A reproducible nonlethal animal model for studying cyanide poisoning.

Previous studies using bolus intravenous injections of sodium cyanide have been used to model the sudden exposure to high concentrations of cyanide that could occur on the battlefield. This study was designed to develop a model that would simulate the type of exposure to cyanide gas that could happen during actual low-level continuous types of exposure and then compare it with the bolus model. Cardiovascular and respiratory recordings taken from anesthetized dogs have been used previously to characterize the lethal effects of cyanide. The intravenous, bolus injection of 2.5 mg/kg sodium cyanide provides a model in which a greater than lethal concentration is attained. In contrast, our model uses a slow, intravenous infusion of cyanide to titrate each animal to its own inherent end point, which coincides with the amount of cyanide needed to induce death through respiratory arrest. In this model, therapeutic intervention can be used to restore respiration and allow for the complete recovery of the animals. After recovery, the same animal can be given a second infusion of cyanide, followed again by treatment and recovery, providing a reproducible end point. This end point can then be expressed as the total amount of cyanide per body weight (mg/kg) required to kill. In this study, the average dose of sodium cyanide among 12 animals was 1.21 mg/kg, which is approximately half the cyanide used in the bolus model. Thus, titration to respiratory arrest followed by resuscitation provides a repetitive-use animal model that can be used to test the efficacy of various forms of pretreatment and/or therapy without the loss of a single animal.

Population pharmacokinetics and pharmacodynamics of pyridostigmine bromide for prophylaxis against nerve agents in humans.

This study was conducted to determine the pharmacokinetics and pharmacodynamics of pyridostigmine given as 30 mg of pyridostigmine bromide every 8 hours in healthy subjects. Plasma pyridostigmine concentration and red blood cell acetylcholinesterase activity were measured in blood samples collected during a 3-week period. Population analysis was performed using standard pharmacokinetic and pharmacodynamic models with the nonlinear mixed-effect modeling software (NONMEM). The pharmacokinetic model that best fit the pyridostigmine plasma levels was a two-compartment open model with first-order absorption, a lag time, and first-order elimination from the central compartment. The pharmacodynamic model that best fit red blood cell acetylcholinesterase activity was an inhibitory Emax model with an effect compartment linked to the central compartment. The results showed that the pharmacokinetics of pyridostigmine bromide are both gender and weight dependent. The pharmacodynamic effect does not lag significantly from the plasma concentration and returns to near normal within 8 hours. With the present dosage regimen of 30 mg every 8 hours, 30% of individuals may not have red blood cell acetylcholinesterase inhibition > 10% at the time of the trough.

Erythrocyte and plasma cholinesterase activity in male and female rhesus monkeys before and after exposure to sarin.

The rhesus monkey (Macaca mulatta) has a menstrual cycle similar to the human. Differences in hormone levels have been demonstrated between the sexes and in females during the menstrual cycle but these differences in terms of organophosphorus toxicity have not been explored. Plasma cholinesterase (ChE/BuChE) and erythrocyte (RBC) acetylcholinesterase (AChE) activity were measured before and after exposure to the organophosphorus compound sarin (11 micrograms/kg, i.v.; 0.75 LD50) in six male and six female rhesus monkeys. After baseline measurements were obtained, sarin was administered to atropinized monkeys to determine in vivo differences between the sexes in their response to sarin. With the baseline values, the intraanimal and intragroup BuChE/AChE variations were found to be minimal. Following sarin intoxication and 2-PAM treatment no significant differences were seen between the sexes in the rate of reactivation of BuChE or AChE by 2-PAM. The rate of aging of sarin phosphonylated RBC AChE between the sexes was also similar. De novo regeneration of RBC AChE and plasma BuChE after sarin intoxication was different between the male and female monkeys. The female plasma BuChE recovery rate was 48% slower than the male recovery rate, while the early (first 63 days) RBC AChE recovery rate was 24.5% faster in the females. In conclusion, there probably are not any clinically significant differences between male and female rhesus monkeys acutely intoxicated with sarin. However, on subsequent exposure clinical differences may be observed due to substantial differences in the rate of de novo synthesis of both plasma BuChE and RBC AChE.

Evaluation of the toxicity, pathology, and treatment of cyclohexylmethylphosphonofluoridate (CMPF) poisoning in rhesus monkeys.

Cyclohexylmethylphosphonofluoridate (CMPF) is an organophosphate cholinesterase inhibitor with military significance. The purpose of these studies was 1) to determine the acute toxicity of CMPF in the male rhesus monkey, 2) to evaluate the efficacy of pyridostigmine (PYR) pretreatment plus atropine and oxime (2-PAM or H16) treatment, and 3) to evaluate the pathological consequences of acute poisoning. An i.m. LD50 of CMPF was estimated using an up-and-down dose selection procedure and 12 animals. The 48-h and 7-day LD50 was 46.6 micrograms/kg, i.m. In the protection experiments, pyridostigmine (0.3-0.7 mg/kg/24 h) was administered by surgically implanted osmotic minipumps for 3-12 days resulting in 21-65% inhibition of erythrocyte acetylcholinesterase activity. Animals were challenged with 5 x L50 CMPF (233 micrograms/kg) and treated with atropine (0.4 mg/kg) and either 2-PAM (25.7 mg/kg) or HI6 (37.8 mg/kg) at the onset of signs or 1 min after challenge. Osmotic pumps were removed within 30 min after agent challenge. Pyridostigmine, atropine, and either 2-PAM or H16 were completely effective against CMPF, saving ten of ten animals in each group. In comparison, three of five animals challenged with 5 x LD50 of soman and treated with atropine and 2-PAM survived 7 days. The primary histologic lesions in the acute toxicity group were neuronal degeneration/necrosis and spinal cord hemorrhage. The CMPF treated groups (total of 20 animals) had minimal nervous system changes with no significant lesion difference resulting from the different oxime therapies. The primary non-neural lesions were degenerative cardiomyopathy and skeletal muscle degeneration which occasionally progressed to necrosis and mineralization.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy evaluation of physostigmine and anticholinergic adjuncts as a pretreatment for nerve agent intoxication.

Pretreatment of nonhuman primates with physostigmine (Phy) and scopolamine or physostigmine and trihexyphenidyl 25 min before exposure to 2 LD50 soman im resulted in complete survival without convulsions or loss of consciousness. When identically pretreated animals were challenged with 5 LD50s of soman followed by atropine and 2-PAM therapy 1 min later, all animals experienced a loss of consciousness for approximately 10 min followed by functional recovery within an additional 20 min. These findings indicated that a pretreatment regimen composed of Phy and cholinolytic is capable of protecting primates from an absolute lethal dose of soman with rapid recovery from incapacitation.

Pharmacokinetics and pharmacodynamics of oximes in unanesthetized pigs.

The pharmacokinetics and cardiovascular pharmacodynamics of two oximes were studied in unanesthetized pigs. Effects of 2-[(hydroxyimino)methyl]-1-methylpyridinium chloride (pralidoxime chloride; 2-PAM Cl; 50 mumol/kg) were compared with those of 1,1-methylene bis[4(hydroxyiminomethyl) pyridinium] dichloride (methoxime; MMB-4; 100 mumol/kg). Cardiopulmonary parameters were monitored and plasma concentrations of oximes were determined from arterial blood samples taken at intervals over a period of 5 hr postinjection. Plasma concentrations for both oximes were fitted to standard pharmacokinetic models using the computer program PCNONLIN. Average pharmacokinetic parameters were determined for each oxime. Only mild to moderate physiological side effects were detected following intramuscular administration. 2-PAM Cl was more rapidly absorbed and distributed in the blood than MMB-4. Although the latter had a slight lag time to attain detectable levels in the blood, retention time was longer than that of 2-PAM Cl.

Effects of physostigmine on the cardiopulmonary system of conscious pigs.

Physostigmine, as a pretreatment candidate for nerve agent poisoning, was examined for cardiopulmonary side effects. Cardiovascular and pulmonary parameters were monitored in unanesthetized domestic pigs which received pulmonary arterial infusion of 5 micrograms/kg/min physostigmine salicylate for 2 hr. A level of 74% inhibition of red blood cell (RBC) acetylcholinesterase (AChE) activity was attained in 45 min, and this level of carbamylation increased only slightly during the remaining infusion period. In addition to this large change in AChE activity, minor changes were observed in hematocrit, heart rate, body temperature, mean aortic pressure, pulmonary arterial wedge pressure, and pulmonary artery pressure. Typically, these parameters showed a trend toward elevated levels. Blood gases, pH, respiratory rate, tidal and minute volume, cardiac output, nonelastic resistance, and dynamic compliance were not significantly different from baseline values. The unanesthetized pig responds to physostigmine in a manner similar to that reported for other species and appears to be a suitable model for evaluating cardiopulmonary effects of cholinesterase inhibitors.

The effect of 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide on tissue cholinesterase and carboxylesterase activities of the rat.

The dose-response (0.1 to 1000 mg/kg sc) effects of 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide (CBDP; a metabolite of the organophosphorus compound tri-o-cresylphosphate) on total cholinesterase (ChE) and carboxylesterase (CaE) activities in tissues from the rat were examined. Doses of CBDP greater than 1.0 mg/kg inhibited CaE activity maximally (greater than 99%) in plasma and lung, two important sites for detoxification of organophosphorus toxicants. A biphasic dose-dependent inhibition of ChE activity was seen in all tissues; the ED50 values showed a difference of two orders of magnitude between the first and the second phases of the dose-response curves. CBDP inhibited the blood esterases in the order plasma CaE much greater than plasma ChE much greater than red blood cell (RBC) ChE. The biphasic dose-response curve and preferential inhibition of the blood esterases may reflect the inhibition of butyrylcholinesterase in preference to acetylcholinesterase in these tissues. At doses of CBDP below 1.0 mg/kg, plasma, RBC, and brain regional ChE activities were inhibited by less than 10%, whereas at doses above 2.0 mg/kg, ChE activities were inhibited substantially (up to 80% in plasma, up to 60% in RBC, and greater than 90% in brain regions). On the basis of these results, a dose of CBDP between 1.0 and 2.0 mg/kg should prove useful as a pretreatment for studies of OP toxicity in the rat.

Carbamoylated enzyme reversal as a means of predicting pyridostigmine protection against soman.

The inhibition of human and mammalian red blood cell (RBC) cholinesterase (AChE) in whole blood in the presence of added pyridostigmine has been examined. After the addition of pyridostigmine to animal and human blood, red cells were separated from plasma at varying intervals and their enzyme activity measured. An apparent rate constant (ke) was derived for the reaction sequence in which carbamate is released from AChE inhibited by pyridostigmine. The constant is a complex of the rates of decarbamoylation and reinhibition of AChE in the blood sample. Rate constants were also determined for the spontaneous reactivation (ks) of carbamoylated AChE in the species studied. Values of Ks were greater than Ke in corresponding species but varied little between species. Pretreatment of animals with pyridostigmine is known to be an effective therapy against organophosphorus compounds, including soman. The ranking of ke values in mammalian blood was the same as that for the protection against soman in animals: monkey greater than guinea-pig greater than rabbit greater than rat (ke = 0.15, 0.07, 0.05, 0.02 h-1, respectively). Since ke for human blood (0.20 h-1) was greater than that of monkey, pyridostigmine pretreatment would be expected to be an effective prophylaxis for soman in humans.

The effects of blood flow and detoxification on in vivo cholinesterase inhibition by soman in rats.

The in vivo time course of cholinesterase inhibition was measured in brain, lung, spleen, hind limb skeletal muscle, diaphragm, intestine, kidney, heart, liver, and plasma of rats receiving 90 micrograms/kg soman, im. This dose of soman produced severe respiratory depression and transient hypertension, but no significant changes in the cardiac output or heart rate of anesthetized rats. The rate and maximal extent of in vivo cholinesterase inhibition by soman varied widely among the tissues. Although cardiac output was unchanged by soman administration, the blood flow in heart, brain, and lung (bronchial arterial flow and arteriovenous shunts) was increased, whereas blood flow in spleen, kidney, and skeletal muscle was decreased. The relative importance of tissue blood flow, tissue levels of cholinesterase and acetylcholinesterase, and tissue levels of soman-detoxifying enzymes (diisopropyl-fluorophosphatase and carboxylesterase) in determining the in vivo rate and maximal extent of cholinesterase inhibition was examined by multiple regression analysis. The best multiple regression model for the maximal extent of cholinesterase inhibition could explain only 63% of the observed variation. The best multiple regression model for the in vivo rate of cholinesterase inhibition contained three independent variables (blood flow, carboxylesterase, and cholinesterase) and could account for 94% of the observed variation. Of these three variables blood flow was the most important, accounting for 79% of the variation in the in vivo rate of cholinesterase inhibition. This suggests that it may be possible to use a flow-limited physiological pharmacokinetic model to describe the kinetics of in vivo cholinesterase inhibition by soman.

Correlation of 2-PAM plasma levels after organophosphate intoxication.

In order to investigate the pathophysiological effects of organophosphorus poisoning on therapeutic compounds, multiples of the LD50 for sarin and soman were injected into guinea pigs after which 2-PAM and atropine sulfate were administered intramuscularly. It was found that intoxication by sarin or soman in therapeutically treated animals resulted in modifications of plasma oxime concentrations. Peak plasma (CPmax) concentrations and areas under the curve (AUC) between 1 to 10 min after the injection of 2-PAM were altered in a systematic fashion which correlated to the multiple of the LD50 (r greater than 0.9498). The effect of the sarin and soman was studied at fractions of the LD50, i.e. 0.7s, 1.5, 2.2, 2.8, 5.6, 11.2. The influence of the organophosphorus poisons on the plasma concentration of 2-PAM is biphasic. Multiples of the LD50 equal to or below 2.2 tend to decrease, whereas multiples equal to or above 2.8 tend to increase CPmax and AUC in different groups of animals. Changes in the plasma concentrations of 2-PAM may result from alterations in blood flow rates and patterns which would alter the distribution and elimination of 2-PAM.

Leaching of zinc compound from rubber stoppers into the contents of automatic atropine injectors.

This report describes how a material within the cartridge of an automatic injector contaminated its contents. On prolonged storage, a formulation that contained atropine produced lethality in mice. The toxic material originated from zinc compounds that were present in the rubber stopper and plunger of the container and that subsequently leached into the formulation. The contents of cartridges that contained greater than or equal to 0.75 mg/mL of solubilized zinc were lethal to at least 20% of the mice tested; those that contained 0.42 mg/mL showed no lethality. The problem resulted from the physicochemical properties of the rubber, not the concentration of zinc used in the vulcanization process.

Plasma free cyanide and blood total cyanide: a rapid completely automated microdistillation assay.

Techniques are presented which provide direct measurement of both free cyanide (CN-) in plasma and total CN- in whole blood. Loss of total CN- from blood is prevented by conversion to cyanmethemoglobin. Both free and total CN- are assayed by a completely automated method providing readout 17 minutes after sampling. No prior isolation technique is required and sensitivity is adjustable to cover a broad range of CN- concentrations from 1 to 4000 uM. Precision of blood CN- values from 2 to 2500 uM is within +/- 2.3%. No interference results from thiocyanate or thiosulfate at a concentration of approximately 1 mM.

The stability of sarin and soman in dilute aqueous solutions and the catalytic effect of acetate ion.

The hydrolysis of aqueous solutions of organophosphonate compounds such as sarin and soman is a critical factor in biochemical and therapeutic studies. The ability to dilute and transport these compounds with assurance of their purity is imperative to the integrity of such studies. An enzymic method has been used to measure low levels of organophosphonates. Maximum stability data were determined in aqueous and isotonic saline solutions. Acetate ion was found to significantly accelerate hydrolysis.1

A completely automated fluorometric blood cyanide method: a specific assay incorporating dialysis and distillation.

Discrete blood samples can be assayed at a rate of 20/hr. By means of a double lumen catheter, venous or arterial blood can be monitored continuously.

Temporal intrapersonal physiological variability of cholinesterase activity in human plasma and erythrocytes.

Erythrocyte and plasma cholinesterase activities were measured biweekly in one group of 22 subjects for a year and daily for three weeks in another group of nine men. The average range [i.e., (range/mean) X 100] of activity of erythrocyte cholinesterase in men during a year was 8% and during three weeks was 5%. For plasma, the corresponding values were 25% and 12%. The average ranges for erythrocyte and plasma cholinesterase activity in women during a year were 12% and 24%. Erythrocyte cholinesterase activity varies less than do hematocrit, hemoglobin, or erythrocyte count.

Influence of age, sex, and oral contraceptives on human blood cholinesterase activity.

We estimated cholinesterase (EC 3.1.1.8) activities in erthrocytes and plasma of 443 men, 188 women not taking oral contraceptives, and 70 women who were taking oral contraceptives. Men in the first six decades of life had higher plasma cholinesterase activity than did women who were not taking oral contraceptives, and these women had higher plasma cholinesterase activity than women taking oral contraceptives. After the age of 60 there was no intersex difference. Activity of erythrocytes from the oral contraceptive group was higher than in the other groups, men had the lowest activity, and there was an increased activity with age in both sexes until the age of 60. These findings suggest that there is no single "normal" value for cholinesterase activity for adults.