The present study was undertaken in order to determine if established or experimental K-oximes can protect from OP (paraoxon) toxicity. Rats that received only the OP were compared with those that had received oxime pretreatment before OP exposure. Best protection from paraoxon-induced mortality was observed after pretreatment with K-48. The other oximes were significantly less efficacious, but still significantly reduced the RR compared to the no-treatment group (paraoxon only). The FDA-approved agent for prophylaxis was least efficacious. The protective efficacy was unrelated to AChE inhibition

Kuca, Kamil

Lorke, Dietrich

Nurulain, Syed M

English

eCommons at Roseman University

ku.ac.aeExperimental Oximes as Organophosphate PreexposureProphylaxisLessons from the Gulf War SyndromeDietrich E. Lorke1, 2, Syed M. Nurulain3, Kamil Kuca41Department of Basic Sciences, College of Medicine, Roseman University of Health Sciences, Las Vegas, Nevada, USA2Department of Anatomy and Cellular Biology, College of Medicine and Health Sciences, Khalifa University, Abu Dhabi, United Arab Emirates3 Bio Science Department, COMSATS Institute of Information Technology, Islamabad, Pakistan4 Department of Chemistry, Faculty of Science, University of Hradec Kralove, Hradec Kralove, Czech RepublicBackgroundMethodsOrganophosphate (OP) acetylcholine esterase (AChE) inhibitors are useful agents as pesticides, but they also represent a serious threat, when abused for terrorist attacks and chemical warfare (nerve agents: sarin, tabun VX, Novichok). The imminent danger of OP nerve agents is demonstrated by the murder of the half-brother of North Korean ruler Kim Jong-un with VX and of the Skripals with Novichok, tabun deployment during the Iraq/Iran war and terrorist attacks with sarin in the Tokyo subway. Moreover, there is evidence that a large number of soldiers participating in the 1991 Gulf War has been exposed to OP AChE inhibitors. It is estimated that about 100,000 personnel came in contact with low levels of nerve agents, when munitions at the Khamisiyah bunker in Southern Iraq, containing 8.5 tons of cyclosarin and sarin, detonated in March 1991. In addition, over 41,000 service members were exposed to high levels of OPC pesticides sprayed to contain vector-borne diseases. Toxicological appraisal of the resulting consequences (gulf war syndrome) is further complicated by the fact that 250,00 soldiers received the carbamate pyridostigmine bromide prophylactically to protect them from imminent nerve agent exposure. It is also assumed that nerve agents have been released during the Syrian conflict, since sarin has been detected in environmental samples of rocket impact sites as well as hair, urine and blood of selected patients. 1,400 fatalities were reported in Ghouta in August 2013, and another 80 civilians were killed in February 2017 when Khan Sheikhon in Northern Syria was attacked. OP toxicity is due to inhibition of AChE, the enzyme terminating the synaptic action of acetylcholine, resulting in a cholinergic crisis, which can be fatal due to respiratory failure and/or generalized seizures. Since standard therapy with atropine and established oxime-type enzyme reactivators (pralidoxime, obidoxime) is unsatisfactory, new bispyridinium (K-) oximes have been synthesized at the University of Hradec Kralove, Czech Republic. Better therapeutic results are obtained when reversible AChE inhibitors are administered before OP exposure (1). We have previously tested the prophylactic efficacy of 5 known AChE inhibitors (physostigmine, pyridostigmine, ranitidine, tacrine, K-27), when given as pretreatment before OP exposure. Best prophylactic efficacy was observed for the experimental oxime K-27. The present study was undertaken to compare the efficacy of two established (pralidoxime, obidoxime) and five experimental K-oximes (K-48, K-53, K-74, K-75, K-203) with that of the established pretreatment compound pyridostigmine to protect from the toxic effects of the OP paraoxon.AChE inhibitory activity of the two established, five experimental K-oximes and pyridostigmine was quantified in vitro by determining their IC50, using Worek’s (2) modification of Ellman’s technique on rat erythrocyte AChE. The relative risk of death (RR) over time was estimated by Cox (3) survival analysis in rats. There were 9 experimental groups. The first group was administered the OP paraoxon at 3 dosages:  1 µmol= 272 µg (1.05 mg/kg average body weight ≈ LD70), 2 µmol= 544 µg (2.11 mg/kg average body weight), 3 µmol= 816 µg (3.16 mg/kg average body weight), diluted in 500 µl saline solution. Groups 2-9 first received i.p. injections of pyridostigmine or the oxime diluted in 500 µl saline solution) at equitoxic dosages (25% of LD01, see Table 1) and thereafter (30 min later) a paraoxon injection at the same 3 dosages. The OP and the oxime were injected at anatomically distinct sites, thereby minimizing the risk of interaction between the OP, the AChE inhibitor and the oxime in the peritoneal cavity. Additional control groups received only the oxime, but no OP injections. The animals were monitored for 48 hours, and mortality was recorded at 10 min, 30 min, 1, 2, 3, 4, 24 and 48 hours. Throughout the entire experiments, the principles outlined in the “Guide for the Care and Use of Laboratory Animals” (National Academy of Sciences’ Institute for Laboratory Animal Research) were observed. All studies were performed with the approval of the Institutional Review Board (FMHS Animal Research Ethics Committee; AE/18/09). AChE inhibition: In vitro testing (Table 1, last column) showed that pyridostigmine was the strongest AChE-inhibitor (IC50= 0.33 µM), followed by K-74 (IC50= 66 µM), K-53 (IC50= 83 µM), K-75 (IC50= 101 µM) and obidoxime (IC50= 193 µM). Pralidoxime (IC50= 412 µM) and K-48 (IC50= 643 µM) only weakly inhibited rat erythrocyte AChE activity. Mortalities: Survival of the experimental animals depended both upon the substance used for pretreatment and the paraoxon dosage. In contrast, the mortality rate of control rats that had only received equitoxic doses of the pretreatment compounds (pyridostigmine, pralidoxime, obidoxime, K-48, K-53, K-74, K-75, K-203), but no paraoxon, was 0%, i.e. all rats survived.Survival analysis: Fig. 2a shows the relative risk (RR) of death at the eight time points (10 min, 30 min, 1, 2, 3, 4, 24 and 48 h), estimated by Cox analysis in pretreated animals. It was compared with animals that had received paraoxon alone, but no pretreatment (no treatment group: RR = 1), and was adjusted for paraoxon dose (high/low). Statistical comparison (Fig. 2b) between the different pretreatment regimens was performed on the cumulative relative risk, i.e. the area under the RR time curve.Based on these data, best protection from paraoxon-induced mortality was observed after pretreatment with the experimental oxime K-48 (RR=0.10), which was significantly (p ≤ 0.05) more efficacious than pretreatment with K-74 (RR=0.21), pralidoxime (RR=0.26) and pyridostigmine (RR=0.31). Obidoxime (RR=0.13) was the second most efficacious compound, being statistically significantly better than pralidoxime and K-74. Marked reduction in mortality (to 20-26%) was also achieved by pretreatment with K-203 (RR=0.20), K-74 (RR=0.21), K-75 (RR=0.24) and pralidoxime (RR=0.26). K-53 (RR=0.30) and pyridostigmine (RR=0.31) were the least efficacious compounds, but still statistically significantly reduced the relative risk of death (Fig. 2b). ResultsReferences[1] Lorke, D.E. & Petroianu, G.A. (2019) Reversible cholinesterase inhibitors as pretreatment for exposure to organophosphates. J Appl Toxicol 39:101-116. [2] Worek, F., Mast, U., Kiderlen, D., Diepold, C. & Eyer, P. (1999) Improved determination of acetylcholinesterase activity in human whole blood. Clin Chim Acta, 288, 73-90.[3] Cox, D.R. (1972) Regression models and life tables. J R Statist Soc B, 34, 189-220.[4] Lorke, D.E. & Petroianu, G.A. (2009) Minireview: does in-vitro testing of oximes help predict their in-vivo action after paraoxon exposure? J Appl Toxicol, 29, 459-469.[5] Lorke, D.E., Hasan, M.Y., Nurulain, S.M., Sheen, R., Kuça, K. & Petroianu, G.A. (2007) Entry of two new asymmetric bispyridinium oximes (K-27 and K-48) into the rat brain: comparison with obidoxime. J Appl Toxicol, 27, 482-490.The authors gratefully acknowledge the generous funding by the Sheikh Hamdan Bin Rashid Al Maktoum Award for Medical Science and the support by grant MH CZ-DRO (UHHK, 00179906).ConclusionsThe five experimental oximes (K-48, K-53, K-74, K-75, K-203), the two established oximes (pralidoxime, obidoxime) and the carbamide pyridostigmine all afford protection from paraoxon-induced mortality, when given as pretreatment. Best outcome is achieved if K-48 is administered prophylactically before paraoxon exposure. Our data also support the hypothesis that protective efficacy is unrelated to AChE inhibition and indicate that the tested experimental oximes may be considered promising alternatives to the FDA-approved pretreatment compound pyridostigmine. AbstractThe present study was undertaken in order to determine if established or experimental K-oximes can protect from OP (paraoxon) toxicity. Rats that received only the OP were compared with those that had received oxime pretreatment before OP exposure. Best protection from paraoxon-induced mortality was observed after pretreatment with K-48. The other oximes were significantly less efficacious, but still significantly reduced the RR compared to the no-treatment group (paraoxon only). The FDA-approved agent for prophylaxis was least efficacious. The protective efficacy was unrelated to AChE inhibition.Fig. 1: Chemical structures of the organophosphorus compound paraoxon, the investigated carbamate and oxime-type acetylcholinesterase (AChE) inhibitors evaluated as potential prophylactic agents. The carbamate pyridostigmine is a potent cholinesterase inhibitor which does not penetrate the blood-brain barrier. It is the only FDA-approved compound for prophylaxis if soman exposure is imminent. K-48, K-53, K-74, K-75, and K-203 belong to a group of newly-developed oxime-type AChE reactivators with promising in vitro and in vivo characteristics. The oximes pralidoxime (2-PAM) and obidoxime are already employed clinically. Oximes, in addition to reactivating phosphylated AChE, also weakly inhibit AChE activity. In our experiment, pralidoxime and obidoxime were administered as chlorides, all K-oximes as bromidesTable 1: Chemical and biological parameters of the investigated carbamate (pyridostigmine) and oximes (pralidoxime, obidoxime, K-48, K-53, K-74, K-75 and K-203) tested as pretreatment agents. Column 2 lists their molecular weight, columns 3-5 the doses injected intraperitoneally (i.p.) for pretreatment before paraoxon exposure. Values are given in µmol/animal (column three), mg/animal (column four) and in mg/kg average body weight (column five). The injected dose is approximately ¼ the LD01. Column 6 lists their LD50 and LD01 values for i.p. application in rats (4). Column 7 lists their IC50 values for AChE inhibition tested on human (pyridostigmine) and rat (remaining values) red blood cells. NA: not assessed. Fig. 2a: Relative risks (RR) of death estimated by (Cox, 1972) analysis in animals exposed to intraperitoneal (i.p.) injections of paraoxon and adjusted for paraoxon dose (high/low) for each of the time points examined (10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 24 h, 48 h). The protective effect of pyridostigmine, two established oximes (pralidoxime, obidoxime) and five experimental oxime-type AChE reactivators (K-48, K-53, K-74, K-75 and K-203), administered prophylactically 30 min before paraoxon exposure, was compared with no pretreatment (paraoxon alone, RR = 1). The injected dose of the compounds used as pretreatment was approximately ¼ of the LD01. Significant protection was achieved by all compounds tested. The most efficacious compounds were K-48 and obidoxime, reducing cumulative mortality to about 10% (RR= 0.10 - 0.13). 2b: table to the right lists the cumulative relative risks (RR) of death, including 95% confidence interval (CI) assessed by determining the area under the RR-time curve. Listed are mean values ± standard deviations (SD). Statistical differences compared to the reference group (only paraoxon and no pretreatment) were tested by the Mann-Whitney U-Test.a b

Experimental Oximes as Organophosphate Preexposure Prophylaxis.  Lessons from the Gulf War Syndrome

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Experimental Oximes as Organophosphate Preexposure Prophylaxis. Lessons from the Gulf War Syndrome

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