Richard P. Solana

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.

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.

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.

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.

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.

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 bischloroethylnitrosourea. 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.

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.

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.

Effects of Subacute Pretreatment Uite Carbamate Togetiieb 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.

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.