In vitro and in vivo characterization of recombinant human butyrylcholinesterase (Protexia) as a potential nerve agent bioscavenger.

Previous studies in rodents and nonhuman primates have demonstrated that pretreatment with cholinesterases can provide significant protection against behavioral and lethal effects of nerve agent intoxication. Human butyrylcholinesterase (HuBuChE) purified from plasma has been shown to protect against up to 5 x LD50s of nerve agents in guinea pigs and non-human primates, and is currently being explored as a bioscavenger pretreatment for human use. A recombinant form of HuBuChE has been expressed in the milk of transgenic goats as a product called Protexia. Protexia was supplied by Nexia Biotechnologies (Que., Canada) as a purified solution with a specific activity of 600 U/mg. Initial in vitro studies using radiolabeled 3H-soman or 3H-DFP (diisopropyl fluorophosphate) demonstrated that these inhibitors specifically bind to Protexia. When Protexia was mixed with soman, sarin, tabun or VX using varying molar ratios of enzyme to nerve agent (8:1, 4:1, 1:1 and 1:4, respectively), the data indicated that 50% inhibition of enzyme activity occurs around the 1:1 molar ratio for each of the nerve agents. Protexia was further characterized for its interaction with pyridostigmine bromide and six unique carbamate inhibitors of cholinesterase. IC50 and Ki values for Protexia were determined to be very similar to those of HuBuChE purified from human plasma. These data suggest that Protexia has biochemical properties very similar to those HuBuChE when compared in vitro. Together these data the continued development of the goat milk-derived recombinant HuBuChE Protexia as a potential bioscavenger of organophosphorus nerve agents.

Putative role of proteolysis and inflammatory response in the toxicity of nerve and blister chemical warfare agents: implications for multi-threat medical countermeasures.

Despite the contrasts in chemistry and toxicity, for blister and nerve chemical warfare agents there may be some analogous proteolytic and inflammatory mediators and pathological pathways that can be pharmacological targets for a single-drug multi-threat medical countermeasure. The dermal-epidermal separation caused by proteases and bullous diseases compared with that observed following exposure to the blister agent sulfur mustard (2,2'-dichlorodiethyl sulfide) has fostered the hypothesis that sulfur mustard vesication involves proteolysis and inflammation. In conjunction with the paramount toxicological event of cholinergic crisis that causes acute toxicity and precipitates neuronal degeneration, both anaphylactoid reactions and pathological proteolytic activity have been reported in nerve-agent-intoxicated animals. Two classes of drugs already have demonstrated multi-threat activity for both nerve and blister agents. Serine protease inhibitors can prolong the survival of animals intoxicated with the nerve agent soman and can also protect against vesication caused by the blister agent sulfur mustard. Poly (ADP-ribose) polymerase (PARP) inhibitors can reduce both soman-induced neuronal degeneration and sulfur-mustard-induced epidermal necrosis. Protease and PARP inhibitors, like many of the other countermeasures for blister and nerve agents, have potent primary or secondary anti-inflammatory pharmacology. Accordingly, we hypothesize that drugs with anti-inflammatory actions against either nerve or blister agent might also display multi-threat efficacy for the inflammatory pathogenesis of both classes of chemical warfare agent.

Protective action of the serine protease inhibitor N-tosyl-L-lysine chloromethyl ketone (TLCK) against acute soman poisoning.

Soman-poisoned rats display cholinergic crisis, a systemic mast cell degranulation characteristic of anaphylactic reactions and an excitotoxin-like sequential seizure and neuronal degeneration. The protection of guinea pigs from soman lethality by prophylactic administration of the serine protease inhibitor suramin suggests a possible proteolytic component in soman poisoning. The present study tested the effect of N-tosyl-L-lysine chloromethyl ketone (TLCK), an inhibitor of trypsin-like serine proteases, on soman-induced toxic signs (convulsions, righting reflex) and survival time. Nine control guinea pigs receiving 2 x LD(50) (56 microg kg(-1), s.c.) of soman immediately followed by a therapeutic dose of atropine sulfate (17.4 mg kg(-1) i.m.) experienced severe convulsions, and 8/9 lost the righting reflex. Six of these nine animals expired within 65 min; the three remaining animals survived 24 h to termination of the experiment. When a second group of animals were given TLCK (12 mg kg(-1), i.p.) 30 min prior to a 2 x LD(50) soman challenge and atropine-sulfate therapy, 5/9 experienced convulsions and only 3/9 lost the righting reflex. All nine animals survived beyond 4 h, with six surviving to 24 h. Compared with soman controls, prophylaxis with TLCK significantly prevented the loss of righting reflex (P = 0.05) and enhanced 4-h survival (P = 0.005). Although, convulsions were reduced and 24-h survival was improved in TLCK-treated animals, these results were not statistically significant. The protection from soman toxicity by chemically distinct protease inhibitors such as suramin and TLCK suggests a role for pathological proteolytic pathways in soman intoxication.

Development of an immunoassay for diagnosis of exposure to toxic organophosphorus compounds.

Currently, diagnosis of exposure to toxic low-molecular-weight compounds is effected by the use of chromatographic techniques. Such an approach is limited by the need for expensive equipment and sample clean-up before carrying out the analysis. To overcome those drawbacks, we have been involved in the development of an immunoassay for diagnosis of exposure to toxic organophosphorus compounds such as pinacolylmethyl phosphonofluoridate (soman), which is a chemical warfare agent. Prior estimates suggested that it is necessary to be able to detect soman at a concentration below 2.5 x 10(-7) M. Using four previously developed monoclonal antibodies, an enzyme-linked immunosorbant assay (ELISA) was used to optimize assay conditions and identify the antibody with the highest apparent affinity. The minimum required assay time was 2.0-2.5 h with no loss in sensitivity. To determine the specificity of the highest affinity antibody, a competitive inhibition enzyme immunoassay (CIEIA) was performed with six structural analogs of soman. The IC50 values for these analogues were 5 x 10(-7) M for 4-nitrophenylpinacolylmethylphosphonate, 8 x 10(-7) M for dipinacolylmethylphosphonate, 2 x 10(-6) M for diisopropylmethylphosphonate, 3 x 10(-5) M for 4-nitrophenylmethyl(phenylphosphinate) and 6.5 x 10(-5) M for 4-nitrophenylethyl(phenyl)phosphinate. 4-Nitrophenyl-di(n-butyl)phosphinate did not inhibit binding. Those inhibitors with branched alkyl side-chains, similar to the soman molecule, were effective inhibitors. Compounds, which contained predominately aromatic groups, were poor inhibitors. We are continuing to probe the binding specificity of the monoclonal antibody to determine its utility in further assay development. Our present results suggest that the antibody chosen may have the appropriate specificity and affinity for immunodiagnosis of exposure to soman.

Organophosphate skin decontamination using immobilized enzymes.

We previously demonstrated that a combination of cholinesterase (ChE) pre-treatment with an oxime is an effective measure against soman and sarin. We describe here a novel approach for the preparation of covalently linked ChEs which are immobilized to a polyurethane matrix. Such preparation of ChE-sponges enhances the stability and usefulness of the enzymes in non-physiological environments. The ChE-sponges, which can be molded to any form, can effectively be used to remove and decontaminate organophosphates (OPs) from surfaces, biological (skin or wounds) or otherwise (clothing or sensitive medical equipment), or the environment. The ChE-sponges retained their catalytic activity under conditions of temperature, time, and drying where the native soluble enzyme would rapidly denature, and can be reused in conjunction with oximes many times. The ChE-sponge in the presence of oxime repeatedly detoxified OPs such as DFP or MEPQ. These developments in ChE technology have extended the applicability of OP scavengers from in vivo protection, to a variety of external detoxification and decontamination schemes. In addition to treatment of OP-contaminated soldiers, the ChE-sponge could protect medical personnel from secondary contamination while attending chemical casualties, and civilians exposed to pesticides or highly toxic nerve agents such as sarin.

Hypothesis for synergistic toxicity of organophosphorus poisoning-induced cholinergic crisis and anaphylactoid reactions.

The neurotoxicity of organophosphorus (OP) compounds involves the inhibition of acetylcholinesterase (AChE), causing accumulation of acetylcholine (ACh) at synapses. However, cholinergic crisis may not be the sole mechanism of OP toxicity. Adverse drug reactions caused by synergistic toxicity between drugs with distinct pharmacological mechanisms are a common problem. Likewise, the multiple pharmacological activities of a single molecule might also contribute to either toxicity or efficacy. For example, certain OP compounds (e.g. soman) exhibit anti-AChE activity and also act as secretagogues by inducing mast cell degranulation with associated autacoid release and anaphylactoid reactions. Anaphylactoid shock can produce a lethal syndrome with symptoms of respiratory failure and circulatory collapse similar to the physiological sequelae observed for OP poisoning. Moreover, the major classes of drugs used as antidotes for OP intoxication can affect anaphylaxis. Acetylcholine can act as an agonist of autacoid release, and autacoids such as histamine can augment soman-induced bronchial spasm. In concert with the demonstrably critical role of cholinergic crisis in OP toxicity, the precepts of neuroimmunology indicate that secondary adverse reactions encompassing anaphylactoid reactions may complicate OP toxicity.

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.

The role of glutamate-199 in the aging of cholinesterase.

Aging of organophosphate-conjugated acetylcholinesterase results from the loss of an alkoxy group with concomitant stabilization of the conjugate to spontaneous or nucleophile-induced deacylation. We have examined the kinetics of aging in a pinacolylmethylphosphonofluoridate (soman)-inhibited mutant enzyme in which the glutamate (E199) located at the amino-terminal to the active-site serine (S200) was converted to glutamine (Q). For wild type enzyme, the soman-acetylcholinesterase conjugate aged immediately, giving rise to a form of enzyme resistant to reactivation by oximes. In contrast, the E199Q mutant enzyme was largely resistant to aging and could be reactivated by oximes. Since the pH dependence for aging was not altered appreciably, the primary influence of the loss of charge appears to be on the intrinsic rate of aging. The negative charge on E199 likely imparts an inductive effect on the conjugated organophosphate to facilitate removal of the alkoxy group.

Catalytic antibodies hydrolysing organophosphorus esters.

Transition state stabilization is considered one means by which enzymes reduce free energy of activation. The transition state of phosphonic acid anhydrides acted on by OPA hydrolase is postulated to be pentacoordinate, which ordains either a square pyramid or a trigonal bipyramid structure. The advent of catalytic monoclonal antibodies has provided a system in which these assumptions can be tested. By immunizing mice with the protein conjugate of a trigonal bipyramid transition state analog, we have produced hybridomas secreting monoclonal antibodies which hydrolyze phosphonates. To date, activity has been shown toward pinacolyl methylphosphonofluoridic acid (soman). Preliminary results suggest that the antibody is an IgG2a with kappa light chain character. Our results support the trigonal bipyramidal transition state for this group of enzymes.

Monoclonal antibodies against soman: characterization of soman stereoisomers.

Hybridomas were produced which expressed monoclonal anti-soman antibodies as determined by microtiter enzyme-linked-antibody immunoassay (EIA). Each of these antibodies was titrated using a competitive inhibition enzyme immunoassay (CIEIA) with a variety of test ligands. The ligands used included soman (a racemic mixture), sarin, tabun, and each of the four stereoisomers of soman (C+ P+, C+ P-, C-P+ and C-P-). In all cases the antibodies tested exhibited IC50 values of 10(-4)-5 x 10(-6) M for soman. When sarin or tabun was used as a ligand, the antibodies exhibited no cross reactivity. All of the antibodies cross reacted with the four soman stereoisomers. A second group of hybridomas were produced which expressed monoclonal antibodies against CsPs-soman. These antibodies were used to make preliminary absolute chiral assignments to the four soman stereoisomers.

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.

Transport of cyanide into guinea pig cardiac mitochondria.

The transport of cyanide (CN) into cells has been presumed to be by passive diffusion. Recently, there have been reports that CN, in the form of an anion, may enter the cell by active or facilitated transport. To characterize the mechanism(s) and kinetics of CN movement across the cell membrane, we measured the rate of 14CN (Na salt) uptake into guinea-pig mitochondria. Initial velocities of CN movement into mitochondria were determined at time points ranging from 10-100 msec and at CN concentrations ranging from 1 microM-10 mM using a rapid filtration device. A Hofstee plot of the data suggests that an active or facilitated transport predominates at lower CN concentrations (less than 10 microM), whereas passive diffusion of CN predominates at higher CN concentrations. The kinetic constants for the active phase transport were Jmax = 0.9 pmol/ms and Kt = 14 microM. These results suggest that a large portion of CN movement across the cell membrane is due to an active or facilitated transport phenomenon.

Effect of an anticholinesterase compound on the ultrastructure and function of the rat blood-brain barrier: a review and experiment.

Soman, an organophosphorous irreversible inhibitor of acetylcholinesterase, was studied for its effect on the rat blood-brain barrier (BBB) during the first 24 h of intoxication. Young adult male Sprague-Dawley rats, injected with Evans blue-dye and surviving a subsequent single convulsive dose of soman (114 micrograms/kg, 0.9LD50), presented focal and diffuse penetration of dye in areas of brain normally considered protected by the BBB. Invasion was widest during the first hour when signs of excitation, respiratory distress and convulsions peaked and was absent at 24 h. During this time period, cholinesterase inhibition, as measured by enzyme assay, persisted in brain and blood at 10% and 6% of control values respectively. Brains of nonconvulsing animals and animals pretreated with nembutal (45 mg/kg, I.P.) or with diazepam (10 mg/kg, I.P.) were free of extravasated dye. A ranking of dye-breached brain areas suggested that cerebellar and cerebral cortex were most frequently involved while brain stem was rarely stained. Ultrastructural analysis of breached areas with horseradish peroxidase as a tracer molecule, revealed that the probable subcellular mechanism of the induced breach was enhanced vesicular transport, a mechanism similarly described for seizure. Consequences of the breach were emphasized with the detection of significantly elevated levels of an exogenously administered quaternary compound, 3H-hexamethonium. These findings present additional evidence that an anticholinesterase-induced breach of the rat blood-brain barrier is convulsive dependent, demonstrates BBB mechanisms similar to that of seizure, and can allow CNS penetration of blood-borne drugs and circulatory proteins that normally would be slowed or excluded by an intact BBB.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic subcellular localization of cresylbenzodioxaphosphorin oxide (CBDP)-sensitive soman binding sites.

The toxicity of the organophosphorus poison soman (pinacolylmethylphosphonofluoridate) is attributable to its irreversible inhibition of the enzyme acetylcholinesterase. In addition, soman binds irreversibly to a number of noncholinesterase tissue binding sites which appear to be its major means of in vivo detoxification. This study was conducted to determine the hepatic subcellular localization of these sites. Subcellular fractions of liver from male Sprague-Dawley rats (200-250 g) were prepared by differential and isopycnic density gradient centrifugation. The binding of [14C]soman to these subcellular fractions was determined in the presence and absence of cresylbenzodioxaphosphorin oxide (CBDP), a compound that binds irreversibly to the noncholinesterase soman binding sites. Crude fractionation of liver homogenates into nuclear, mitochondrial, microsomal, and soluble fractions revealed that 78% of the total CBDP-sensitive binding activity was localized in the nuclear and microsomal fractions. Further purification of these fractions indicated that all of the homogenate binding activity could be accounted for in the purified microsomal fraction. When purified liver microsomes were solubilized and fractionated on linear sucrose gradients, 90% of the CBDP-sensitive soman binding activity cosedimented with carboxylesterase activity which suggests that these binding sites are carboxylesterase.

Effects of repeated injection of sublethal doses of soman on behavior and on brain acetylcholine and choline concentrations in the rat.

The effects of repeated exposure to a sublethal dose (60 micrograms/kg; 0.4 LD50) of soman on brain regional acetylcholine (ACh) and choline (Ch) levels, spinal cord cholinesterase (ChE) activity and on water consumption, body weight and gross behavioral changes were examined. Male rats were dosed once a week or three times a week and at 24 h after 2, 4 or 6 weeks of dosing, selected brain tissues and behavior were examined. During the 6-week period, there was no difference between control and soman-dosed rats in water consumption or body weight under either treatment regimen. The animals treated once a week adapted to this exposure regimen well. They exhibited no change in the levels of ACh or Ch in any of the brain areas when examined at the end of 2, 4 or 6 weeks, nor did they show any obvious signs of poisoning. The total ChE activity fluctuated between 70 and 100% of control. When treated three times a week, however, survivors (90%) of the soman-treated rats developed signs that progressed in severity to a hyper-reactivity syndrome which consisted of an exaggerated reaction to mild tactile stimuli. Brain ACh levels did not change and ChE activity showed inhibition of 40, 58 and 75% when measured at 2, 4 and 6 weeks, respectively. At the end of 6 weeks, the levels of Ch, except in the striatum, were significantly elevated in brainstem, cerebral cortex, hippocampus, midbrain, and cerebellum (52%, 147%, 68%, 46%, and 91%, respectively), indicating that Ch metabolism in neuronal membranes may be altered following more frequent low-dose soman exposures.

Partial characterization of an enzyme that hydrolyzes sarin, soman, tabun, and diisopropyl phosphorofluoridate (DFP).

The properties of a rat liver enzyme that hydrolyzes organophosphorus (OP) inhibitors of cholinesterases were studied. The rates of hydrolysis of OP inhibitors were determined by continuous titration of released hydrogen ions, using a pH stat method. Centrifugation of homogenates at 205,000 g for 30 min demonstrated that the activity was in the soluble fraction. Hydrolysis of sarin, soman, and diisopropyl phosphorofluoridate (DFP), but not of tabun, was stimulated by the addition of Mn2+ and Mg2+. Hydrolysis of sarin greater than soman greater than tabun greater than DFP. Unlike other OP hydrolases that preferentially hydrolyze the non-toxic isomers of soman, this enzyme hydrolyzed all four soman isomers at approximately the same rate. This result was obtained in vitro by gas chromatographic analysis of enzyme-catalyzed soman hydrolysis and confirmed in vivo by demonstrating reduced toxicity in mice of soman partially hydrolyzed by this enzyme. Km and Vmax were determined by fitting V vs [S] to a hyperbolic function using regression analysis. Km values ranged from 1.1 mM for soman to 8.9 mM for tabun. Vmax values ranged from 54 nmol/min/mg protein for DFP to 2694 for sarin. The enzyme was stable for at least 2 months at -90 degrees but was inactivated by heating at 100 degrees for 5 min. Elution profiles from gel filtration by high pressure liquid chromatography showed that the hydrolytic activity for the OP inhibitors eluted in a single peak, suggesting that a single enzyme was responsible for the observed hydrolysis. Further purification and characterization of this enzyme should prove useful for the development of methods for detection, detoxification, and decontamination of these cholinesterase inhibitors.

Specific soman-hydrolyzing enzyme activity in a clonal neuronal cell culture.

An enzymatic activity that specifically hydrolyzes the highly toxic organophosphorus anticholinesterase compound soman (pinacolyl methylphosphonofluoridate) has been identified and partially characterized in the clonal neuronal neuroblastoma-glioma hybrid NG108-15 cell line. Using the whole cell homogenate as the enzyme source and 1 mM substrate, the relative rate of hydrolysis of two other toxic anticholinesterase compounds sarin (isopropyl methylphosphonofluoridate) and tabun (ethyl-N-dimethyl phosphoramidocyanidate) is approximately one-tenth the rate of hydrolysis of soman, while DFP (diisopropyl phosphorofluoridate), paraoxon (p-nitrophenyl diethylphosphate), and a phosphinate PNMPP (p-nitrophenyl methyl (phenyl) phosphinate) are not hydrolyzed. Analysis of the kinetics of soman hydrolysis reveals two components of the enzyme activity with different affinities and reaction rates. Unlike previously reported enzymes of this type, this enzyme lacks chiral specificity and thus hydrolyzes both toxic and non-toxic soman stereoisomers at equal rates. The enzyme activity is stable at low temperature, found almost exclusively in the soluble fraction of these cells, and enhanced significantly by Mn2+ and by chemical differentiation of these cells in culture. The results suggest possible application of this enzyme for soman detection and/or detoxication, and use of the NG108-15 cell line to study the natural function(s) of enzymes of this type.

A pharmacodynamic model for soman in the rat.

A pharmacodynamic model for inhibition of acetylcholinesterase (AChE) by soman was developed to describe the intertissue differences in AChE inhibition, the dose response of AChE to inhibition by soman, and the effect of differences in xenobiotic metabolism on soman toxicity. Based on the principles of physiological pharmacokinetics, this pharmacodynamic model consisted of a set of mass balance equations that included parameters for blood flow, tissue volumes, soman metabolism, tissue/plasma partition coefficients, initial AChE levels, and the rate constant for AChE inhibition. Sensitivity analysis of the model revealed that variation of the soman metabolism parameter in plasma was the most important determinant of variation in the inhibition of brain AChE by soman.

Effect of carboxylesterase inhibition on carbamate protection against soman toxicity.

The ability of the carbamates pyridostigmine and physostigmine to protect against the lethal effects of soman, an extremely toxic anticholinesterase agent, was measured in rats, guinea pigs and rabbits. Pharmacologically equivalent doses of these carbamates that inhibited 70% of the blood acetylcholinesterase in each species were injected i.m. 25 min before s.c. injection with soman. Pretreatment with either carbamate, in combination with 17.4 mg/kg of atropine, produced protection against soman toxicity in all species. When protection was expressed as the ratio between the soman LD50 values in carbamate-protected animals and control animals, this protective ratio varied 3-fold between species (2.1-6.1 for pyridostigmine; 2.2-6.6 for physostigmine). When protection was expressed as the difference in the soman LD50 values between carbamate-protected animals and control animals, this protective difference was consistent among species (126 +/- 19 micrograms/kg). Species variation in protective ratios was observed largely because the control LD50 values defining soman toxicity in unprotected animals varied among species (20 micrograms/kg in rabbits, 28 micrograms/kg in guinea pigs and 126 micrograms/kg in rats). The species variation of the soman LD50 values in control animals was eliminated by pretreating animals with cresylbenzodioxaphosphorin oxide, which reduced the species variation in soman detoxification. The LD50 values for soman in cresylbenzodioxaphosphorin oxide-treated animals (9.8-15.6 micrograms/kg) did not differ significantly between species. Similarly, protective ratios for carbamates against soman in cresylbenzodioxaphosphorin oxide-treated animals were also clustered in a narrow range (8.5-11.4 for pyridostigmine; 9.0-13.4 for physostigmine) that did not differ significantly, regardless of species or carbamate.(ABSTRACT TRUNCATED AT 250 WORDS)

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.