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Suicidal ingestion of organophosphorus (OP) or carbamate (CM) compounds challenges health care systems worldwide, particularly in Southeast Asia. The diagnosis and treatment of OP or CM poisoning is traditionally based on the clinical appearance of the typical cholinergic toxidrome, e.g. miosis, salivation and bradycardia. Yet, clinical signs might be inconclusive or even misleading. A current case report highlights the importance of enzymatic assays to provide rapid information and support clinicians in diagnosis and rational clinical decision making. Furthermore, the differentiation between OP and CM poisoning seems important, as an oxime therapy will most probably not provide benefit in CM poisoning, but—as every pharmaceutical product—it might result in adverse effects. The early identification of the causing agent and the amount taken up in the body are helpful in planning of the therapeutic regimen including experimental strategies, e.g. the use of human blood products to facilitate scavenging of the toxic agent. Furthermore, the analysis of biotransformation products and antidote levels provides additional insights into the pathophysiology of OP or CM poisoning. In conclusion, cholinesterase activities and modern analytical methods help to provide a more effective treatment and a thorough understanding of individual cases of OP or CM poisoning. [ABSTRACT FROM AUTHOR]

etylcholinesterase (AChE) is the primary target of organophosphorus compounds (OP). The investigation into interactions between AChE, OP and oximes in vitro may be affected by the experimental conditions, e.g. by the buffer system. Hence, it was tempting to investigate the Michaelis-Menten kinetics and the inhibition and reactivation kinetics of paraoxon-ethyl, sarin, soman and VX in the presence of phosphate, MOPS, Tyrode and TRIS buffer with human AChE. Compared to phosphate buffer, the inhibition and reactivation kinetics of human erythrocyte AChE were markedly changed by TRIS and in part by MOPS, whereas Tyrode showed similar results to phosphate buffer. These results indicate an effect of the tested buffers on the properties of AChE, and an interaction between OP and oximes has to be considered for the design of in vitro studies and may impair the comparison of data from different laboratories. In view of the comparability of human in vitro kinetic data determined with phosphate buffer with data from human OP poisoning, it seems to be a suitable buffer for the investigation into interactions between AChE, OP and oximes. [ABSTRACT FROM AUTHOR]

Sulfur mustard (SM) is a bifunctional alkylating agent. Its primary toxic consequence is severe skin damage with blisters, occurring after skin contact. These vesicant properties of SM have been linked to cell death of proliferating keratinocytes in the basal layer of the skin. Catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP-1) has been demonstrated to be a major event in response to high levels of DNA damage, and PARP-1 activation may be part of apoptotic signaling. In other contexts, overstimulation of PARP-1 triggers necrotic cell death because of rapid consumption of its substrate, β-nicotinamide adenine dinucleotide (NAD+) and the consequent depletion of ATP. These findings prompted us to evaluate whether SM induces apoptosis in keratinocytes like HaCaT cells and to determine whether blocking of PARP enzyme activity with 3-aminobenzamide (3AB) can influence the mode of cell death. HaCaT cells were exposed to SM (10-1,000 μM; 30 min) and then cultivated in SM-free medium with or without 3AB for up to 48 h. This treatment resulted in a time and SM dose-dependent increase of apoptotic cell death characterized by PARP-1 cleavage and DNA fragmentation during the experimental period. After just 45 min of exposure to 1 mM SM, we observed a significant increase in PARP-1 activity in HaCaT cells. About 6 h after exposure, intracellular ATP levels were diminished by 22%, which seemed to be completely prevented by the addition of 3AB directly after exposure. However, 18 h later, this 3AB effect on the SM concentration-dependent loss of ATP was no longer detectable. Interestingly, the effect of SM on total cell viability was not changed by 3AB. However, the mode of cell death was influenced by 3AB exhibiting an increase of apoptotic cells and a concomitant decrease of necrotic HaCaT cells during the first 24 h after SM exposure. Our results indicate that SM concentrations of 1 mM or higher induce a prominent PARP activation leading to ATP depletion and necrosis. In contrast, lower concentrations of SM cause minor PARP activation and, especially, PARP-1 cleavage by caspase 3 without ATP depletion. Because ATP is required for apoptosis, we suggest that ATP acts as an early molecular switch from apoptotic to necrotic modes of SM-induced cell death, at least at high concentrations (≥1 mM). Thus, the observed early proapoptotic effect of 3AB at lower SM concentrations may point to the influence of ATP-independent cell-death regulating mechanisms. [ABSTRACT FROM AUTHOR]

The direct reaction of seven pyridinium oximes with the organophosphorus compounds (OPCs) crotylsarin, cyclosarin, and VX was studied by spectrophotometry. This method allows to quantify different parameters: (a) the half-life times ( t 1/2) of the oxime-OPC reactions on the basis of the changes in the absorption at the zwitterion (betaine) peak maximum, (b) the first- and second-order rate constants ( k 1, k 2), and (c) the maximum reaction velocities ( v max). The results of the study show that the reaction velocity of the nerve agents with any of the oximes investigated decreased in the order crotylsarin > cyclosarin > VX. The comparison of the reaction rates of the three therapeutically used oximes (2-PAM, obidoxime, HI 6) with the respective OPC gave the highest rate for crotylsarin and cyclosarin with obidoxime and to a similar degree with HI 6, while in the case of VX the most reactive oxime was HI 6. The reaction velocity of the nerve agents with the monopyridinium oxime 2-PAM was lower as compared to the bispyridinium oximes (obidoxime, HI 6). The results obtained with the two sarin analogues indicate that the direct reaction with 2-PAM, obidoxime, or HI 6 could be used for non-corrosive decontamination purposes, especially, if sensitive biological surfaces like skin, mucous membranes, or wounds are considered. However, in view of the concentrations of nerve agents and oximes, which could be expected during OPC poisoning in man, the maximum reaction velocities would not be high enough to contribute markedly to the detoxication of nerve agents in vivo. [ABSTRACT FROM AUTHOR]

The reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) by oximes results inevitably in the formation of highly reactive phosphyloximes (POX), which may re-inhibit the enzyme. An impairment of net reactivation by stable POX was found with 4-pyridinium aldoximes, e.g. obidoxime, and a variety of OP compounds. In this study the effect of organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA) and diisopropylfluorophosphatase (DFPase) on obidoxime-induced reactivation of human acetylcholinesterase (AChE) inhibited by different OPs was investigated. Reactivation of paraoxon-, sarin-, soman- and VX-inhibited AChE by obidoxime was impaired by POX-induced re-inhibition whereas no deviation of pseudo first-order kinetics was observed with tabun, cyclosarin and VR. OPH prevented (paraoxon) or markedly reduced the POX-induced re-inhibition (VX, sarin, soman), whereas OPAA and DFPase were without effect. Additional experiments with sarin-inhibited AChE indicate that the POX hydrolysis by OPH was concentration-dependent. The activity of OP-inhibited AChE was not affected by OPH in the absence of obidoxime. In conclusion, OPH may be a valuable contribution to the therapeutic regimen against OP poisoning by accelerating the degradation of both the parent compound, OP, and the reaction product, POX. [ABSTRACT FROM AUTHOR]

A great number of structurally different organophosphorus compounds (OPs) was synthesized in the past decades to be used as pesticides or chemical warfare agents. Methyl-fluorophosphonylcholines were found to be highly toxic OPs and the acetylcholinesterase (AChE) reactivator pralidoxime was shown to be unable to reactivate inhibited AChE. In the course of the development of more effective AChE reactivators, we have determined the reactivation rate constants of various oximes with human AChE inhibited by methylfluorophosphonylcholine (MFPCh), methylfluoro-β-phosphonylcholine (MFPβCh) and methylfluorophosphonylhomocholine (MFPhCh). In addition, we investigated the potential influence of aging phenomena on the oxime efficacy. Human AChE inhibited by MFPCh, MFPβCh or MFPhCh was extremely resistant towards reactivation by oximes. Nevertheless, the newer compounds, HLö 7 and HI 6, were substantially more potent reactivators than obidoxime and pralidoxime. The low oxime efficacy was not due to rapid aging since no decrease in reactivatability was found over 96 h at 37°C. Within this period a substantial spontaneous reactivation was observed, with MFPCh >MFPβCh >MFPhCh, which did not follow pseudo-first-order kinetics. In conclusion, the unexpected results, i.e., high resistance of inhibited AChE towards oxime reactivation and aging, and much lower resistance towards spontaneous reactivation, calls for further experiments at a molecular level for a better understanding of the interactions among AChE, its inhibitors and reactivators. [ABSTRACT FROM AUTHOR]

The well-documented efficacy of HI 6 dichloride in reactivating acetylcholinesterase (AChE) inhibited by nerve agents is curtailed by its poor water-solubility at temperatures below 10°C. This drawback can be circumvented by using HI 6 dimethanesulfonate, which has been developed in our laboratory. Since investigations on the efficacy of this new entity are lacking, it has been proposed that some bridging experiments be performed, aimed at demonstrating reactivator equivalence in vitro. The reactivating properties of the two salts were compared on human erythrocyte AChE inhibited with paraoxon, sarin, cyclosarin and agent VX. The comparison was extended to cynomolgus erythrocytes exposed in vitro for sarin and VX. Finally, mouse diaphragm preparations were circumfused with sarin, cyclosarin and VX and reactivation by each of the HI 6 salts was examined in muscle homogenates. AChE activity in erythrocyte suspensions was monitored by a modified Ellman procedure, muscle AChE by a radiometric assay. In all models tested no differences between the HI 6 salts could be detected (P=0.05). From these data, equipotency in AChE reactivation by the two HI 6 salts can be anticipated. [ABSTRACT FROM AUTHOR]

Standard treatment of poisoning by organophosphates (OP) includes the administration of an antimuscarinic agent, e.g. atropine, and of an acetylcholinesterase (AChE) reactivator (oxime). The presently available oximes, obidoxime and pralidoxime (2-PAM), are considered to be insufficient for highly toxic OPs, e.g. sarin. In the past decades numerous oximes were prepared and tested for their efficacy in OP poisoning, mostly in animal experiments. However, data indicate that the reactivating potency of oximes may be different in humans and animal species, which may hamper the extrapolation of animal data to humans and may pose a problem in the drug licensing of new compounds. In order to provide data for a better evaluation of the reactivating potency of oximes, experiments were undertaken to determine the reactivation rate constants of several oximes with human, rabbit, rat and guinea-pig AChE inhibited by the OPs sarin, cyclosarin and VX. The results show marked differences among the species, depending on the inhibitor and on the oxime, and indicate that the findings from animal experiments need careful evaluation before extrapolating these data to humans. [ABSTRACT FROM AUTHOR]

The potential of obidoxime and other pyridinium-4-aldoximes to reactivate dimethyl- and diethylphosphorylated cholinesterases is markedly restricted by the inevitable formation of rather stable phosphoryl oximes (POXs) with high anticholinesterase activity. This effect is hardly seen with very dilute enzyme preparations, but becomes significant at physiological enzyme concentrations. Human plasma with the butyrylcholinesterase irreversibly blocked by soman was able to stimulate obidoxime-induced reactivation of concentrated erythrocyte acetylcholinesterase (EryAChE) to the same extent as was observed with a dilute preparation, suggesting phosphoryl oxime-destroying capacity. The inactivating factor, which was tentatively termed POX-hydrolase, had (1) a molecular weight of >100 kDa; (2) required Ca2+, which could not be substituted by Zn2+ or Mg2+; and (3) lost its catalytic activity reversibly in the presence of ethylenediaminetetraacetic acid (EDTA). The enzyme activity varied widely (20-fold) among different subjects and did not follow the activity pattern of human serum paraoxonase (PON1). Rabbit plasma with its particularly high paraoxonase content showed only weak POX-hydrolase activity. These data suggest POX-hydrolase to be a different entity. POX-hydrolase was most active with the putative phosphoryl-obidoxime from paraoxon-ethyl, less with the product from paraoxon-methyl and least with that from diisopropylfluorophosphate. The analogue TMB-4 reacted similarly to obidoxime. The putative phosphonyl oximes arising by the reaction of obidoxime with nerve agents were apparently not cleaved. The variation in POX-hydrolase activity may additionally contribute to the variable response to oxime therapy in patients with organophosphate insecticide poisoning. [ABSTRACT FROM AUTHOR]

The reactivation of organophosphate-inhibited acetylcholinesterase (AChE) by oximes inevitably results in the formation of highly reactive phosphoryloximes (POX), which are able to re-inhibit the enzyme. In this study, the dependence of POX formation on AChE concentration was investigated with sarin-inhibited human erythrocyte AChE (EryAChE). A marked dependence was found with obidoxime but not with the experimental oxime HI 6, suggesting great differences in the decomposition rates of the respective POXs. At a physiological erythrocyte content the reactivation of EryAChE was markedly affected by POX with obidoxime and pralidoxime (2-PAM) but not with the newer oximes HI 6 and HLö 7. Addition of extensively dialysed, sarin-treated human plasma reduced the reactivation by obidoxime and 2-PAM even more. Obidoxime and 2-PAM were superior to HI 6 and HLö 7 in reactivating butyrylcholinesterase (BChE). This effect was pronounced in diluted plasma, but was obscured in concentrated plasma, probably because of re-inhibition by the generated POX. Addition of native erythrocytes to sarin-treated plasma resulted in marked inhibition of EryAChE in the presence of obidoxime, suggesting a higher affinity of the POX for EryAChE. The results indicate that obidoxime and 2-PAM may reactivate sarin-inhibited AChE insufficiently due to re-inhibition by the POX formed. In addition, the re-inhibition of EryAChE may be aggravated by the POX that is produced during BChE reactivation. These reactions must be regarded as therapeutically detrimental and disqualify those oximes which are capable of forming stable POX by reactivation of BChE. [ABSTRACT FROM AUTHOR]

Human poisoning by organophosphates bearing two methoxy groups, e.g. by malathion, paraoxon-methyl, dimethoate and oxydemeton-methyl, is generally considered to be rather resistant to oxime therapy. Since the oxime effectiveness is influenced not only by its reactivating potential but also by inhibition, aging and spontaneous reactivation kinetics, experiments were performed with human acetyl- (AChE) and butyrylcholinesterase (BChE) to determine the respective kinetic constants. The efficacy of obidoxime in reactivating dimethylphosphoryl-AChE was 40, 9 and 3 times higher than of HI 6, pralidoxime and HLö 7, respectively. Aging ( t 1/2 3.7 h) and spontaneous reactivation ( t 1/2 0.7 h) occurred concomitantly, with the portion of the aged enzyme being dependent on the presence of excess inhibitor. Calculation of steady-state AChE activity in the presence of inhibitor and oxime revealed that obidoxime was superior to pralidoxime. In addition, organophosphate concentrations up to 10−6 M (paraoxon-methyl) and 10−4 M (oxydemeton-methyl) could be counteracted at clinically relevant oxime concentrations (10 μM). These data indicate that oximes may effectively reactivate human dimethylphosphoryl-AChE. Failure of oximes may be attributed to megadose intoxications and to prolonged time intervals between poison uptake and oxime administration. The potency of the oximes to reactivate dimethylphosphoryl-BChE was much lower and the spontaneous reactivation slower ( t 1/2 9 h), while aging proceeded at a comparable rate. Thus, BChE activity determination for diagnosis and therapeutic monitoring may give no reliable information on AChE status. [ABSTRACT FROM AUTHOR]

Cyclohexylmethylphosphonofluoridate␣(cyclosarin) is a highly toxic organophosphate, which was shown to be rather resistant to conventional oxime therapy. To give more insight into the inhibition, reactivation and aging kinetics, human acetyl-(AChE) and butyrylcholinesterase (BChE) were inhibited by cyclosarin ( k 2 of 7.4 and 3.8 * 108 M−1 min−1, respectively; pH 7.4, 37 °C) and reactivated with obidoxime, pralidoxime and three experimental oximes. The new oxime HLö 7 (1-[[[4-aminocarbonyl)-pyridinio]-methoxy]-methyl]-2,4-bis-[(hydroxyimino)methyl] pyridinium dimethanesulphonate) was shown to be superior to the other oximes. At oxime concentrations anticipated to be relevant in humans, obidoxime and pralidoxime were extremely weak reactivators of AChE. Aging velocity of BChE was almost fourfold higher compared to AChE (ka of 0.32 h−1 and 0.08 h−1, respectively). A substantial spontaneous reactivation was observed with AChE. These results support previous in vivo findings that obidoxime and pralidoxime are insufficient antidotes in cyclosarin poisoning. By contrast, HLö 7 was shown to be an extremely potent reactivator of human AChE and BChE,␣which supports its position as a broad-spectrum oxime. [ABSTRACT FROM AUTHOR]

The treatment of poisoning by highly toxic organophosphorus compounds (nerve agents) is unsatisfactory. Until now, the efficacy of new potential antidotes has primarily been evaluated in animals. However, the extrapolation of these results to humans is hampered by species differences. Since oximes are believed to act primarily through reactivation of inhibited acetylcholinesterase (AChE) and erythrocyte AChE is regarded to be a good marker for the synaptic enzyme, the reactivating potency can be investigated with human erythro cyte AChE in vitro. The present study was undertaken to evaluate the ability of various oximes at concentrations therapeutically relevant in humans to reactivate human erythrocyte AChE inhibited by different nerve agents. Isolated human erythrocyte AChE was inhibited with soman, sarin, cyclosarin, tabun or VX for 30 min and reactivated in the absence of inhibitory activity over 5–60 min by obidoxime, pralidoxime, HI 6 or HLö 7 (10 and 30 μM). The AChE activity was determined photometrically. The reactivation of human AChE by oximes was dependent on the organophosphate used. After soman, sarin, cyclosarin, or VX the reactivating potency decreased in the order HLö 7 > HI 6 > obidoxime > pralidoxime. Obidoxime and pralidoxime were weak reactivators of cyclosarin-inhibited AChE. Only obidoxime and HLö 7 reactivated tabun-inhibited AChE partially (20%), while pralidoxime and HI 6 were almost ineffective (5%). Therefore, HLö 7 may serve as a broad-spectrum reactivator in nerve agent poisoning at doses therapeutically relevant in humans. [ABSTRACT FROM AUTHOR]

The new bispyridinium oximes HI 6 and HLö 7 are promising antidotes against poisoning by highly toxic organophosphorus compounds, i.e. nerve agents. Until now, their ability to reactivate pesticide inhibited human acetylcholinesterase (AChE) has not been elucidated. For this purpose human erythrocyte AChE (EC 3.1.1.7) was inhibited (30 min) by chlorfenvinphos, dichlorvos, dicrotophos, heptenophos, mevinphos, monocrotophos, paraoxon, phosphamidon, trichlorfon, malaoxon, omethoate, oxydemeton-methyl or methamidophos by 85-98% of control. After removal of excess inhibitor, obidoxime, pralidoxime (2-PAM), HI 6 or HLö 7 (10, 30 or 100 μmol/l) were added and the AChE activity was measured spectrophotometrically at various times thereafter (5-60 min). The oximes significantly, but not completely, reactivated organophosphate inhibited AChE. The velocity and extent of reactivation were dependent on the oxime and its concentration. In all cases obidoxime was superior to the three other oximes, followed by HLö 7, 2-PAM and HI 6. In most cases obidoxime and HLö 7 were most effective at 10 or 30 μmol/l while 2-PAM and HI 6 needed 100 μmol/l. These data suggest that 2-PAM, HI 6 and HLö 7 are less patent than obidoxime in reactivating human AChE inhibited by organophosphate pesticides. [ABSTRACT FROM AUTHOR]

HLö 7 dimethanesulfonate (1-[[[4-(aminocarbonyl)pyridinio] methoxy] methyl] -2,4-bis [(hydroxyimino) methyl]pyridinium dimethanesulfonate) is a broad-spectrum reactivator against highly toxic organophosphorus compounds. The compound was synthesized by a new route with the carcinogenic bis(chloromethyl)ether being substituted by the non-mutagenic bis(methylsulfonoxymethyl)ether. The very soluble dimethanesulfonate of obidoxime was also prepared by this way. HLö 7 dimethanesulfonate is the first water-soluble salt of HLö 7 that should be suitable for the wet/dry autoinjector technology, because aqueous solutions of HLö 7 are not very stable (calculated shelf-life 0.2 years when stored at 8°C, 1 M solution, pH 2.5). The crystalline preparation contains 96% of the syn/syn-isomer, less than 2% of the syn/anti-isomer and some minor identified by-products. HLö 7 was very efficient in reactivating acetylcholinesterase (AChE) blocked by organophosphates as long as ageing did not prevent dephosphylation. HLö 7 was superior to HI 6 (1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2-[(hydroxyimino)methyl]pyridinium dichloride) in reactivating soman and sarin-inhibited AChE from erythrocytes, and literature data indicate that HLö 7 exceeds HI 6 by far in reactivating tabun-inhibited AChE. In atropine-protected, soman-poisoned mice HLö 7 was three times more potent than HI 6 (protective ratio 5 versus 2.5), and in sarin-poisoned mice HLö 7 was 10 times more potent than HI 6 (protective ratio 8 for both oximes). In atropine-protected guinea-pigs HLö 7 was less effective than HI 6 (protective ratio: 2.3 versus 5.2 for soman; 5.2 versus 6.8 for sarin; 4.3 versus 3.8 for tabun). The mean survival time of anaesthetized guinea-pigs exposed to 5 LD soman (6.3 min) was increased by atropine (27 min) and atropine + HLö 7 (57 min). HLö 7 alone did not prolong the survival. The most impressive effect of HLö 7 was on respiration: 3 min after i.v. injection of HLö 7 and atropine, the depressed respiration increased rapidly to 60% of control and remained at that level during the observation period (60 min). With atropine alone, respiration recovered only slowly. Behavioural and physiologic parameters were determined in atropine-protected mice exposed to a sublethal soman dose. The running performance was significantly improved by HLö 7. Even central symptoms, e.g. hypothermia and convulsions, were decreased markedly by HLö 7 (evaluation 60 min after poisoning). The pharmacokinetic data for HLö 7 in male beagle dogs are similar to those of HI 6. After i.v. injection: t = 5 min; t = 46 min; V = 0.24 1/kg; Cl = 3.7 ml x min x kg; Cl= 3.2 ml x min x kg; renal excretion of unchanged HLö 7 = 86%. After i. m. injection: t = 14 min; t = 48 min; Vd = 0.27 1/kg; Cl= 3.9 ml x min x kg; Cl= 2.7 ml x min x kg; renal excretion of unchanged HLö 7 = 76%; bioavailability >95%. Plasma protein binding was <5%; HLö 7 did not permeate into red cells. A dose of 20 μmol/kg was well tolerated both after i.v. and i.m. administration. In anaesthetized dogs (chloralose) HLö 7 i.v. (20 (imol/kg) showed marginal hypotensive effects, whereas 50 μmol/kg resulted in decreased mean blood pressure (−15%) and blood flow (−30%) without reflex tachycardia. One out of four dogs developed a circulatory shock syndrome with anuria. Respiration varied only transiently. Blood gases and pH were not influenced. Similar cardiovascular effects were observed in anaesthetized (urethane) guinea-pigs. In isolated guinea-pig hearts (Langendorff) sinus and ventricular heart rate were not influenced by HLö 7 <500 μM. HLö 7 antagonized both carbachol and nicotine effects. Red cell AChE was inhibited by HLö 7 by up to 50%; C about 100 μM. Previously, HLö 7 was shown to block ganglionic transmission (IC= 500 μM), probably due to ion-channel blockade. These data indicate that HLö 7 combines ganglion blocking, anticholinergic and indirect cholinergic properties like other bispyridinium compounds. The results suggest that HLö 7 may be tolerated by man at a dose of 10 μmol/kg. Vital functions are not expected to be impaired. At such a dose (250-500 mg), which can be injected by an autoinjector, HLö 7 is expected to be superior to HI 6. [ABSTRACT FROM AUTHOR]