Oximes TMB-4 Research Articles

Repetitive antidotal treatment is crucial in eliminating eye pathology, respiratory toxicity and death following whole-body VX vapor exposure in freely moving rats.

Exposure to the chemical warfare nerve agent VX is extremely toxic, causing severe cholinergic symptoms. If not appropriately treated, death ultimately ensues. Based on our previously described whole-body vapor exposure system, we characterized in detail the clinical outcome, including respiratory dynamics, typical of whole-body exposure to lethal doses of VX vapor in freely moving rats. We further evaluated the efficacy of two different antidotal regimens, one comprising a single and the other repeated administration of antidotes, in countering the toxic effects of the exposure. We show that a 15min exposure to air VX concentrations of 2.34-2.42mg/m3 induced a late (15-30min) onset of obvious cholinergic signs, which exacerbated over time, albeit without convulsions. Marked eye pathology was observed, characterized by pupil constriction to pinpoint, excessive lacrimation with red tears (chromodacryorrhea) and corneal damage. Respiratory distress was also evident, characterized by a three-fourfold increase in Penh values, an estimate of lung resistance, and by lung and diaphragm histological damage. A single administration of TAB (the oxime TMB-4, atropine and the anticholinergic and antiglutamatergic benactyzine) at the onset of clinical signs afforded only limited protection (66% survival), with clinical deterioration including weight loss, chromodacryorrhea, corneal damage, increased airway resistance and late death. In contrast, a combined therapy of TAB at the onset of clinical signs and repeated administration of atropine and toxogonin (ATOX) every 3-5h, a maximum of five i.m. injections, led to 100% survival and a prompt recovery, accompanied by neither the above-described signs of eye pathology, nor by bronchoconstriction and respiratory distress. The necessity of recurrent treatments for successful elimination of VX vapor toxicity strongly supports continuous penetration of VX following termination of VX vapor exposure, most likely from a VX reservoir formed in the skin due to the exposure. This, combined with the above-described eye and respiratory pathology and absence of convulsions, are unique features of whole-body VX vapor exposure as compared to whole-body vapor exposure to other nerve agents, and should accordingly be considered when devising optimal countermeasures and medical protocols for treatment of VX vapor exposure.

A physiologically based pharmacokinetic model for the oxime TMB-4: simulation of rodent and human data

Multiple oximes have been synthesized and evaluated for use as countermeasures against chemical warfare nerve agents. The current U.S. military and civilian oxime countermeasure, 2-[(hydroxyimino)methyl]-1-methylpyridin-1-ium chloride (2-PAM), is under consideration for replacement with a more effective acetylcholinesterase reactivator, 1,1'-methylenebis{4-hydroxyiminomethyl}pyridinium dimethanesulfonate (MMB-4). Kinetic data in the scientific literature for MMB-4 are limited; therefore, a physiologically based pharmacokinetic (PBPK) model was developed for a structurally related oxime, 1,1'-trimethylenebis{4-hydroximinomethyl}pyridinium dibromide. Based on a previous model structure for the organophosphate diisopropylfluorophosphate, the model includes key sites of acetylcholinesterase inhibition (brain and diaphragm), as well as fat, kidney, liver, rapidly perfused tissues and slowly perfused tissues. All tissue compartments are diffusion limited. Model parameters were collected from the literature, predicted using quantitative structure-property relationships or, when necessary, fit to available pharmacokinetic data from the literature. The model was parameterized using rat plasma, tissue and urine time course data from intramuscular administration, as well as human blood and urine data from intravenous and intramuscular administration; sensitivity analyses were performed. The PBPK model successfully simulates rat and human data sets and has been evaluated by predicting intravenous mouse and intramuscular human data not used in the development of the model. Monte Carlo analyses were performed to quantify human population kinetic variability in the human evaluation data set. The model identifies potential pharmacokinetic differences between rodents and humans, indicated by differences in model parameters between species. The PBPK model can be used to optimize the dosing regimen to improve oxime therapeutic efficacy in a human population.

Magnesium sulfate treatment against sarin poisoning: dissociation between overt convulsions and recorded cortical seizure activity

Sarin, a potent organophosphate cholinesterase inhibitor, induces an array of toxic effects including convulsions. Many antidotal treatments contain anticonvulsants to block seizure activity and the ensuing brain damage. Magnesium sulfate (MGS) is used to suppress eclamptic seizures in pregnant women with hypertension and was shown to block kainate-induced convulsions. Magnesium sulfate was evaluated herein as an anticonvulsant against sarin poisoning and its efficacy was compared with the potent anticonvulsants midazolam (MDZ) and caramiphen (CRM). Rats were exposed to a convulsant dose of sarin (96 μg/kg, im) and 1 min later treated with the oxime TMB4 and atropine to increase survival. Five minutes after initiation of convulsions, MGS, CRM, or MDZ were administered. Attenuation of tonic-clonic convulsions was observed following all these treatments. However, radio-telemetric electro-corticography (ECoG) monitoring demonstrated sustained seizure activity in MGS-injected animals while this activity was completely blocked by MDZ and CRM. This disrupted brain activity was associated with marked increase in brain translocator protein levels, a marker for brain damage, measured 1 week following exposure. Additionally, histopathological analyses of MGS-treated group showed typical sarin-induced brain injury excluding the hippocampus that was partially protected. Our results clearly show that MGS demonstrated misleading features as an anticonvulsant against sarin-induced seizures. This stems from the dissociation observed between overt convulsions and seizure activity. Thus, the presence or absence of motor convulsions may be an unreliable indicator in the assessment of clinical status and in directing adequate antidotal treatments following exposure to nerve agents in battle field or terror attacks.

The antidotal efficacy of the bispyridinium oximes K027 and TMB-4 against tabun poisoning in mice

A toxic effect of highly toxic nervous agents is irreversible inhibition of vitally important enzyme acethylcholinesterase (AChE). Inhibition of AChE results in accumulation of acetylcholine (ACh) at the synaptic cleft of the cholinergic neurons, leading to overstimulation of cholinergic receptors. The highly toxic nature of tabun has been known for many years, but there are still serious limitations to the antidotal therapy. In this paper a bispyridinium compound K027 [1-(4-hydroxyiminomethylpyridinium)-3-(-4-carbamoylpyridinium) propane dibromide] was tested as potential antidote in tabun poisoned mice. Oxime TMB-4 was included for comparison. The therapeutic efficacy of applied antidotal regimens was tested as pretreatment given 15 min before tabun poisoning and/or as therapy given 1 min after tabun poisoning. Using oxime K027 (25% of its LD 50) plus atropine as both, pretreatment and therapy, we showed that this combination can protect mice 8 times better than the therapy alone. Under these experimental conditions we confirmed good antidotal efficacy of K027. Moreover, its low acute toxicity is as much as beneficial effect in contrast to high toxicity of currently used TMB-4.

Transient and reversible nephrotoxicity of sarin in rats

Organophosphate (OP) poisoning, which inhibits cholinesterase activity, leads to severe cholinergic symptoms. Effective and quick management of these symptoms is considered critical to the clinical outcome. Acute renal damage following exposure to OP insecticides has been reported. Similar complications might occur following exposure to OP nerve agents, however, this subject has been studied only sporadically. In the present study, the effect of the nerve agent sarin on renal function was examined in rats. A single dose of sarin ( approximately 0.9 LD(50)) led to a significant reduction (of 45%) in renal function during the first 2 days post exposure, as exhibited by evaluation of the glomerular filtration rate, through measuring the clearance of ( 99m)Tc-DTPA. The urine volume was reduced by 50%, the urine specific gravity increased to 104% of the control value and massive hematuria and glucosuria were recorded 24-48 h post exposure. In addition, around 60% decrease in urine electrolytes was monitored during the first 2 days following exposure, with a recovery after 8 days. Post mortem gross inspection of the bladder, 24 h post exposure, revealed severe edema and hemorrhage. Treatment with the muscarinic antagonist atropine and the oxime TMB-4, at excessive doses administered 1 min post exposure, did not prevent most renal impairments. It has been concluded that sarin caused an acute renal dysfunction, possibly accompanied by bladder damage. These impairments were reversible, recovered spontaneously within 3-8 days, and were probably related to the state of shock and hypovolemia caused by the poisoning. However, if renal impairments are left unattended, they might contribute to the overall toxic manifestation and as a result aggravate the clinical state of intoxicated casualties.

Effects of Repeated Oral Postnatal Exposure to Chlorpyrifos on Cholinergic Neurochemistry in Developing Rats

The neurochemical effects of repeated postnatal exposure to chlorpyrifos (CPS) were studied in developing rats. Rats were gavaged daily from postnatal day (PND) 1-21 with CPS in corn oil starting at 1.5 mg/kg (low dosage group) and increasing gradually to 3 mg/kg and then to 6 mg/kg (high dosage group). Brain cholinesterase (ChE) activity was significantly inhibited on PND 6, 12, 22, and 30, with maximum inhibition on PND 6 of 49 and 59% and recovering to 18 and 33% on PND 30 in the low and high dosage groups, respectively. On PND 22 and 30, 94% or greater of the inhibited ChE could not be reactivated by the oxime TMB-4 in both treatment groups, indicating aging of the phosphorylated ChE. Total muscarinic acetylcholine receptors (mAChR) were reduced in a dose-related manner on PND 12 and 22, with substantial recovery by PND 30. M1/M3 mAChR were significantly reduced on PND 6 and 12 only in the high dosage group, and on PND 22 in both groups, while M2/M4 mAChR were reduced in the high dosage group on PND 22 and 30. On PND 30 choline acetyltransferase activity and vesicular acetylcholine transporter levels were decreased by 12 and 22%, respectively, only in the high dosage group. High-affinity choline transporter levels were decreased at all time points in the high dosage group and on PND 6, 22, and 30 in the low dosage group. The results presented here demonstrate that repeated postnatal exposures to CPS result in transient reductions of mAChR and more persistent alterations of presynaptic cholinergic neurons. In addition, the long-term reduction of brain ChE activity observed following repeated postnatal exposure to CPS is attributable to permanent inactivation or "aging" of the enzyme.

Behavioral Comparison of the Oximes TMB-4, 2-PAM, and HI-6 in Rats Using Operant Conditioning

It has recently been shown that oximes can amplify the ability of cholinesterases to scavenge organophosphorus (OP) agents. Since both OP agents and oximes can disrupt performance, behavioral evaluation of bioscavenger therapies using oximes can be hindered. Therefore, we investigated the ability of three oximes, administered alone, to disrupt performance. The effects of trimedoxime bromide (TMB-4) (3.16-56.2 mg/kg), pralidoxime chloride (2-PAM) (10.0-237.1 mg/kg), and, 1-([[4-amincarbonyl)pyridino]-methoxy]-methyl)-2,4-bis[(hydroxyimino)methyl] pyridinium dichloride monohydrate (HI-6) (10.0-237.1 mg/kg) were evaluated in rats using a variable-interval 56 (VI 56) s schedule of food reinforcement. Under control conditions, the VI 56 s schedule produced a constant rate of responding (i.e., lever-pressing). All three oximes produced dose-dependent decreases in responding, and the largest doses of TMB-4 and 2-PAM produced complete or nearly complete suppression of responding in all rats. Only the largest dose of HI-6 suppressed responding. Analysis of the dose-effect functions demonstrated that TMB-4 was substantially more potent than 2-PAM, which was slightly more potent than HI-6, for producing response suppression. These results establish doses of each oxime that will not contribute to disruption of responding, and thus, facilitate future evaluation of bioscavenger therapies against OP toxicity. Published by Elsevier Science Inc., 1997

Cardiotonic drugs inhibit purified mammalian acetylcholinesterase

Oxime- and non-oxime-related drugs, as well as cardiotonic drugs (CDs), have been used to treat the effects of organophosphorus (OP) poisoning. We conducted our experiments to determine what effects CDs may have on acetylcholinesterase (AChE), and how CDs interact with other treatment drugs as well as with OP-inhibited AChE. True AChE (EC 3.1.1.7) was purified from fetal bovine serum, and enzyme activity was measured according to Ellman et al. The CDs coumingine, cassaine, proscillaridin and convallatoxin were incubated with AChE at 550 microM at pH 7.6 and 25 degrees C. The CD ouabain was incubated with AChE at 500 microM. The CDs inhibited AChE by 97%, 89%, 10%, 7% and 6%, respectively. The mean AChE activities for these experiments, except for ouabain, were significantly different (P = 0.05) from their controls, as determined by the two-tailed Student's t-test. In a separate experiment, the oxime TMB-4.2Br (100 microM), which did not inhibit AChE, increased the inhibitory effect of proscillaridin from 4% to 11% (a 3.7-fold increase). When AChE was inhibited 39% with 37 nM VX, the addition of proscillaridin increased the inhibition to 51% (a 1.3-fold increase). When TMB-4 was added to the proscillaridin- and VX-inhibited AChE mixture, the inhibition decreased from 50% to 32% (a 0.37-fold decrease), whereas TMB-4 alone added to VX-inhibited AChE decreased the inhibition from 39% to 24% (a 0.38-fold decrease). The results show that TMB-4 increases the inhibition of AChE by proscillaridin. However, TMB-4 decreases the inhibition of AChE by VX and proscillaridin combined.(ABSTRACT TRUNCATED AT 250 WORDS)

Reactivation of acetylcholinesterase inhibited by methamidophos and analogous (di)methylphosphoramidates.

Reactivation of electric eel acetylcholinesterase (AChE) inhibited by MeO(NH2)P(O)SMe (methamidophos) and by MeS(NH2)P(O)SMe was studied at pH 7.5 and 25 degrees C. The former inhibited enzyme shows a rather rapid spontaneous reactivation (t1+2 = 3.7 h); this reactivation is accelerated by 1 micrometer of the bispyridinium oximes TMB4 and obidoxine, and, to a lesser extent, by the monopyridinium oximes P2S and its 1-benzyl analogue (benzyl-P2A). The latter inhibited enzyme shows rapid aging (t1+2 = 0.6 h). Reactivation with 1 mM of the bispyridinium oximes is incomplete and reactivation with 1 mM of the monopyridinium oximes proceeds very slowly. These large differences between the properties of the two inhibited enzymes indicate that the methylthio group is the leaving group during inhibition of AChE by methamidophos. Additional support is afforded by the observation of induced aging of the former inhibited enzyme by thiourea. Upon comparison of the reactivation of AChE inhibited by methamidophos with that of AChE inhibited by an N-methyl analogue, crufomate, and an N,N-dimethyl analogue, tabun, it appears that the rate of spontaneous reactivation decreases with increasing alkylation of the P-NH2 group. Whereas benzyl-P2A is somewhat less active than P2S for reactivation of ACHE inhibited by methamidophos, it is superior to P2S for reactivation of ACHE inhibited by crufomate and also superior to P2S and to the bispyridinium oximes for AChE inhibited by tabun.

In vitro reactivation and “ageing” of tabun-inhibited blood cholinesterases : Studies with n-methylpyridinium-2-aldoxime methane sulphonate and N,N'-trimethylene bis (pyridinium-4-aldoxime) dibromide

Tabun-inhibited acetyl- and butyrylcholinesterases from human and horse blood were stored at varying conditions and the degree of reactivation after incubation with the oximes TMB-4 and P2S was measured. The influence of the concentration of the oxime upon reactivation was studied. When possible, rate constants for the reactivation were calculated and compared with rate constants for the transformation of the phosphorylated enzyme to a non-reactivable form. Reactivation of human erythrocyte and plasma cholinesterase with P2S was shown to be very slow. Reactivation with TMB-4 was more rapid, but decreasing (he concentration of TMB-4 below 10 −3M resulted in incomplete reactivation. At therapeutical concentrations of the reactivator (∼2·5 × lO −3 M) at least 75 per cent of the enzyme activity was rcstored. Phosphorylated plasma cholinesterase from human blood was shown to “age” much more rapidly than phosphorylated plasma cholinesterase from horse blood and to have a promoting effect upon the “ageing” of phosphorylated, purified horse serum cholinesterases. The “ageing” of the purified serum cholinesterase preparations was found to be slower than that of the corresponding untreated plasma enzyme.