions. With both carbamates, trihexyphenidyl gave similar protective benefits against VX. Scopolamine, however, under the conditions used herein, failed to act beneficially with Pyr against VX.

Estimation and analysis of the concentration-response surfaces associated with multiple-agent combinations.

Chinese hamster cells (V79) were treated with ethylnitrosourea (ENU) and cis-diamminedichloroplatinum(II) (DDP) alone and in combination. Sister chromatid exchanges (SCEs) were quantified as measures of genotoxicity of the two agents. The combination experiment employed a factorial design in which cells were treated, in various concentration combinations, with both agents simultaneously. Response surface methodology, using a polynomial model based on a negative binomial distribution of SCE events, was employed for analysis of the interactions of the two genotoxic agents. The negative binomial distribution, a generalization of the Poisson distribution, is required since SCEs are discrete variables which, under the conditions of these experiments, have a distribution which exhibits extra-Poisson variability. The model of the ENU/DDP combinations indicated an increasingly less-than-additive effect resulting from increasing concentrations of each agent in the combination. The analysis of these experiments demonstrates the usefulness of a powerful statistical procedure for evaluating the biological effects resulting from exposure to multiple cytotoxic agents. The methodology can be used with many other types of endpoints and is not limited by the number of treatment agents.

Effects of subacute administration of physostigmine on blood acetylcholinesterase activity, motor performance, and soman intoxication.

Subacute administration of carbamates is under study as pretreatment against soman, a toxic anticholinesterase agent. In this study, the sustained release of physostigmine salicylate (Phy) in rats was achieved via osmotic minipumps; each pump contained 2 ml of Phy solution (0.4, 10, or 50 mg/ml) and delivered 2.5 microliter/hr for 28 days. At the corresponding dosage rates, rat whole blood acetylcholinesterase (AChE) activity was suppressed by approximately 11, 42, and 66%, respectively. These levels of Phy administration caused no decrement in performance on an accelerating rotarod (ARR) when tested between Days 3 and 27 of the 28-day exposure. The highest level of Phy caused a mean weight loss of 11% initially, with recovery by the 17th day. On Day 27 the rats were given 0.08 mg/kg, im, of scopolamine (SCP), 30 min before exposure to soman (58 micrograms/kg; 1 LD50, iv). In combination with SCP, the two highest levels of Phy prevented lethality and a decrement in ARR performance by soman, while the lowest level showed 40% lethality after soman and the survivors exhibited partial recovery to their own presoman control performance by 24 hr. These results suggest that, in a pretreatment mode, 42-66% inhibition of AChE by sustained exposure to Phy, with an acute dose of cholinolytic, would suffice to protect against lethality and motor performance decrement by a toxic level of soman.

Physostigmine (alone and together with adjunct) pretreatment against soman, sarin, tabun and VX intoxication.

A pretreatment for organophosphorus (OP) anticholinesterase (e.g., soman) intoxication should prevent lethality and convulsions (CNV) at 2 LD50s and be behavioral-decrement-free when given alone. Behavioral-deficit-free pretreatment regimens (PRGs) for guinea pigs consisted of Physostigmine (0.15 mg/kg, im) and adjunct. Adjuncts [mg/kg, im] tested were akineton [0.25], aprophen [8], trihexyphenidyl [2], atropine [16], azaprophen [5], benactyzine [1.25], cogentin [4], dextromethorphan [7.5], ethopropazine [12], kemadrin [1], memantine [5], promethazine [5], scopolamine [0.08] and vontrol [2]. PRGs were given 30 min before soman (60 micrograms/kg, sc; 2 LD50s) or other OP agents. Animals were then observed and graded for signs of intoxication, including CNV at 7 time points and at 24 hr. Physostigmine alone reduced the incidence of CNV and lethality induced by 2 LD50s of soman by 42 and 60%, respectively. All of the PRGs tested abolished lethality and 12 shortened recovery time to 2 hr or less. Also, PRGs including azaprophen or atropine prevented CNV. When selected PRGs were tested against intoxication by sarin, tabun or VX, the efficacy was generally superior to that for soman. The data show that several PRGs are effective against soman intoxication in guinea pigs.

Protection by butyrylcholinesterase against organophosphorus poisoning in nonhuman primates.

Butyrylcholinesterase (BuChE) was examined as an in vivo exogenous scavenger for highly toxic organophosphorus (OP) poisons. Protection studies with equine BuChE were carried out in rhesus monkeys trained to perform a Serial Probe Recognition task. The pharmacokinetics of equine BuChE administered i.v. in rhesus monkeys revealed an elimination T1/2 of approximately 620 hr. Animals given 503 nmol of BuChE i.v. and then challenged with 220 to 260 nmol of soman (two LD50; a lethal dose in untreated animals) all survived with no clinical signs of OP poisoning. Serial Probe Recognition performance was depressed after enzyme administration and at 1 hr postsoman. However, all monkeys performed the task at base-line levels at 8 hr after soman and throughout the remainder of the experimental period. Two different monkeys each were given two doses of sarin, 183 nmol/dose (one LD50) after 460 nmol of BuChE. No signs were observed. A third group of monkeys given 253 or 340 nmol (three and four LD50, respectively) of soman after 460 nmol of BuChE required 1 mg/kg of atropine i.v. 10 min postsoman, but recovered completely within 24 hr. Our results indicate that BuChE has the required properties to function as a biological scavenger to protect against the pharmacological and behavioral toxicity of OP poisons.

Evaluation of the interaction of three genotoxic agents in eliciting sister-chromatid exchanges using response surface methodology.

Chinese hamster cells (V79) were treated with ethylnitrosourea (ENU), bischloroethylnitrosourea (BCNU), and cis-diamminedichloroplatinum(II) (DDP) alone and in combination. Sister chromatid exchanges (SCEs) were quantitated as measures of genotoxicity of the three agents. The combination experiment employed a factorial design in which cells were treated, in various concentration combinations, with all agents simultaneously. Response surface methodology using a polynomial model in treatment variables to approximate the mean the distribution of SCE events was employed for analysis of the interactions of the three genotoxic agents. Due to unequal variances of the number of SCEs in the various treatment groups, a weighted least-squares analysis was used to estimate the parameters of the dose-response relationship. The single-agent results suggest that the DDP concentration-response curve has a much steeper slope than the ENU and BCNU curves, and is concave downward as compared to the relatively linear concentration-response curves of ENU and BCNU. The combination results suggest that ENU and DDP are involved in a negative interaction. The BCNU/DDP interaction, the ENU/BCNU interaction, and the three-factor interaction are not statistically significant. The analysis of these data demonstrates the usefulness of a statistical procedure for evaluating the biological effects resulting from exposure to multiple cytotoxic agents. The methodology can be used with many other types of endpoints and is not limited by the number of treatment agents.

The evaluation of biological interactions using response surface methodology.

Response surface methodology was employed in the statistical analysis of the combination exposures of genotoxic agents, bischloroethylnitrosourea with cis-diamminedichloroplatinum (II) and cis-diamminedichloroplatinum (II) with X rays. The measured endpoint in each case was sister chromatid exchanges in V79 Chinese hamster cells. The combination experiments employed a factorial design in which cells were treated, in various concentration combinations, with two agents simultaneously. Bis-chloroethylnitrosourea and cis-diamminedichloroplatinum (II) each exhibited curvilinear concentration-related increases in sister chromatid exchanges. X rays exhibited a dose-dependent increase in sister chromatid exchanges. For the cis-diamminedichloroplatinum (II)/X ray combinations, response surface methodology indicates a less-than-additive interaction, suggested by the non-parallel concentration-response curves of one agent at varying concentrations of the other, and a slight dose-dependent increase in sister chromatid exchanges due to X rays alone. Both cis-diamminedichloroplatinum and bis-chloroethylnitrosourea exhibited concentration-related increases in sister chromatid exchanges, cis-diamminedichloroplatinum (II) being 8-10 times (dependent on what level of effect was compared) more potent than bis-chloroethylnitrosourea. For the cis-diamminedichloroplatinum (II)/bis-chloroethylnitrosourea combinations, an increasingly less-than-additive interaction was detected. The analysis of these combinations demonstrates the strength of response surface methodology, a collection of mathematical and statistical techniques for detecting, analyzing and describing the biological effects resulting from exposures to multiple cytotoxic agents. The descriptive ability of these procedures is shown to be useful in that it leads to the suggestion of hypotheses regarding mechanisms of action.

Successful pretreatment/therapy of soman, sarin and VX intoxication.

Chemical pretreatment is effective against a 2 LD50 challenge of soman, sarin or VX or a 5 LD50 challenge of tabun. Chemical pretreatment followed by post challenge therapy should be effective against greater levels of agent. Such tests in guinea pigs are reported here; pretreatment regimens (PRGs) consisted of physostigmine (0.15 mg/kg, im) and an adjunct. The adjuncts [mg/kg, im] used were aprophen [8], atropine (AT)[16], azaprophen (AZA)[5], benactyzine [1.25], benztropine (BT) [4], scopolamine [0.08] and trihexyphenidyl [2]. Pretreatment was given 30 min before, and atropine (16 mg/kg, im) and 2-PAM (25 mg/kg, im) therapy (T) at one min after, 5 LD50s of agent. Results indicate that, all of the PRG+T regimens, except BT-not tested with T, prevent lethality by soman; trihexyphenidyl and scopolamine (the only adjuncts used therein) regimens each prevent lethality by sarin and VX. Against soman, all PRG+T regimens (vs PRG only) may shorten the median recovery time to 2 hrs or less. Even without therapy, the PRGs containing AT, AZA or BT prevent lethality by 5 LD50s of soman; however, used alone, only the PRG containing AZA reduces the incidence of convulsions at this level of soman.

Comparing the response surfaces of two cholinolytics when used in combination with physostigmine as a pretreatment against organophosphate challenge.

Physostigmine (PHY) is currently being evaluated as a potential pretreatment against nerve agent challenge. Although it might provide increased protection against nerve agent-induced incapacitation, it is nonetheless behaviorally toxic itself. Addition of a cholinolytic adjunct is expected to significantly reduce this behavioral toxicity. The efficacy of pretreatment regimens (PRG) (composed of PHY in combination with either scopolamine (SCP) or trihexyphenidyl (artane; ART] was evaluated in guinea pigs challenged with soman (98 ug/kg, sc; 3.5 LD50) to determine the dose of PHY and adjunct required for optimal efficacy as well as the better of the two adjuncts. Three different endpoints were measured on each animal: (1) whether or not the animal survived up to 24 hours post challenge, (2) severity of incapacitation, and (3) time to recovery. The survival data revealed no significant differences between the two adjuncts and PHY against soman, but the data suggests that a pretreatment combination of PHY and ART is likely to give a greater therapeutic index than one containing SCP. Furthermore, since ART is less toxic behaviorally, it would most likely be better tolerated as a pretreatment with PHY in non-agent exposed subjects. The findings in this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents. In conducting the work described in this report, the investigators adhered to the "Guide for the Care and Use of Laboratory Animals" as promulgated by the Committee on Revision of the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Resources, National Research Council.

Evaluation of a two-drug combination pretreatment against organophosphorus exposure.

A pretreatment combination of physostigmine and azaprophen (6-methyl-6-azabicyclo[3.2.1]octan-3-ol-2,2-diphenylpropionate), a novel cholinolytic, was evaluated for its ability to minimize soman-induced incapacitation and lethality in guinea pigs. This was accomplished by using response surface methodology to model and analyze the combination, varying physostigmine from 0 to 194 micrograms/kg, azaprophen from 0 to 5 mg/kg, and soman from 30 to 150 micrograms/kg. One hundred percent survival was achieved against 5 LD50 of soman using as little as 100 micrograms/kg of physostigmine in the presence of 5 mg/kg azaprophen. Both survival and soman-induced incapacitation were similarly affected by this pretreatment combination. For both endpoints, greater efficacy was achieved with the combination than could be achieved with either component alone (therapeutic synergism). This suggests that such a pretreatment combination may prove very efficacious against soman-induced lethality and incapacitation in higher species.

Efficacy comparison of scopolamine and diazepam against soman-induced debilitation in guinea pigs.

The efficacy of diazepam (DZ) and scopolamine (SCP), in combination with atropine (ATR)+oxime therapy, against soman-induced seizure/convulsive activity and associated brain damage has been demonstrated, but the efficacy of each against the incapacitating effects of soman has not been addressed. Thus, the therapeutic efficacies of SCP (5 doses; 0-0.86 mg/kg) and DZ (5 doses; 0-5 mg/kg), when each was used in conjunction with ATR (3 doses; 0.5-8 mg/kg) + 2-PAM (25 mg/kg) therapy, were compared in groups of pyridostigmine pretreated guinea pigs exposed to 1.6, 2.0, 2.5, or 3.2 LD50s of soman. Response surface methodology was employed to describe the relationship between soman-induced incapacitation and the ATR/DZ or ATR/SCP dosages. Incapacitation was measured by toxicity scores assigned by three graders to test animals at 60 min postsoman. Results show that as the dosage of SCP increased, the mean toxicity scores decreased. Also, within the indicated dose ranges used, the efficacy of SCP was not dependent on the presence of ATR. In contrast, ATR alone was found to be more effective than when combined with DZ at any dose, and indicates that DZ might be temporarily contributing to soman-induced incapacitation. These findings suggest that in guinea pigs, SCP could replace ATR or DZ, or both, as therapy against soman-induced incapacitation.

Assessing the efficacy of azaprophen and physostigmine as a pretreatment for soman-induced incapacitation in guinea pigs by response-surface modeling.

Physostigmine (PHY) has the advantage over pyridostigmine of minimizing OP-induced incapacitation because it penetrates into the CNS. However, physostigmine is behaviorally toxic at relatively low concentrations. It is anticipated that this could be offset by a cholinolytic to prevent behavioral deficit due to the carbamate pretreatment alone. The therapeutic efficacy of physostigmine/azaprophen pretreatment therapy was evaluated in soman-challenged guinea pigs. Response surface methodology was employed to describe the relationship of the pretreatment combination with duration of incapacitation. The significance of the combination relative to PHY alone was evaluated in addition to dose combinations that yield optimal time to recovery. Analysis of the fitted response surface indicated that combination pretreatment with these compounds significantly reduces the time to recovery after soman challenge versus pretreatment with PHY alone.

Evaluation of the efficacy of two carbamates, physostigmine and pyridostigmine, when used in conjunction for protection against organophosphate exposure.

Recent studies have shown that pretreatment with either pyridostigmine (PYR) or physostigmine (PHY) followed by atropine-oxime therapy is very effective in reducing the lethality of nerve agents. The therapeutic efficacy of a PHY and PYR combination pretreatment was evaluated in guinea pigs challenged with two LD50s of soman. Endpoints measured were percentage of acetylcholinesterase inhibition induced by the pretreatment and survival up to 24 hr postchallenge. Response surface methodology was employed to describe the relationship between each endpoint and the pretreatment combination. Although both carbamates contributed to blood acetylcholinesterase inhibition, PHY alone protected as well as the optimal dose of the combination.

Efficacy comparison of scopolamine (SCP) and diazepam (DZ) against soman-induced lethality in guinea pigs.

Diazepam (DZ) and scopolamine (SCP) are known to be beneficial when each is used in combination with atropine (AT) + oxime therapy against intoxication by soman, but the efficacy of each might be expected to vary with the dosage of AT. Thus the therapeutic efficacy of SCP (5 doses; 0-0.86 mg/kg) versus DZ (5 doses; 0-5 mg/kg), when used in conjunction with AT (3 doses; 0.5-8 mg/kg) + 2-PAM (25 mg/kg) therapy, was tested in groups of pyridostigmine pretreated guinea pigs exposed to 1.6, 2.0, 2.5 or 3.2 LD50s of soman. Response surface methodology was employed to describe the relationship between lethality and the AT/DZ or AT/SCP dosages. Results show that within the indicated dose ranges used, the efficacy of SCP is not dependent on the presence of AT, whereas AT is needed for DZ to maintain the lowest probability of death. These findings suggest that in guinea pigs SCP could supplement AT or replace DZ as therapy against nerve agent intoxication.

Protection of rhesus monkeys against soman and prevention of performance decrement by pretreatment with acetylcholinesterase.

The ability of acetylcholinesterase from fetal bovine serum (FBS AChE) to protect against soman, a highly toxic organophosphorus (OP) compound, was tested in rhesus monkeys. Intravenous administration of FBS AChE produced a minimal behavioral effect on the serial probe recognition task, a sensitive test of cognitive function and short-term memory. Pharmacokinetic studies of injected FBS AChE indicated a plasma half-life of 40 hr for FBS AChE in monkeys. Both in vitro and in vivo titration of FBS AChE with soman produced a 1:1 stoichiometry between organophosphate-inhibited FBS AChE and the cumulative dose of the toxic stereoisomers of soman. Administration of FBS AChE protected monkeys against the lethal effects of up to 2.7 LD50 of soman and prevented any signs of organophosphate intoxication, e.g., excessive secretions, respiratory depression, muscle fasciculations, or convulsions. In addition, monkeys pretreated with FBS AChE were devoid of any behavioral incapacitation after soman challenge, as measured by the serial probe recognition task. Compared to the current multicomponent drug treatment against soman, which does not prevent the signs or the behavioral deficits resulting from OP intoxication, use of FBS AChE as a single pretreatment drug provides significantly effective protection against both the lethal and the behavioral effects of soman.