Israel Institute for Biological Research, Ness Ziona, Israel
Received July 23, 2003; accepted October 2, 2003
ABSTRACT
Human butyrylcholinesterase (HuBChE) is a drug candidate for protection against organophosphates (OP) intoxication. A mathematically based model was validated and employed to better understand the role of the endogenous HuBChE in detoxification of OPs and to estimate the dose of exogenous HuBChE required for enhancing protection of humans from lethal exposure to OPs. The model addresses the relationship between the HuBChE dose needed to maintain a certain residual activity of human acetylcholinesterase (HuAChE) and the following parameters: (1) level and duration of exposure, (2) bimolecular rate constants of inhibition of HuAChE (kA) and HuBChE (kB) by OPs, and (3) time elapsed from enzyme load. The equation derived for the calculation of HuBChE dose requires the knowledge of kA/kB in human blood and the rate constant of HuBChE elimination. Predictions of HuBChE doses were validated by in vitro experiments and data of published human studies. These predictions highlight two parameters that are likely to decrease the calculated dose: (1) the rapid consumption of the less toxic isomers of OPs in human plasma, and (2) the volume of distribution of HuBChE that appears significantly greater than the volume of plasma. The first part of the analysis of the proposed model was focused on acute bolus exposures and suggests that upper limit doses of 134, 115, and 249 mg/70 kg are sufficient to protect RBC AChE above 30% of baseline activity following a challenge with 1 LD50 VX, soman, and sarin, respectively. The principles of the validated model should be applicable for advanced predictions of HuBChE dose for protection against continuous exposures to OPs.
Key Words: human; acetylcholinesterase; butyrylcholinesterase; organophosphates; theoretical model; inhibition.
Inhibition of acetylcholinesterase (AChE; EC 3.1.1.7) of physiologically important organs is the major cause of the manifestation of cholinergic crisis following acute exposure to toxic doses of organophosphates (OP). The full protection of animals against multiple LD50 doses of OPs by pretreatment with human butyrylcholinesterase (HuBChE; EC 3.1.1.8) alone has been demonstrated in the last decade (Allon et al., 1998; Ashani et al., 1991
; Brandeis et al., 1993
; Raveh et al., 1997
). Results suggest that enhancement of HuBChE activity by exogenous administration of purified enzyme would significantly improve human plasma ability to detoxify OPs. Thus, HuBChE is a viable drug candidate for further development as a prophylactic antidote against OP-based pesticides and nerve agent toxicity. A model for estimation of HuBChE dose that is required to confer protection against OPs under a variety of exposure scenarios is essential for the design of safety evaluation of HuBChE in clinical trials. It is also intended to provide scientific ground for the recommendation of a dose regimen to minimize risks from exposure to OPs.
One of the advantages of the scavenger strategy for protection against OP toxicity is the defined biochemical end point of challenge detoxification by the protecting enzyme. This parameter enabled a reasonable animal-to-human (ATH) extrapolation of prophylaxis with HuBChE (Ashani et al., 1998) and offered the development of theoretical models for the estimation of prophylactic doses in humans. The ATH extrapolation uses protective ratio values that were determined in rodents weighing 20400 g (Allon et al., 1998
; Raveh et al., 1993
) and 10 kg monkeys (Raveh et al., 1997
). However, in view of the dependence of OP's LD50 on body weight and the difficulties in validating the predicted dose in human by use of larger animals, an independent approach was deemed important. A second shortcoming of the ATH model is the lack of information on the fate of human AChE (HuAChE) and the residual levels of the OP.
Comprehensive physiologically based pharmacokinetic models for the prediction of inhibition of AChE in human by diisopropylfluorophosphate (DFP), parathion (Gearhart et al., 1994), and O,O-diethyl-O-[3,5,6-trichloro-2-pyridyl]-phosphorothioate (chlorpyrifos; Timchalk et al., 2002
) were developed by the optimization of multicompartmental model equations. These studies used numerous physiological and biochemical parameters needed for model development and were obtained either by direct measurements or computer simulations fitted to experimental data. Sweeny and Maxwell (1999)
proposed a theoretical model, based on single-compartment kinetics, that described OP toxicity surfaces. These surfaces reflected the combined contribution of an OP hydrolase and carboxylesterase (CaE) to the reduction of OP toxicity in rats. More recently, the same authors were using a similar approach to predict protective ratios of OPs in mice pretreated with stoichiometric and catalytic scavengers (Sweeny and Maxwell, 2003
).
A considerable detoxification of OPs in humans is processed by plasma HuBChE, which is a universal scavenger of potent OP-based inhibitors of AChE. Its enhancement by exogenous administration of purified enzyme is expected to increase plasma ability to protect from OP intoxication. This study was undertaken to assess the HuBChE dose essential for adequate protection. A simple set of equations was derived to address the question of how the rate of inhibition of HuBChE and HuAChE and the rate of elimination of HuBChE from the circulation affect the loading dose of HuBChE. Validation was based on the (1) in vitro determination of the rate constants of inhibition of HuBChE and HuAChE in human blood by a variety of OPs, (2) reassessment of reported findings on the biosynthesis and elimination rate constant of HuBChE from the circulation, (3) published data of human studies, and (4) prophylaxis experiments in HuBChE-treated animals.
MATERIALS AND METHODS
O-isopropyl methylphosphonofluoridate (sarin), O-pinacolyl methylphosphonofluoridate (soman), and O-ethyl-S-(2-N,N-diisopropylaminoethyl) methylphosphonothiolate (VX) were obtained from the Organic Chemistry Department of the Israel Institute for Biological Research. O,O-diethyl-p-nitrophenylphosphate (paraoxon) and the nonionic detergent n-octyl ß-D-glucopyranoside were purchased from Sigma Chemical Co. (St Louis, MO).
Human blood was collected above heparin, and plasma was separated from RBC by 10 min centrifugation at 1000 x g. HuBChE was purified from outdated human plasma by procainamide-Sepharose 4B gel affinity chromatography (Grunwald et al., 1997). Specific activity was 710 units/mg using assay conditions specified below.
Enzyme assays.
The activity of RBC HuAChE and plasma HuBChE was determined by the method of Ellman et al. (1961) using 1 mM acetylthiocholine iodide (ATC) and 1 mM butyrylthiocholine iodide (BTC), respectively, as substrates. Assays were carried out in 50 mM phosphate buffer, pH 8.0, at 25°C. Under these conditions, the hydrolysis rate ratio of BTC/ATC by HuBChE is 1.9, and BTC-catalyzed hydrolysis of HuAChE is negligible. Thus, the assay with two substrates permitted the calculation of the activity of each enzyme in whole blood. Nonspecific increase at 412 nm due to the interaction of blood constituents with Ellman's reagent 5,5`-dithio-bis (2-nitrobenzoic acid, DTNB) and nonenzymic substrate hydrolysis were subtracted.
Measurements of ChEs inhibition in whole blood.
Whole blood samples (1 ml) containing 5.46.2 and 1.92.2 units/ml of HuAChE and HuBChE, respectively, were preincubated at 37°C and spiked with OP's stock solutions to produce a final concentration of inhibitors ranging from 10 to 100 nM. Blood samples were shaken gently in a thermostatic bath at 37°C. At certain time intervals, 50 µl blood were diluted in 0.95 ml deionized water, vortexed to complete hemolysis, and kept in an ice-water bath until assayed. Residual activity of AChE and BChE was carried out by 20-fold dilution of the hemolized blood into Ellman's assay mixture. Activity measurements were run in duplicates for ATC and BTC.
Plasma-free HuBChE/RBC AChE in saline.
Inhibition of ChEs by OPs was repeated as described above in reconstituted HuBChE/RBC AChE in plasma-free saline (pH 7.4). Human RBC were washed three times with twice the volume of saline, and the washed RBC were resuspended in a sufficient amount of saline to reconstitute the original blood volume. Concentrated stock solution of purified HuBChE was diluted in the RBC saline. Final activities were 5.7 and 2.1 units/ml of HuAChE and HuBChE, respectively.
Detergent solubilization of RBC.
To compare rates of inhibition of HuAChE in intact erythrocytes with a solubilized preparation, washed RBC were treated with 2% w/v of the nonionic detergent n-octyl ß-D-glucopyranoside and the clear solution diluted in saline to match the activity of a similar dilution of intact RBC. n-Octyl ß-D-glucopyranoside did not affect the rates of inhibition of AChE from bovine fetus serum or BChE of human plasma.
Determination of the bimolecular rate constants in human plasma.
The separated plasma that contained approximately 60100 nM HuBChE was treated by stepwise addition of aliquots of 30 µM soman solution until HuBChE activity decreased to 1015% of its baseline activity. Soman-treated plasma contained free 510 nM active sites of HuBChE that enabled production of conditions of first-order kinetic (kobs) of inhibition by OPs, which progressed at reasonable rates to allow for an accurate determination. Fresh aqueous stock solutions of approximately 850 µM OPs in deionized water were diluted into the soman-treated plasma preincubated at 37°C. At suitable time intervals, plasma was diluted 60-fold into the assay cuvette containing 0.33 mM DTNB, and residual enzyme activity was monitored at 412 nm by use of 1 mM BTC, as described above. A blank cuvette contained DTNB and the same amount of plasma sample. Enzyme activity of soman-treated plasma remained unchanged throughout the entire experiment. The bimolecular rate constant kB was determined from the slope of the straight lines obtained by plotting kobs versus [OP].
Basic considerations of model development.
The governing equations that describe the time course of inhibition of RBC AChE ([A], Equation 1), plasma BChE ([B], Equation 2
), and the concentration of [OP] in blood (Equation 3
) following exposure to OPs are as follows:
![]() | (1) |
![]() | (2) |
![]() | (3) |
where [A] is the RBC AChE concentration; [B] is the concentration of HuBChE; kA and kB are the bimolecular rate constants of the inhibition of RBC AChE and plasma BChE, respectively; [OP] is the concentration of organophosphates in blood; and [OP]in is the rate of OP blood concentration produced by the challenge.
The value of [B]o at t = 0 prior to the OP challenge is given by the sum of [B]exog and [B]endg, which are the concentrations of the exogenous and endogenous HuBChE, respectively. The residual [B]exog following enzyme load may be calculated, to a good approximation, provided , the first-order elimination rate constant of the enzyme from the circulation, is known. This is done under the assumption that the native (both exogenous and endogenous HuBChE) and the phosphorylated enzyme are cleared at the same rate. With these assumptions, the following two major equations were derived (see Supplementary data):
![]() | (4) |
![]() | (5) |
where t is the exposure time, T is the time elapsed from HuBChE loading, and A0 is the initial concentration of RBC AChE in blood. The parameters and ß are the fractions of residual HuAChE and HuBChE activity, respectively, and they decrease as functions of time. It should be clarified that ß is defined as the ratio of the residual activity of HuBChE to the activity of the enzyme recorded prior to administration of the challenge and is given by Equation 6
.
![]() | (6) |
The term [B]endg is either the endogenous activity of the usual phenotype that has the same kB as the exogenous enzyme or the mutant enzyme. It was assumed that in the case of an endogenous nonactive mutant (population at high risk), its kB is substantially lower than that of the exogenous enzyme and the denominator is approximated by . A similar approximation is obtained for the usual phenotype when T is sufficiently long, while for very short T the denominator is given by the sum of [B]exog + [B]endg.
The mass balance of OP and enzymes given by Equation 4, together with Equation 5
, provide a straightforward way to estimate the amount of [B]exog required to maintain the value of
at, or above, a predetermined level, throughout the entire exposure period.
RESULTS
The Ratio of kA/kB
As seen from Equation 5, regardless of OP concentration or time of exposure, the ratio of the active fraction of the scavenger (ß) to that of the protected enzyme RBC AChE (
) depends only on kA and kB. Thus, kA/kB is a crucial parameter that needs to be determined to estimate and fit the dose of HuBChE to a given exposure scenario. It can be evaluated in vitro in human blood at 37°C by increasing the concentration of the inhibitor and measuring residual activity of the two enzymes. A plot of log
versus log ß should provide a straight line that passes through the origin and has a slope of kA/kB. Results are shown in Figure 1A
for paraoxon, soman, sarin, and VX and summarized in Table 1
together with data reported on the individual rate constants of kA and kB in buffer solutions at 25 and 26°C. When plasma was replaced by saline, the plot of log
versus log ß resulted in a curved line (Fig. 1B
), and a similar pattern was observed when the endogenous enzyme was enhanced by exogenous HuBChE (Fig. 1C
).
|
|
Since kB is used to estimate the time to scavenge the OP by the recommended dose of HuBChE, it was determined directly in undiluted plasma and the results are shown in Table 1. Of the three nerve agents, VX was the least potent inhibitor, and the t1/2 values shown in Table 2
for this OP represent an upper limit for the rate of removal of all three OPs from blood.
|
![]() | (7) |
Equation 7 constitutes the upper limit of the HuBChE load ([B]exog) that is required to completely remove the OP from the circulation while maintaining HuAChE at or above a threshold
value. Substituting
kB/kA for ß in Equation 7
yields the following:
![]() | (8) |
A simple case to examine the influence of kA/kB on [B]exog is offered when T is <24 h and, hence, . It is clear that the higher the kB/kA ratio, the smaller the slope. For example, when
= 0.3 (70% reduction of RBC-AChE activity) and kB/kA >2, the slope approaches the limit of unity as follows:
![]() | (9) |
Thus, toxic doses of an OP that inhibit HuBChE more than two-fold faster than HuAChE can be detoxified by a stoichiometric ratio of HuBChE:OP. By contrast, when kB/kA <0.5, a substantial stoichiometric excess of HuBChE is required to maintain at 0.3 or above, as shown three-dimensionally in Figs 2
and 3
. The dependence of the HuBChE dose on [OP]0 as
increases is greater for VX than soman and sarin due to differences in kB/kA (Fig. 2
). Soman and sarin surfaces are almost covered due to the close proximity of the corresponding kB/kA ratio. When residual RBC AChE is set at a fixed value, for example
= 0.3, an increase in the rate of detoxification of the OP by HuBChE relative to HuAChE reduces the HuBChE dose that is required for complete removal of the inhibitor and keeps
above 0.3. This is illustrated by the tendency to produce a surface with a moderate inclination upon increase of kB/kA (Fig. 3
). Figs 4
and 5
are two-dimensional contour graphs prepared for facile prediction of HuBChE load for any pair of
-VX and
-soman dose, respectively.
|
|
|
|
![]() | (10) |
Equation 10 can be examined against a specific need to protect RBC AChE from a concentration of VX that corresponds to a 50% lethality dosage (225 µg/70 kg, or 843 nmol, based on LCt50 of VX 15 mg/min/m3; Hartman, 2002
). Using 1 mg HuBChE
12 nmol and 20 mg/70 kg of endogenous enzyme in a population that carries the usual phenotype of the enzyme (Lockridge and Masson, 2000
), a maximal dose of 214 mg HuBChE is required to ascertain that AChE activity remains above 30% until the OP in blood is completely removed by HuBChE. Essentially, similar considerations with respect to soman (kA/kB = 1.65;
0.3; LD50, iv, 280 µg/70 kg or 1.55 µmol; Wolfe et al., 1992
) theoretically suggest a loading dose of 228 mg/70 kg. Calculation shows that 548 mg/70 kg HuBChE would be required to protect against inhaled 1 LD50 sarin (kA/kB = 1.49;
0.3; 1 LD50, 525 µg/70 kg or 3.75 µmol; Hartman, 2002
). To further demonstrate the convenience of Equation 8
, Table 2
summarizes the results of estimates of the required dose of HuBChE against VX exposure in individuals who lack enzyme activity in plasma and those who represent the average activity of the usual HuBChE phenotype (Bendg = 20 mg).
Calculation of the Time Course of the Detoxification
So far, the model addressed the stoichiometric characteristic of protection, namely, the concentration of HuBChE ([B]0) that is required to completely remove [OP]0. To ascertain that the recommended dose can detoxify a substantial amount of the OP within the time scale of blood circulation in human, the t1/2 of the reaction of [B] + [OP] was obtained by using the standard second-order kinetic equation and substituting the value of [OP]0 from Equation 7 after setting [A]0 to zero, as follows:
![]() | (11) |
Representative t1/2 values for VX are shown in Table 2. It should be pointed out that Equation 11
does not take into account the detoxification of OP by RBC AChE, the estimated values are upper limits, and t1/2 values are expected to be even shorter than the calculated figures. Yet, the maximal reduction in time cannot exceed two-fold, and this will be apparent when kA[A]
kB[B]. For a dose >100 mg HuBChE, the time to remove 50% of the challenge is within the arm-to-aorta transit time of blood in human (1025 s; Francois et al., 2003
; Puskas and Schuierer, 1996
).
Evaluation of
The stability of HuBChE in human circulation is a key parameter in the approximation of the length of its therapeutic levels in blood. Data for of human are limited to a small number of studies that were re-evaluated by the use of Equation 12
. The latter expression describes the rate of approach to a steady state during regeneration of activity following inhibition of human plasma HuBChE by OPs, and it also holds for enzyme load and its subsequent disappearance from the circulation (see Supplementary).
![]() | (12) |
where [B]0, [B]t, and [B]endg are the activities of HuBChE at time zero, t, and , respectively, following the cessation of OP administration. According to Equation 12
, the first-order rate constant of the approach to steady state for the reappearance of enzymic activity reflects the rate constant of elimination
rather than the zero-order rate constant of de novo synthesis. Re-evaluation of the literature data in accordance with Equation 12
is summarized in Table 3
, and an average value of
was assigned at 0.069 ± 0.009 (SD) d-1. The prediction of the initial amount of HuBChE that would be required to provide protection of RBC AChE at any given time T following a single load of the enzyme is shown in Figure 6
for VX. For example, the protective efficacy of an initial load of 250 mg HuBChE for VX decays from 260 to 135 µg over 10 days (48% reduction), whereas for 50 mg load the reduction is significantly smaller (30%). This behavior is attributed to the contribution of the endogenous level of HuBChE (20 mg/70 kg).
|
|
|
|
A different source for substantiating Equation 8 arises from the consistency of the current conclusions with results of protection observed in four species (Ashani et al., 1998
). Yet, no data on RBC AChE levels were reported and results can serve only as an indirect support of the proposed model. A systematic study across four species revealed that a constant molar ratio of exogenous HuBChE to VX of 1.2 was sufficient to protect against 1.4 to 2.1 LD50 of VX in mice, rats, guinea pigs, and monkeys. The ATH extrapolation suggested that 200 mg HuBChE should protect against an iv dose of 0.9 LD50 VX in human. Table 2
shows that 200 mg HuBChE are expected to preserve RBC AChE above 30% of control at VX dose near its inhaled 1 LD50 value (Hartman, 2002
).
Equation 5
The studies by Sidell and Groff (1974) and Marrs et al. (1996)
did not report accurate values of reduction of human plasma HuBChE following VX injections, and it can only be said that kA/kB obtained in vitro here are consistent with their in vivo data (kA/kB <5.8). The effect of sarin on cholinesterases activity of human blood provides distinct validation of Equation 5
. Variable single oral doses of sarin in three subjects resulted in depression of HuBChE and HuAChE activity (Grob and Harvey, 1958
) from which kA/kB values were calculated as 1.42, 1.37, and 1.37. Similarly, injection of sarin into the brachial artery of two individuals resulted in kA/kB values of 1.33 and 1.47. These findings are in excellent agreement with the ratio of kA/kB = 1.49, determined in vitro at 37°C.
DISCUSSION
Prophylaxis with HuBChE is based on rapid neutralization of OPs in the blood circulation before they can reach target organs, and RBC AChE, the first HuAChE pool to interact with the OP, represents such a target site. Pretreatment with exogenous enzyme is expected to protect RBC AChE in a manner that depends on specific kinetic parameters and stoichiometric considerations. The same enzymic characteristics are expected at cholinergic synapses.
The ongoing controversy on blood-target tissue relationship of AChE levels following OP intoxication reflects a composite correlation between the manifestation of toxic symptoms and depression of RBC AChE activity (Bueters et al., 2003; Ellin, 1982
; Jimmerson et al., 1989
; Marrs et al., 1996
; Padilla et al., 1994
). By contrast, there is a general agreement that residual AChE in target tissues such as brain and muscles is well correlated with the severity of symptoms following acute intoxication with OPs such as sarin or soman, and inhibition should be well above 65% to produce visible toxic signs (Bueters et al., 2003
; Meeter and Wolthuis, 1968
; Tonduli et al., 1999
). In fact, it was suggested that the critical brain and diaphragm AChE activity essential for the maintenance of physiological responses is less than 10% of the target enzyme pool (Meeter and Wolthuis, 1968
; Sweeny and Maxwell, 1999
, 2003
). Bueters et al. (2003)
proposed that preserving neuronal levels of enzyme activity at 1015% of the original activity should protect from manifestation of OP toxicity.
Despite the apparent controversy that stems from animal experiments, published human studies with OPs such as sarin (Grob and Harvey, 1958) and VX (Marrs et al., 1996
; Sidell and Groff, 1974
) suggest that the value of 30% residual activity of RBC AChE (70% depression of baseline) is not associated with severe toxic signs. Thus, it was reasonable to assume that for acute bolus exposures to toxic doses of OPs, the estimation of the dose of exogenous HuBChE that can maintain residual RBC AChE at 30% of baseline activity can set a limit to the extent of survivability of critical AChE levels in target organs.
The proposed model disregards nonspecific degradation and clearance of OPs and the spontaneous reactivation of the inhibited cholinesterases. Since these pathways are likely to act in favor of preserving AChE activity at physiologically important sites, they are considered as extra protection factors of the recommended dose.
To provide guidelines for the selection of appropriate HuBChE dosage, Equations 4 and 5
were derived on assumptions of a single-compartment kinetic model and represent a general upper limit expression that is applicable for both acute bolus and continuous exposures. Equation 4
is essentially similar to the equation derived by Sweeny and Maxwell (1999)
, who used a single-compartment model for the prediction of toxicity in rats that use high levels of endogenous CaE and OP hydrolase for the detoxification of OPs. Recently, Sweeny and Maxwell (2003)
used the same strategy to estimate protective ratios in mice exposed to OPs by extending the mass balance equation to include exogenously administered CaE and catalytic scavengers. Equation 4
was derived here with consideration of HuBChE as the major detoxification component of human blood. For acute bolus administration of the challenge, the general expression of Equation 4
is approximated by Equation 8
and constitutes a straightforward and simple relationship between the dose of the protecting enzyme, the kinetic constants of inhibition of HuAChE and HuBChE, and the necessary level of protection of RBC AChE.
The ratio of kB/kA is readily available from direct in vitro measurements of inhibition of ChEs in human blood, and the duration of therapeutic levels of HuBChE after a single load can be determined by using the elimination rate constant of HuBChE that corresponds to t1/2 of 10 days.
The finding that the model predictions are consistent with animal experiments can also be viewed as support of the animal-to-human extrapolation model and will increase the reliability of the prediction of prevention of toxic effects in HuBChE-treated humans intoxicated under different exposure patterns.
Validation by use of published data of human studies and animal experiments calls attention to important factors that were not included in the derivation of the OP mass balance shown in Equations 4, 7
, and 8
due to a lack of sufficient data. These are the apparent rapid decomposition of the less toxic isomers of sarin, soman, and probably VX in human blood and the potential of a volume of distribution of HuBChE in humans that significantly exceeds the volume of plasma. Two studies indicated that the steady state volume of distribution of HuBChE in humans might range between 1418% of body weight compared with 5% of plasma volume (Garry et al., 1974
; Ostergaard et al., 1988
). It appears that the potential of endogenous HuBChE to detoxify OPs is significantly greater than the usually cited values, and this could be used, in part, to explain a discrepancy encountered during validation of the mass balance model equation against in vivo data. On the whole, validation confirms that Equation 8
provides upper limits of the HuBChE dose necessary for protection. The model can be applied for quantitative evaluation of the overall impact of endogenous HuBChE of various populations on their susceptibility to OPs. For example, calculations summarized in Table 2
highlight the differences between a population that is at high risk (kB x Bendg
0) and that containing the average activity of the usual phenotype. From the point of view of risk assessment, the assumptions used offer significant safety factors that compensate for uncertainty associated with OP intoxication.
In addition to the experimental validation of Equation 5, the ratio of kA/kB is of special interest since it is a useful parameter for various purposes. With VX it revealed a small yet significant deviation from the predicted ratio. At first, this was attributed to the fact that VX is protonated at pH 7.4 and the charged molecule encounters a diffusion barrier during its transfer from the aqueous layer into the hydrophobic environment of RBC AChE. However, the rate constants for the inhibition of AChE of both intact and detergent-solubilized RBC by VX were found to be practically the same; more experiments are required to clarify this observation.
Another observation worth mentioning was made with sarin, soman, and VX. All three methylphosphonates are racemic mixtures of optical isomers that vary widely in their anticholinesterase potency (Benschop et al., 1984; Boter and Van Dijk, 1969
; Hall et al., 1977
). The finding that log
/log ß plots for racemic sarin, soman, and VX gave straight lines is at odds with the requirement set for the derivation of Equation 5
, because, in contrast to kA, kB values of the two stereoisomers of sarin and VX are only 4- to 10-fold apart and that of four stereoisomers of soman varies between 1.6 and 170 (Van der Schans et al., 2002
). Under such circumstances a curvature was expected in the log-log plot. It is possible to reconcile the apparent discrepancy on the reported rapid enzymatic hydrolysis of the less toxic isomers of sarin and soman (and presumably on the rapid consumption of considerable fraction of VX) in mammalian blood, including human plasma (Christen and Van Den Muysenberg, 1965
; De Bisschop et al., 1987
; De Jong et al., 1988
). Such an explanation is strongly supported by the curved lines shown in Figure 1B
, where plasma of human blood was replaced with saline containing HuBChE, and Figure 1C
, where the endogenous enzyme was enhanced by exogenous HuBChE. In the latter case, the increase of HuBChE concentration appears to compete with the enzymatic detoxification of the OPs. To the best of our knowledge, this is the first experimental demonstration that confirms the non-ChE degradation of one of the VX isomers in human plasma.
Finally, based on the finding that only part of the racemic mixture of OPs requires rapid detoxification by HuBChE in blood and regarding the potential volume of distribution of HuBChE, it is reasonable to suggest that the upper limit doses of 134, 115, and 249 mg/70 kg are sufficient to protect RBC AChE above 30% of baseline activity following a challenge with 1 LD50 VX, soman, and sarin, respectively. Since the time to reduce to half the initial blood levels of 1 LD50 dose of all three OPs in HuBChE-pretreated individuals is less than 10 s, it is highly likely that the recommended amount of HuBChE should prevent manifestation of severe toxic signs in human.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Y. Alexander for critically reviewing the manuscript of this article. The following symbols and nomenclature are used throughout the text: [A], RBC-AChE concentration; A0, initial concentration of RBC-AChE in blood; [B], concentration of HuBChE; [B]endg, endogenous concentration of plasma HuBChE; [B]exog, concentration of plasma HuBChE produced by exogenous administration of enzyme; , the fraction of active RBC-AChE; ß, the fraction of active HuBChE; t, time of exposure; T, time elapsed from loading of HuBChe;
, first order constant of elimination of HuBChe from blood; kA, bimolecular rate constant of inhibition of RBC-AChE by organophosphates; kB, bimolecular rate constant of inhibition of HuBChE by organophosphates; [OP], concentration of organophosphates in blood; [OP]in, the rate of build-up of organophosphates concentration in blood.
NOTES
1 To whom correspondence should be addressed at Israel Institute for Biological Research, PO Box 19, Ness Ziona, Israel. Fax: +972 8-9381432. E-mail: shlomi{at}math.iibr.gov.il.
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