Murray G. Hamilton

HI-6 therapy of soman and tabun poisoning in primates and rodents.

The bis-pyridinium oxime HI-6, in conjunction with atropine, was found to offer significant protection against multiple LD50 challenges with the organophosphorus compounds soman and tabun. In adult rhesus macaques, the therapeutic administration of HI-6 with atropine and diazepam protected three of four animals from the lethal effects of 5 × LD50 of soman and three of three animals from 5 × LD50 of tabun. However, when toxogonin was substituted for HI-6 in the therapeutic mixture, all three animals poisoned with 5 × LD50 of soman died. In rats, the 24 h protective ratios against tabun and soman with HI-6 were 2 and 3.5, respectively, whereas in guinea pigs these values were between 4 and 6 for both agents. No evidence was obtained for acetylcholinesterase (AChE) reactivation by HI-6 in tissue from tabun-poisoned rodents or following soman or tabun in primate plasma. The results underscore the significant therapeutic benefit of HI-6 in primates, a species specific efficacy against tabun, and argue for some mechanism of action of HI-6 at least partly unrelated to AChE reactivation.

Pharmacological evidence for an ω-conotoxin, dihydropyridine-insensitive neuronal Ca2+ channel

Inactivation of N-type voltage-sensitive Ca2+ channels (VSCC) with ω-conotoxin (ω-CgTx) in tissue obtained from chicken brain produces a concentration dependent (0.01–0.1 μM) inhibition of K−-stimulated Ca2+ influx (ΔK+) the rise in [Ca2+]1 and acetylcholine (ACh) release. In identical preparations from rat brain, Ca2+ influx and the rise in [Ca2+]1 were only marginally affected by much higher (1–10 μM) concentrations of ω-CgTx. The release of ACh, however, was inhibited to the same degree with similar amourts of ω-CgTx as those used in chicken brain. An L-type VSCC inhibitor failed to affect any of these parameters alone, or to augment the effect of ω-CgTx. The results suggest that almost all the VSCC in chicken brain are of the N type and that these channels regulate neurotransmitter release. In rat brain, on the other hand, Ca2+ channels resistant to N- or L-type blockers account for almost 75% of the measurable Ca2+ influx and rise in [Ca2+]1. The conspicuous dissociation between the regulation of Ca2+ influx and ACh release demonstrated in rat brain by using ω-CgTx, suggest that neurotransmitter release is governed by only a small proportion of strategically located N-type, ω-CgTx sensitive. VSCC in the presynaptic terminal.

Stimulation of Ca2+ influx through ATP receptors on rat brain synaptosomes: identification of functional P2X7 receptor subtypes

ATP receptors of the P2X class have previously been identified on autonomic nerve endings and on a limited population of CNS neurons. In the present study P2X receptors on mammalian cortical synaptosomes have been identified by a variety of functional and biochemical studies. In choline buffer ATP analogues caused concentration/time dependent Ca2+ influx. Relative to the effects caused by ATP, benzoylbenzoyl ATP (BzATP) was about seven times more active than ATP while 2‐me‐S‐ATP and ATPγS were much less active. α,β‐me‐ ATP and β,γ‐me‐ATP were virtually inactive. In sucrose buffer, relative to choline buffer, the activity of BzATP was more than doubled while activity in sodium buffer was reduced. Moreover, the P2X antagonists PPADS or Brilliant Blue G both significantly attenuated influx. These observations suggest the presence of P2X receptors on synaptosomes which subserve Ca2+ influx. This activity profile of the ATP analogues and the response to blocking agents are characteristic of responses of P2X7 receptors. Influx was unaffected by the VSCC inhibitors ω‐CTx‐MVIIC and (−) 202 – 791, indicating that ATP induced Ca2+ influx occurred primarily through P2X receptors. P2X7 receptor protein was identified by Western blotting and immunohistochemical staining. Purified preparations were devoid of significant concentrations of GFAP or the microglial marker OX‐42 but contained greatly enriched amounts of syntaxin and SNAP 25. The various pharmacological and biochemical studies were all consistent with the presence of functional P2X7 receptors.

Comparative protective effects of HI-6 and MMB-4 against organophosphorous nerve agent poisoning

The oximes pralidoxime (2-PAM), its dimethanesulphonate salt derivative P2S, and obidoxime (toxogonin) are currently licensed and fielded for the treatment of chemical warfare (CW) organophosphorous (OP) nerve agent poisoning. While they are effective against several of the identified threat CW OP agents, they have little efficacy against others such as soman (GD) and cyclosarin (CF). In addition, they are also significantly less effective than other investigational oximes against the nerve agent known as Russian VX (RVX). Among the oximes currently being investigated, two in particular, HI-6 (asoxime) and MMB-4 (ICD-039, methoxime) have been proposed as replacement therapies for the currently licensed oximes. HI-6 has been safely used in individuals to treat OP insecticide poisoning, as well as in human volunteers, although its efficacy against OP nerve agent poisoning in humans cannot be demonstrated due to ethical considerations. It is currently available for use in defined military settings in Canada, Sweden and the Czech Republic, and is also under development in a number of other countries. The oxime MMB-4 has not yet been studied clinically, but is fielded by the Czech Republic, and is being developed by the United States armed services as a replacement for the currently fielded 2-PAM. This review compares the effectiveness of HI-6 and MMB-4 against nerve agent threats where comparisons can be made. HI-6 has been demonstrated to be generally a superior reactivator of nerve agent inhibited enzyme, particularly with human and non-human primate derived enzyme, and has also shown better protective effects against the lethality of most OP agents in a variety of species. Both compounds appear to be clearly superior to the available oximes, obidoxime and 2-PAM.

Magnetic resonance imaging predicts neuropathology from soman-mediated seizures in the rodent

Intoxication by the organophosphate compound soman causes prolonged seizures that lead to neuropathology in the brain. This MRI-based study describes the temporal and spatial evolution of brain pathology that follows soman-induced convulsions. We observed significant decreases in apparent diffusion coefficients (ADC; 23% below control) of the hippocampus and thalamus by 12 h after soman treatment. The ADC then returned to near normal values in all regions at 24 h but declined again during the next 7 days. These data suggest that the initial cellular degradation may be resolved but is ultimately followed by regional cellular remodeling. T2 relaxa- tion values declined significantly at 12 h (37% decrease) returning to near normal values by 24 h. These data lend detail to the model suggesting that injured tissues experience an edematous influx that is resolved by 24 h. The imaging data was fully supported by histopathological comparisons where moderate cell loss and swelling within the hippocampus and piriform cortex was observed. This is the first report providing excellent temporal and spatial resolution of emerging soman-mediated, seizure-induced neuropathology using MRI with histological correlation.

Distribution of the bispyridinium oxime [14C] HI-6 in male and female rats

Female rats poisoned with multiple LD50s of soman or tabun have been shown previously to respond to the protective effects of HI-6 more positively than male rats. This present study was designed first to determine the distribution pattern and concentration of [14C] HI-6 in rats, and secondly, to determine the possibility that HI-6 might be located in high concentrations in critical tissues in the female as opposed to the male. To these ends, [14C] HI-6 was administered to groups of male and female rats and its radiolabelled distribution determined by whole body autoradiography and/or by measurement of its actual concentration, by scintillation spectrometry. The experiments were repeated in the presence of 2 × LD50 soman and supporting therapy with atropine. In both sexes, HI-6 levels were highest in the kidney, followed in order by cartilage >plasma >liver >heart >lung > diaphragm >brain and spinal cord. The relative distribution in the two sexes was confirmed by both methods and was not significantly altered in the presence of soman and atropine. The lack of a measurable difference in tissue distribution of [14C] HI-6 derived radioactivity between males and females suggested that the hormone-dependent difference in the protective effects previously observed was not due to selective accumulation of [14C] HI-6 in organs believed to be important in its therapeutic activity, such as brain or diaphragm.

Pharmacological identification of a novel Ca2+ channel in chicken brain synaptosomes

Ca2+ influx was measured in rat and chicken brain synaptosomes in the presence of a number of pharmacological tools which have recently been used to define voltage-sensitive Ca(2+)-channel (VSCC) types. In chicken brain synaptosomes. VSCCs which, because of their sensitivity to inhibition by omega-conotoxin (omega-CgTx), are thought to be exclusively N-type, the P-type VSCC polyamine inhibitor FTX (from Agelenopsis aperta venom; 1 microliters/ml), its synthetic analogue, sFTX (1-5 mM) and the polypeptides AgaIVA (IC50 0.29 microM) and omega-CgTx MVIIC (IC50 0.0022 microM) inhibited 70-100% of the measurable K+ stimulated Ca2+ influx. The prototypical N-channel VSCC inhibitor omega-CgTx GVIA (IC50 0.014 microM), Cd2+ (50 microM) and diluted venom from Hololena curta (1:2,500) also caused complete or almost complete, inhibition of Ca2+ influx. In comparable studies using rat brain synaptosomes, sFTX (1-10 mM) caused a dose-dependent reduction of Ca2+ influx, while FTX (1 microliters/ml) and AgaIVA (IC50 0.02 microM) completely inhibited Ca2+ influx. Similar to the findings in chicken synaptosomes, Cd2+ (50 microM) and H. curta (1:2,500 dilution) both inhibited K+ stimulated influx by > 80% whereas omega-CgTx (1 microM) only caused a maximum 25% inhibition. Both sFTX and its congener spermine, inhibited [125I]omega-CgTx binding to rat and chicken synaptosomal membranes. These results strongly implicate P-type channels as the major VSCC in rat brain. The results also clearly demonstrate a heretofore unrecognized, novel, FTX/AgaIVA/omega-CgTx GVIA/omega-CgTx MVIIC-sensitive VSCC in chicken brain.

Identification of noradrenaline in venom from the funnel-web spider Hololena curta

Venom from the funnel-web spider Hololena curta was added to the Krebs-Henseleit solution bathing isolated ring preparations of rat thoracic aorta, suspended in water-jacketed organ baths, for the purpose of tension recordings. Hololena curta venom at dilutions of 1:100,000 to 1:1000 caused a marked vasoconstriction, which was completely inhibited by the alpha 1 adrenoceptor antagonist prazosin (1 microm). The vasoconstriction appears to be due to the direct effects on alpha 1 adrenoceptors of a venom constituent, which we have identified using HPLC/ECD as the catecholamine, noradrenaline.