In vitro degradation of the stereoisomers of soman in guinea-pig, mouse and human skin

The fate of the four stereoisomers of soman [C(-)P(+), C(+)P(+), C(+)P(-) and C(-)P(-)] was studied by incubating 10 microM C(+/-)P(+/-)-soman at pH 7.4 and 37 degrees for various periods in the presence or absence of homogenates (1:10 and 1:20 w/v) of guinea-pig, mouse or human skin. The remaining concentrations of the soman isomers were determined gas chromatographically. Similar rates of spontaneous (non-enzymatic) hydrolysis (K = 0.005 min-1) were found for the four isomers of soman. Hydrolysis of the toxic (C(+/-)P(-)-isomers is not accelerated in the presence of the skin homogenates. In contrast, the non-toxic C(+/-)P(+)-isomers are enzymatically hydrolysed. As the amount of proteins present in the homogenates varied the rate constants for enzyme hydrolysis per protein concentration were calculated. Except for the high hydrolysis rate constant of greater than 0.127/min.g.l for C(+)P(+) in human skin, these values were almost similar (0.031-0.045/min.g.l) for the skin homogenates tested. Irreversible binding sites for the four soman-stereoisomers are only found in homogenates of mouse skin; 122-195 pmol soman-isomer are bound per mg protein. After preincubation of mouse homogenate with 10 microM soman during 18 hr at 0-4 degrees no further binding of the isomers was detected. It is concluded that skin of the three species tested does not contain enzymes that degrade the toxic C(+/-)P(-)-isomers of soman, whereas phosphorylphosphatase activity for the C(+/-)P(+)-isomers is present in the skin of all three species. Binding sites for all four soman isomers are only present in mouse skin.     

Stereoisomers of soman (pinacolyl methylphosphonofluoridate): inhibition of serum carboxylic ester hydrolase and potentiation of their toxicity by CBDP (2-(2-methylphenoxy)-4H-1,3,2-benzodioxaphosphorin-2-oxide) in mice

The interaction of C(+/-)P(+/-)-soman (pinacolyl methylphosphonofluoridate) and its individual stereoisomers with serum carboxylic-ester hydrolase and potentiation of their toxicity by a carboxylic-ester hydrolase inhibitor CBDP (2-(2-methylphenoxy)-4H-1,3,2-benzodioxaphosphorin-2-oxide) was investigated. C(+/-)P(+/-)-Soman and the individual stereoisomers all inhibited purified mouse serum carboxylic-ester hydrolase to different degrees. C(+/-)P(+/-)-Soman and the C(-)P(-)- and C(+)P(-)-isomers had Ki values of 30.6, 18.7, and 35.7 nM, respectively, and C(-)P(+)- and C(+)P(+)-isomers had Ki values of 1412 and 2523 nM, respectively. In toxicity experiments CBDP (0.5 mg/kg; iv 1 hr prior to soman) pretreatment potentiated the toxicity of C(+/-)P(+/-)-, C(+)P(-)-, and C(-)P(-)-soman to a similar degree. Thus, it appears that the toxic stereoisomers of soman have a similar affinity for mouse serum carboxylic-ester hydrolase, and CBDP pretreatment does not enhance selectively the toxicity of one stereoisomer over the other.     

Isolation, anticholinesterase properties, and acute toxicity in mice of the four stereoisomers of the nerve agent soman

The four stereoisomers of the nerve agent pinacolyl methylphosphonofluoridate (soman), designated as C(+)P(+), C(+)P(-), C(-)P(+), and C(-)P(-), have different toxicologic properties due to stereospecific interactions in living organisms. We report the isolation of these stereoisomers with more than 99% optical purity. This result was realized by means of (i) complete optical resolution of pinacolyl alcohol, (ii) synthesis of C(+)- and C(-)-soman from the (+)- and (-)-enantiomers of the alcohol, (iii) optimalization of conditions for stereospecific inhibition of alpha-chymotrypsin with the P(-)-isomers of C(+)- and C(-)-soman, followed by isolation of the C(+)P(+)- and C(-)P(+)-isomers, (iv) isolation of the C(+)P(-)- and C(-)P(-)-isomers after incubation of C(+)- and C(-)-soman, respectively, in rabbit plasma, which hydrolyzes stereospecifically the P(+)-isomers. The bimolecular rate constants for inhibition of electric eel acetylcholinesterase (AChE) at pH 7.7, 25 degrees C, are at least 3.6 X 10(4) larger for the P(-)- than for the P(+)-isomers. The enzyme inhibited with C(+)P(-)-soman is much more effectively reactivated with the oximes HI-6, HGG-42, and obidoxime than AChE inhibited with C(-)P(-)-soman. The LD50 values (sc, mice) are in accordance with the P(-)/P(+) ratio of inhibition rates of AChE, i.e. 99, 38, greater than 5000, greater than 2000, 214, 133, and 156 micrograms/kg for C(+)P(-)-, C(-)P(-)-, C(+)P(+)-, C(-)P(+)-, C(+)-, C(-)-soman, and "soman", respectively. The relative LD50 values of the C(-)P(-)- and C(+)P(-)-isomers do not correspond with the small differences in their rates of inhibition of AChE, indicating that such small rate ratios may be overruled by other stereospecific effects, e.g., in vivo rates of detoxification.     

Stereoselective phosphonylation of human serum proteins by soman

Phosphonylation has been reported as part of the degradation of soman in human serum. The concentration of phosphonylation sites can be quantified by comparing the degradation in serum, preincubated with soman (all sites occupied), with the degradation in serum not preincubated. The mean value of 73 nM of phosphonylation sites is in agreement with the concentration of active sites of butyrylcholinesterase (EC 3.1.1.8.), which is known to be phosphonylated by soman. Hence, it is concluded that butyrylcholinesterase accounts for all the phosphonylation sites present in human serum. The stereoselectivity of the reaction was investigated by using epimeric pairs of soman, in casu C(+)P(+/-)- and C(-)P(+/-)-soman. In a first approach enzymatic hydrolysis was blocked and the ratios of phosphonylation rate constants, C(+)P(+)/C(+)P(-) and C(-)P(+)/C(-)P(-), were determined to be 0.15 and 0.31, respectively. In a second approach, in untreated serum, the bimolecular phosphonylation rate constants of C(+)P(-)- and C(-)P(-)-soman were determined, neglecting their small hydrolysis rate and taking advantage of the fast enzymatically catalysed disappearance of their respective P(+)-epimeric counterparts. Values for C(+)P(-)- and C(-)P(-)-soman are 3.6 X 10(7) and 0.6 X 10(7) M-1.min-1, respectively. Using a combination of both approaches, a relative ranking of phosphonylation rates of the four isomers was found to be C(+)P(-) much greater than C(+)P(+) approximately equal to C(-)P(-) greater than C(-)P(+).     

Phosphonylation of purified human, canine and porcine cholinesterase by soman. Stereoselective aspects

Cholinesterases (EC 3.1.1.8, acylcholine acylhydrolase) from the sera of man, dog and pig were purified 400-600-fold using a combination of ion-exchange and affinity chromatography. In a first approach, phosphonylation by soman was studied by using the half-resolved epimers C(+)P(+/-)-soman and C(-)P(+/-)-soman. The degradation of soman at the nanomolar level was followed in time by determining the remaining soman by capillary gas chromatography with NP detection. In the three sera investigated the P-(-)-epimer phosphonylates at a higher rate than its corresponding P(+)-counterpart and the stereoselectivity is greater for the C(+)-epimers than for the C(-)-epimers. Individual soman isomers were isolated from C(+)- and C(-)-epimers and quantified by gas chromatography. Second-order rate constants were determined for the phosphonylation of purified cholinesterase by isolated soman isomers. The C(+)P(-)-isomer has the highest phosphonylation rate for the three species; the other toxic isomer, C(-)P(-), has a five to ten-fold lower rate. The overall stereoselectivity is more marked in human cholinesterase than in canine. Porcine serum cholinesterase is phosphonylated by the P(-)-isomers at a slightly higher rate than the human enzyme.     

Hydrolysis of the four stereoisomers of soman catalyzed by liver homogenate and plasma from rat, guinea pig and marmoset, and by human plasma

Stereoselective hydrolysis at pH 7.5 and 37 degrees of C(+/-)P(+/-)-soman by liver homogenate and plasma from rat, guinea pig and marmoset, and by human plasma is studied by using the four single stereoisomers. The fast hydrolysis of the C(+/-)P(+)-isomers is monitored titrimetrically, whereas the decay of the much slower reacting C(+/-)P(-)-isomers is followed by gas chromatographic determination of the residual concentration. Values of Km and Vmax are evaluated for the enzymatic hydrolysis of the two relatively nontoxic C(+/-)P(+)-isomers. The plasma enzymes have a high affinity for these isomers (Km: 0.01-0.04 mM); the Km values of the liver enzymes vary between 0.04 and 0.7 mM. Except for rat liver homogenate, only first-order rate constants can be obtained for catalyzed hydrolysis (kc) of the highly toxic C(+/-)P(-)-isomers: most measurements with C(+/-)P(-)-isomer concentrations greater than 0.3 mM are complicated by epimerization to C(+/-)P(+)-isomers, which may conceal enzyme saturation with the C(+/-)P(-)-isomers. The first-order rate constants of catalyzed hydrolysis (Vmax/Km or kc) by all liver homogenates and plasmata decrease in the order: C(+)P(+)- greater than C(-)P(+)- much greater than C(-)P(-)- greater than C(+)P(-)-soman. The highest P(+)-/P(-)-stereoselectivity is found for rat plasma. Rat liver homogenate is more potent than the other liver homogenates in catalyzing the hydrolysis of both the C(+/-)P(+)- and the C(+/-)P(-)-isomers. Rat plasma shows the highest activity for degradation of the C(+/-)P(+)-isomers, but is approximately as active as marmoset and human plasma for degradation of the C(+/-)P(-)-isomers.     

Toxicokinetics of the four stereoisomers of the nerve agent soman in atropinized rats—Influence of a soman simulator

The toxicokinetics of the four stereoisomers of C(+/-)P(+/-)-soman were investigated in anesthetized, atropinized, and artificially ventilated rats at iv doses of 6 (495 micrograms/kg) and 3 LD50. By integration of a thermodesorption/cold trap injector into our GLC analysis, the soman stereoisomers could be followed in rat blood down to a minimum detectable concentration, i.e., 1.5 pg/ml (8.3 pM), 55-fold lower than that published previously. This new detection limit is probably near or below the minimum concentration relevant for survival. Whereas C(+)P(+)-soman disappears in vivo from rat blood within 0.25 min, the toxicokinetics of C(-)P(+)-soman could be described by a two-compartment model, with a biological half-life of 1-1.5 min. The extremely toxic C(+/-)P(-)-isomers could be followed in rat blood for greater than 4 and 2 hr at doses of 6 and 3 LD50, respectively. The toxicokinetics of the P(-)-isomers are best described with a three-compartment model, with terminal half-lives of 40-64 and 16-22 min at doses of 6 and 3 LD50, respectively. Administration of a 13.6-fold molar excess of the soman simulator 1,2,2-trimethylpropyl dimethylphosphinate (PDP) 10 min prior to administration of 6 LD50 of C(+/-)P(+/-)-soman reduces the terminal half-lives of the C(+/-)P(-)-isomers to the values measured at the dose of 3 LD50 without PDP pretreatment. Previous investigations showed that, without PDP pretreatment, rats suffer from endogenous reintoxication 4-6 hr after initially successful therapy, at C(+/-)P(+/)-soman doses greater than or equal to 6 LD50. Both this reintoxication phenomenon due to the presence of toxicologically significant C(+/-)P(-)-soman levels up to 4 hr after intoxication and its antagonism via PDP pretreatment can be understood on the basis of our toxicokinetic measurements. This shows that such investigations can contribute to insight into the toxicology of C(+/-)P(+/-)-soman and to a better treatment of intoxications with this agent.     

Development of a physiologically based model for the toxicokinetics of C(±)P(±)-soman in the atropinized guinea pig

A physiologically based model was developed which describes the in vivo toxicokinetics of the highly reactive nerve agent C(±)P(±)-soman at doses corresponding to 0.8–6 LD₅₀ in the atropinized guinea pig. The model differentiates between the summated highly toxic C(±)P(−)-soman stereoisomers at supralethal doses and the individual nontoxic C(±)P(+)-isomers. Several toxicant-specific parameters for the soman stereoisomers were measured in guinea pig tissue homogenates. Cardiac output and blood flow distribution were measured in the atropinized, anesthetized, and artificially ventilated guinea pig. The model was validated by comparison of the time courses for the blood concentrations of the two pairs of stereoisomers in the guinea pig after i.v. bolus administration with the blood concentrations predicted by the model. The predictions put forward for the summated C(±)P(−)-isomers are in reasonable agreement with the experimental data obtained after doses corresponding to 2 and 6 LD₅₀. In view of large differences in the rates of hydrolysis of the C(±)P(+)-isomers, these two isomers had to be differentiated for satisfactory modeling of both isomers. In order to model the toxicokinetics of C(±)P(−)-soman at a dose of 0.8 LD₅₀, the almost instantaneous elimination of the C(+)P(−)-isomer at that dose had to be taken into account. The sensitivity of the predictions of the model to variations in the parameters has been studied with incremental sensitivity analysis. The results of this analysis indicate that extension to a model involving four individual stereoisomers is desirable in view of large differences in the biochemical characteristics of the two C(±)P(−)- and C(±)P(+)-isomers. Received: 8 July 1996 / Accepted: 30 October 1996     

Stereospecific reactivation by some Hagedorn-oximes of acetylcholinesterases from various species including man, inhibited by soman

Reactivation by bispyridinium mono-oximes (Hagedorn-oximes) and some classical oximes (0.03 or 1mM) was studied in vitro of rat, bovine and human erythrocyte acetylcholinesterase and of electric eel acetylcholinesterase inhibited by soman. Relative reactivating potencies of the oximes are similar for the three inhibited erythrocyte enzymes. In general, Hagedorn-oximes are more potent than the classical oximes. Among the Hagedorn-oximes, HI-6 is the most potent reactivator for the three inhibited enzymes. Relative reactivating potencies for the inhibited erythrocyte acetylcholinesterases and electric eel acetylcholinesterase, however, clearly differ. Since the reactivation experiments were carried out with racemic soman, a mixture of the two inhibited enzymes may be formed, which may cause additional problems in the comparison of various results. In order to get more detailed information on differences between human erythrocyte and electric eel acetylcholinesterase, reactivation of these enzymes inhibited with the P(-)-isomers of C(+)- and C(-)-soman were studied separately. Reactivation appeared to be dependent on the chirality of the alpha-carbon atom in the pinacolyl group. HI-6 is by far the most potent reactivator for the human enzyme inhibited by the two P(-)-isomers. It is suggested that electric eel acetylcholinesterase is not a reliable model for in vitro testing of therapeutic potencies of oximes against soman intoxication in mammals. Rate constants of aging of the four acetylcholinesterases inhibited with racemic soman and of the human and eel enzyme inhibited by the P(-)-isomers of C(+)- and C(-)-soman were also determined. The aging of the inhibited rat enzymes proceeds remarkably slowly (t1/2 = 21 min). The rate of aging is not affected by the chirality on the alpha-carbon atom in the pinacolyl group. Consequences of the present results are discussed in view of extrapolation of reactivation data of a series of reactivators to their relative therapeutic effect, ultimately in man. It is speculated that the more rapid aging of the human inhibited enzyme may hamper oxime-therapy in man more seriously than in rat.     

In vitro degradation of the four isomers of soman in human serum

Starting from racemic soman (1,2,2-trimethylpropyl methylphosphonofluoridate), the degradation of its four stereoisomers in human serum (25 degrees, pH 8.8), has been studied at the nM level. Phosphylation of serum proteins is eliminated by preincubation of the serum with soman. A capillary gas chromatographic method with nitrogen-phosphorous detection allows the separation of the diastereoisomers. The total hydrolysis (enzymatic and non-enzymatic) rate constants of the isomers can then be resolved indirectly on the basis of the important rate difference between P(+) and P(-) isomers. The enzymatic hydrolysis rate constants are obtained by subtracting, for each isomer, the spontaneous (non-enzymatic) rate constant from the total hydrolysis rate constant. The non-enzymatic part of the hydrolysis is obtained from experiments in serum ultrafiltrate (30,000 NMWL). Enzymatic hydrolysis of C(+) P(+) soman occurs so rapidly that only a lower limit of the rate constant can be given. The other enzymatic rate constants are 0.016 min-1 for C(+)P(-), 0.74 min-1 for C(-)P(+) and 0.028 min-1 for C(-)P(-).     

Formation of soman (1,2,2-trimethylpropyl methylphosphonofluoridate) via fluoride-induced reactivation of soman-inhibited aliesterase in rat plasma

After incubation (37°) of rat blood or plasma with the nerve agent soman, (CH₃)₃C(CH₃)C(H)O(CH₃)P(O)F (7.7 μM), for 10 min, only a small amount of this organophosphate (7 or 1%, respectively) is left, as determined enzymatically (acetylcholinesterase) and gas chromatographically. Comparison of the results obtained with both analyses shows that this residual soman consists only of its P(?)-isomers. Incubation (25°) at pH 4.8–6.1 of such soman-treated rat blood or plasma with sodium fluoride (2.5 mM) for 0.5 min leads to (i) a substantial increase of the P(?)-soman concentration, and (ii) a (partial) reactivation of the soman-inhibited aliesterase, proportional to the amount of generated P(?)-soman. These results indicate strongly that added fluoride ions regenerate soman by a reversal of the inhibition reaction. From the relationship between percentage of reactivation and increase of soman concentration the aliesterase concentration in rat plasma is calculated as 2.6 μM.Sodium fluoride has a similar effect in blood taken from rats to which soman was administered intravenously.The increase of the P(?)-soman concentration is higher with higher sodium fluoride concentrations and at lower pH values.In accordance with the absence of aliesterase, addition of sodium fluoride does not induce an increase of the P(?)-soman concentration in soman-treated human plasma.     

Toxicokinetics of soman in cerebrospinal fluid and blood of anaesthetized pigs

The toxicokinetics of the four stereoisomers of the nerve agent C(±)P(±)-soman was analysed in cerebrospinal fluid (CSF) and blood in anaesthetized, spontaneously breathing pigs during a 90-min period after injection of soman. The pigs were challenged with different intravenous (i.v.) doses of C(±)P(±)-soman corresponding to 0.75–3.0 LD₅₀ (4.5, 9.0 and 18 μg/kg in a bolus injection and 0.45 μg/kg per min as a slow infusion). Artificial ventilatory assistance was given if, after soman intoxication, the respiratory rate decreased below 19 breaths/min. Blood samples were taken from a femoral artery and CSF samples from an intrathecal catheter. The concentrations of the soman isomers were determined by gas chromatography coupled with high resolution mass spectrometry. All four isomers of soman were detected in both blood and CSF samples. The relatively non-toxic C(±)P(+) isomers disappeared from the blood stream and CSF within the first minute, whereas the levels of the highly toxic C(±)P(−) isomers could be followed for longer, depending on the dose. Concurrently with the soman analyses in blood and CSF, cholinesterase (ChE) activity and cardiopulmonary parameters were measured. C(±)P(−) isomers showed approx. 100% bioavailability in CSF when C(±)P(±)-soman was given i.v. as a bolus injection. In contrast, C(±)P(−) isomers displayed only 30% bioavailability in CSF after slow i.v. infusion of soman. The ChE activity in blood decreased below 20% of baseline in all groups of pigs irrespective of the soman dose. The effect of soman intoxication on the respiratory rate, however, seems to be dose-dependent and the reason for ventilatory failure and death. Artificial ventilation resulted in survival of the pigs for the time-period studied. Received: 3 March 1998 / Accepted: 5 May 1998     

Effect of pretreatment with CBDP on the toxicokinetics of soman stereoisomers in rats and guinea pigs

Pretreatment of rats and guinea pigs with the specific carboxylesterase inhibitor 2-(o-cresyl)-4H-1 3 2-benzodioxaphosphorin-2-oxide (CBDP) reduces the LD₅₀ of the nerve agent C(±)P(±)-soman in these species to the same range as in primates. This suggests that such CBDP-pretreated animals can be used in investigations that are relevant for prophylaxis and therapy of intoxication with C(±)P(±)-soman in primates including humans. In order to test this hypothesis we have studied the toxicokinetics of the toxic C(±)P(–)-isomers of soman in artificially respirated and CBDP-pretreated rats and guinea pigs at intravenous doses corresponding to 6 × LD₅₀. A comparison of the areas under the curve (AUCs) of the blood levels of C(±)P(–)-soman in pretreated and non-pretreated animals at the same absolute dose shows extreme nonlinearity with dose, indicating that CBDP occupies highly reactive binding sites which are no longer available for sequestration of the soman isomers. The AUCs of C(±)P(–)-soman at equitoxic doses of 6× LD₅₀ are reduced by pretreatment with CBDP from 1683 to 464 ng.min.ml^–1 in rats and from 978 to 176 ng.min.ml^–1 in guinea pigs, which is in the range of the AUC in non-pretreated marmosets at an equitoxic dose (419 ng.min.ml^–1). The blood levels of the C(±)P(–)-isomers in marmosets and CBDP rats are rather similar during the first 7 min, but persist in CBDP rats for 2 h longer at toxicologically relevant levels than in marmosets. The levels of C(±)P(–)-soman in CBDP-pretreated guinea pigs are substantially lower than in marmosets for an initial period of 80 min. Nevertheless, they drop below toxicologically relevant levels approximately 50 min later than in marmosets. Evidently, one should be cautious in considering CBDP, pretreated rats and guinea pigs as substitute primates.This research was supported in part by the US Army Medical Research and Development Command under grants DAMD17-87-G-7015 and DAMD17-90-Z-0034.     

Interaction of Soman with {beta}-Cyclodextrin

Interaction of Soman with ß-CycIodextrin. DESIRE,B., AND SAINT-ANDRE, S. (1986). Fun-dam. Appl. Toxicol. 7, 646-657.Of the following neurotoxic agents, pinacolyl methylphospho-nofluoridate(soman), isopropyl methylphosphonofluoridate (sarin) and ethylN, N-dimethyl-phosphoramidocyanidate (tabun), only soman wasinactivated appreciably at pH 7.40 by ß-cyclodextrin.The interaction of soman, a mixture of four stereoisomers designatedas C(+)P(–), C(–)P(–), C(+)P(+), and C(–)P(+),with cyclodextrins was revealed by methods based on (i) theirreversible inhibition of acetylcholinesterase (AChE) thatis phosphonylated chiefly by P(–)-isomers of racemic somanand (ii) continuous titration of fluoride ions released by somanusing a fluoride-specific electrode. Soman and ß-cyclodextrinform a 1:1 complex. At pH 7.40 and 25°C the dissociationconstant Kd of this complex and the rate constant k2 of cleavageof soman by ß-cyclodextrin are (0.53 ± 0.05)mM and (5.9 ± 0.6) x 10² min¹, respectively. The rateconstant k₂^max for the cleavage of soman by monoionized ß-cyclodextrinhas a value of 2.8 x 103 min¹ and the second order rate constantk₂^max/K_d 5.3 x 106 M^–1 min^–1. Consequently, somanis hydrolyzed about 2500 times faster by the monoanion of ß-cyclodextrin,than by the hydroxide ion. The cleavage of P(–)-somanby ß-cyclodextrin as estimated by AChE inhibitionproceeds apparently at the same rate for the C(–)P(–)-and C(+)P(–)-isomers. However, the release of fluorideions indicated a stereospecific rate of reaction, the P(-Hsomersreacting faster than the P(+)-isomers. At pH 7.40, the inactivationrate of soman by ß-cyclodextrin was as fast in humanplasma in vitro as in Tris buffer. This interaction betweensoman and ß-cyclodextrin, and other data from theliterature, suggests that the introduction of catalytic or noncatalyticgroups on ß-cyclodextrin might possibly make it abetter catalyst for soman inactivation through improvement inthe catalytic or in the binding process.     

Reactivation of nerve agent inhibited human acetylcholinesterases by HI-6 and obidoxime

Acetylcholinesterase was purified from human caudate nucleus and skeletal muscle. The enzyme preparations were used to study aging and reactivation by HI-6 and obidoxime after inhibition by soman and its isomers. HI-6 was found to be the most potent reactivator. For both enzyme preparations a higher reactivatability and a higher rate of aging were observed after inhibition by C+-soman than after inhibition by C(-)-soman. Aging was retarded by propidium diiodide. Reactivation by the two oximes was also studied after inhibition by tabun, sarin and VX. Tissue homogenates were used for this part of the work. Our conclusion is that HI-6 is superior to obidoxime for human acetylcholinesterases inhibited by soman and sarin, while obidoxime is better towards tabun-inhibited enzyme.     

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.     

Structural and stereochemical specificity of mouse monoclonal antibodies to the organophosphorous cholinesterase inhibitor soman

To test the usefulness of immunotherapy in organophosphate poisoning, two mouse monoclonal antibodies were prepared to the chemical warfare agent soman. The antibodies bound reversibly to soman and afforded considerable protection to acetylcholinesterase in vitro. However, they were only marginally effective in preventing the consequences of soman poisoning in mice (these data have been published elsewhere). Since potential for immunotherapeutic usefulness resides in antibody affinity and specificity, we conducted experiments to define these parameters to enable us to maximize them in the production of later antibodies. Interaction of the antibodies (CC1 and BE2) in affinity-purified form with a series of soman analogs in a competitive inhibition enzyme immunoassay was used to assess the contribution to binding affinity of each functional group on the soman molecule. Neither antibody interacted with the -P = S analog of soman or methylphosphonic acid. A decrease in the number of methyl groups on the pinacolyl side chain reduced or eliminated binding with both antibodies while increasing the size of this group had a mixed result. The major metabolite of soman, its basic hydrolysis product, interacted weakly with BE2 and failed to interact with CC1. Alkyl ester group substitution at the fluorine position increased antibody binding up to the symmetrical dipinacolyl analog. Stereochemical specificity was determined by measuring the apparent decrease in the rate of inhibition of cholinesterases (acetylcholine acetylhydrolase, EC 3.1.1.7, or acylcholine acylhydrolase, EC 3.1.1.8) by pure soman stereoisomers in the presence of increasing concentrations of each antibody. CC1 demonstrated specificity that varied as C(+)P(+) less than C(-)P(+) less than C(-)P(-) less than C(+)P(-). Although affinities were much lower, BE2 also showed a preference for the more toxic P(-) isomers.     

Asymmetric fluorogenic organophosphates for the development of active organophosphate hydrolases with reversed stereoselectivity

In order to enhance the enzymatic detoxification rate of organophosphorus (OP) nerve agents we have searched for more active variants of recombinant mammalian paraoxonase (PON1). We have previously identified three key positions in PON1 that affect OP hydrolysis: Leu69, Val346 and His115, that significantly enhance the hydrolysis of cyclosarin (GF), soman, chlorpyrifos-oxon (ChPo), O-isopropyl-O-(p-nitrophenyl)methylphosphonate (IMP-pNP) and diisopropyl fluorophosphate (DFP). GC/FPD analysis compared to residual AChE inhibition assay displayed stereoselective hydrolysis of GF, soman and IMP-pNP, indicating that wild type PON1 and its variant V346A are more active toward the less toxic P(+) optical isomer. In order to obtain new PON1 variants with reversed stereoselectivity, displaying augmented activity toward the more toxic isomer P(-) of nerve agents, we synthesized new asymmetric fluorogenic OPs (Flu-OPs). Six Flu-OPs were prepared containing either ethyl (E), cyclohexyl (C) or pinacolyl (P) alkyl radicals attached to methyl-phosphonyl (MP) moiety analogous to the structure of VX, GF and soman, respectively. The fluorescent moieties are either 3-cyano-4-methyl-7-hydroxy coumarin (MeCyC) or 1,3-dichloro-7-hydroxy-9,9-dimethyl-9H-acridin-2-one (DDAO). The kinetics of AChE and BChE inhibition by these new Flu-OPs display k(i) values 8.5x10(4) to 8.5x10(7) and 5x10(4) to 2x10(6)M(-1)min(-1), respectively. EMP-MeCyC and EMP-DDAO are the most active inhibitors of AChE whereas CMP-MeCyC and CMP-DDAO are better inhibitors of BChE than AChE, indicating accommodation of bulky cyclohexyl group inside the active site of BChE. PMP-MeCyC and PMP-DDAO are the least active inhibitors of both AChE and BChE. CMP-MeCyC and CMP-DDAO were significantly detoxified only by the five-site mutations PON1 variant L69V/S138L/S193P/N287D/V346A. Degradation kinetics of Flu-OPs measured by increase in absorbance of the released fluorogenic group was fit by a two exponential function, indicating faster hydrolysis of the less toxic optical isomer. Interestingly, wt PON1 caused only 50% degradation of racemic EMP-MeCyC, CMP-MeCyC and CMP-DDAO indicating complete hydrolysis of P(+) isomer. This remarkable stereoselectivity was used for the enzymatic separation of the P(-) isomer of CMP-MeCyC. The bimolecular rate constant k(i) for human AChE inhibition by the isolated P(-) isomer of CMP-MeCyC is five-fold larger than that of its P(+) isomer. The marked preference of wt PON1 toward P(+) stereo-isomer of CMP-MeCyC and CMP-DDAO renders their P(-) stereo-isomers suitable for the selection of new OP hydrolase variants with reversed stereoselectivity.     

Toxicokinetics of soman stereoisomers after subcutaneous administration to atropinized guinea pigs

The toxicokinetics of the four stereoisomers of the nerve agent C(±)P(±)-soman were investigated after subcutaneous administration of a 6 LD₅₀ dose (148 μg/kg) to anaesthetized, atropinized, and artificially ventilated guinea pigs. Whereas the relatively nontoxic C(±)P(+)-isomers were not detected in blood, the highly toxic C(±)P(?)-isomers appeared within 1 min in the general circulation and reached maximum levels of 10–15 ng/ml blood within a period of ca. 7 min. In this absorption phase the blood levels of the C(+)P(?)-isomer lag clearly behind those of the C(?)P(?)-isomer. The blood levels of both C(±)P(?)-isomers could be mathematically described using non-linear regression by a three-exponential equation, with one exponential term describing the rapid absorption phase and the other two terms describing distribution and elimination. A comparison with the toxicokinetics of the same isomers upon intravenous administration of the same dose shows that the systemic availability upon subcutaneous administration is in the range of 74–83%. Toxicologically relevant concentrations of the C(±)P(?)-isomers prevail almost twice as long after subcutaneous than after intravenous administration. From a toxicokinetic point of view, subcutaneous administration of C(±)P(±)-soman appears not to be a realistic model for the most relevant route of exposure to C(±)P(±)-soman in case of chemical warfare, i.e. short term respiratory exposure.