br Materials and methods br Results The determination of the

Materials and methods
Results The determination of the intrinsic inhibitory potential of the oximes HI-6 and MMB-4 with human and guinea pig AChE revealed marked differences between both oximes (Table 1). MMB-4 had an only marginal effect on both AChE species, calculated IC50 of >1000μM, while HI-6 resulted in a relevant, concentration-dependent inhibition of human AChE and to less extent of guinea pig AChE. The impact of 100μM HI-6 and 100μM MMB-4 on the inhibition kinetics of soman was tested with human and guinea pig AChE (Fig. 2 and Table 2). It turned out, that MMB-4 had a negligible effect on soman inhibition kinetics with human AChE while HI-6 resulted in a substantial decrease of the calculated inhibition rate constant ki. With guinea pig AChE virtually no difference of ki values in the presence and absence of oximes was recorded. The determined inhibition rate constant were used to calculate the protective index of oximes (PI=ki of soman/ki of soman plus oxime), again demonstrating the substantial attenuation of HI-6 on soman inhibition kinetics of human AChE. It appears that HI-6 can partially shield the enzyme leading to a delayed phosphonylation. These data are in good agreement to the inhibition rate constants determined with the dynamic model (Table 3). Simultaneous perfusion of human AChE with soman and HI-6 resulted in a significant Pyridoxal isonicotinoyl hydrazone of the inhibition rate constant k1 in comparison to perfusion with soman alone. All other combinations did not result in a significant effect on AChE inhibition by soman. Fig. 3 demonstrates the effect of soman and HI-6 perfusion on the inhibition of human AChE. Simultaneous soman and HI-6 perfusion resulted in a slower decrease of AChE activity but could not prevent total AChE inhibition. Reactivation of soman-inhibited human and guinea pig AChE by 100μM HI-6 or MMB-4 was negligible, irrespective of the start of oxime perfusion (Table 4 and Fig. 3). AChE activity at t=150min was less than 1% of control activity with HI-6 and MMB-4 and human AChE and with HI-6 and guinea pig AChE. MMB-4 resulted in a guinea pig AChE activity of less than 2%.
Discussion Previous in vitro studies on the reactivation of soman-inhibited AChE by oximes gave rather variable results. In a number of studies the inhibition of AChE by soman was performed under conditions to prevent premature aging, i.e. pH 9–10 and cooling, with excess soman being removed before adding an oxime (De Jong and Wolring, 1980, Schoene et al., 1983, De Jong and Kossen, 1985, Worek et al., 1998, Luo et al., 2007). An in part remarkable reactivation was observed in these studies, HI-6 being in general the lead compound. Other studies used short (Puu et al., 1986) or undefined incubation times of soman with AChE (Wei et al., 2016). Hence, there is convincing evidence that oximes are able to reactivate soman-inhibited AChE under specific conditions, i.e. prevention of premature aging and absence of excess soman during the reactivation process. Due to the rather high volatility of soman, the most likely exposure route is by inhalation. Determination of the toxicokinetics of soman stereoisomers in guinea pigs after inhalation exposure demonstrated a rapid increase of agent concentrations in blood followed by a decrease with terminal half-times between 8 and 63min, depending on the dose and exposure time (Langenberg et al., 1998). Hence, relevant soman concentrations are most likely present at the time of injection of oxime (and atropine) during emergency treatment or self- and buddy aid. In contrast to above mentioned studies using static test tube protocols the dynamic model allows the continuous perfusion of AChE with soman and oxime thus enabling the application of different perfusion protocols to determine the dynamic changes of AChE activity online. Perfusion of AChE with 100μM HI-6 or MMB-4 had no significant effect on the inhibition of AChE by soman, except with HI-6 and human AChE. This may be attributed to the intrinsic inhibitory potency of HI-6 (IC50 of 218μM) resulting in a transient shielding of the enzyme. Due to the reversible nature of HI-6 inhibition and the higher affinity of soman to the active site of AChE HI-6 appears not able to prevent complete inhibition of the enzyme within short time.