Compared efficacy of diazepam or avizafone to prevent soman-induced electroencephalographic disturbances and neuropathology in primates: relationship to plasmatic benzodiazepine pharmacokinetics.

We performed an experiment to characterize the toxicity of soman in cynomolgus monkeys in which organophosphorus intoxication was followed by treatment with either the current three-drug therapy atropine/pralidoxime/diazepam or a combination of atropine/pralidoxime/avizafone, avizafone being the water soluble prodrug of diazepam. Clinical, electrophysiological, and histological approaches were combined. When benzodiazepines were injected at the similar molar dose of 0.7 micromol/kg, the protection against soman toxicity was better with the atropine/ pralidoxime/diazepam combination than with the atropine/pralidoxime/avizafone one. Pharmacokinetic studies demonstrated that this difference of efficacy could be explained by a lower plasmatic load of diazepam obtained after injection of avizafone at 0.7 micromol/kg, compared to the administration of diazepam at the same molar dose. Moreover, after injection of avizafone, plasmatic levels of diazepam were achieved faster and declined more rapidly than after administration of diazepam. Compared to diazepam given at a dose of 0.7 micromol/kg, injection of 1 micromol avizafone/kg gave a similar plasmatic load of benzodiazepine, but with a lower time to maximum plasma concentration (tmax) and a higher maximum plasma concentration (Cmax) for plasmatic diazepam. We therefore went on to demonstrate that administration of the atropine/pralidoxime/avizafone combination at a dose 1 micromol benzodiazepine/kg to intoxicated monkeys afforded electrophysiological and histological protection similar to that obtained after administration of atropine/pralidoxime/diazepam at a dose of 0.7 micromol diazepam/kg. Reflections on the possible incorporation of avizafone in three-drug emergency treatment are presented.

Memory impairment after soman intoxication in rat: correlation with central neuropathology. Improvement with anticholinergic and antiglutamatergic therapeutics.

The effects of soman, a potent irreversible inhibitor of acetylcholinesterase, on central neuropathology in rats were studied in relation with subsequent spatial memory impairments. In a first step, it was found that, without treatment, neuropathology and learning impairment were observed only in rats which experienced convulsions. Then, treatment consisting of atropine sulfate, and/or TCP and/or NBQX was administered to intoxicated animals at infraanticonvulsant doses to obtain a graded subsequent neuropathology and to appreciate an eventual relation between neuropathology and spatial memory impairment. Thus, a correlation between neuropathology in the hippocampal CA1 region and spatial learning performance was found, the degradation of performance of rat being directly related to the amplitude of their neural damage. A threshold was emphasized : below a certain degree of neural loss, no memory impairment was found. Only treatment with tritherapy (atropine + TCP + NBQX) was able to improve the different parameters of spatial learning, despite no effect on the convulsions of the animals.

Acute soman poisoning in primates neither pretreated nor receiving immediate therapy: value of gacyclidine (GK-11) in delayed medical support.

Organophosphorus (OP) nerve agents are still used as warfare and terrorism compounds. Classical delayed treatment of victims of organophosphate poisoning includes combined i.v. administration of a cholinesterase reactivator (an oxime), a muscarinic cholinergic receptor antagonist (atropine) and a benzodiazepine anticonvulsant (diazepam). The objective of this study was to evaluate, in a realistic setting, the therapeutic benefit of administration of GK-11 (gacyclidine), an antiglutamatergic compound, as a complement to the above therapy against organophosphate poisoning. Gacyclidine was injected (i.v.) in combination with atropine/diazepam/pralidoxime at man-equivalent doses after a 45- or 30-min latency period to intoxicated primates (2 LD50). The effects of gacyclidine on the animals' survival, electroencephalographic (EEG) activity, signs of toxicity, recovery after challenge and central nervous system histology were examined. The present data demonstrated that atropine/diazepam/pralidoxime alone or combined with gacyclidine did not prevent signs of soman toxicity when treatment was delayed 45 min after poisoning. Atropine/diazepam/pralidoxime also did not control seizures or prevent neuropathology in primates exhibiting severe signs of poisoning when treatment was commenced 30 min after intoxication. However, in this latter case, EEG recordings revealed that additional treatment with gacyclidine was able to stop soman-induced seizures and restore normal EEG activity. This drug also totally prevented the neuropathology observed 5 weeks after soman exposure in animals treated with atropine/diazepam/pralidoxime alone. Overall, in the case of severe OP-poisoning, gacyclidine represents a promising adjuvant therapy to the currently available polymedication to ensure optimal management of organophosphate poisoning in man. This drug is presently being evaluated in a human clinical trial for a different neuroprotective indication. However, it should always be kept in mind that, in the case of severe OP-poisoning, medical intervention must be conducted as early as possible.

Heat stress, even extreme, does not induce penetration of pyridostigmine into the brain of guinea pigs.

Stress due to forced swimming was recently shown to allow penetration of pyridostigmine (PYR) into the brain of mice. Accordingly, it was suggested that in troops exposed to emotional stress under conditions of war, as during the Gulf War, the BBB may have unexpectedly become permeable to PYR thus leading to an increased frequency of CNS symptoms. In this study, the entry of PYR into the brain was investigated in guinea pigs subjected to different heat stress levels. In a first group, guinea pigs were maintained at room temperature for 2 hours, their core temperature remaining stable at about 39.8 degrees C. In a second group, animals were placed in a climatic chamber in order to keep their core temperature at 41.5 degrees C for 2 hours. In a third group, animals were subjected to a high ambient temperature (42.6 degrees C) during about 2 hours and developed heatstroke symptoms, their core temperature progressively increasing and reaching around 44.3 degrees C. In each group, the stress of the animals was assessed by measuring the increase of plasma cortisol level. PYR (0.2 mg/kg, s.c.) was injected 90 minutes after beginning the experiment. Penetration of the drug into the brain was examined by measurement of acetylcholinesterase (AChE) activity in the cortex, the striatum and the hippocampus of the animals 30 minutes after PYR administration. A passage of this drug into the brain was also evaluated autoradiographically after i.v. injection of tritiated PYR 90 minutes after the beginning of the experiment (100 microCi/animal). Whatever the group examined, no entry of PYR into the CNS could be detected. Exposure to an ambient temperature at 42.6 degrees C for 2 hours resulted by itself in a partial inhibition of cerebral AChE activity. Our results, which agree with previous data obtained in humans exposed to heat stress, are opposite to the recent research showing a central passage of PYR in mice following a forced swim stress test. This demonstrated that the penetration of PYR into the brain of rodents under stress depends on the experimental conditions used (animal species, nature of the stressor, etc.). Extrapolations to humans of results primarily obtained in rodents about central passage of a drug under stress must thus be done very carefully.

Nerve agent poisoning in primates: antilethal, anti-epileptic and neuroprotective effects of GK-11.

Organophosphorus nerve agents are still in use today in warfare and as terrorism compounds. Classical emergency treatment of organophosphate poisoning includes the combined administration of a cholinesterase reactivator (an oxime), a muscarinic cholinergic receptor antagonist (atropine) and a benzodiazepine anticonvulsant (diazepam). However, recent experiments with primates have demonstrated that such treatment, even when administered immediately after organophosphate exposure, does not rapidly restore normal electroencephalographic (EEG) activity and fails to totally prevent neuronal brain damage. The objective of this study was to evaluate, in a realistic setting, the therapeutic benefit of administration of GK-11 (gacyclidine), an antiglutamatergic compound, as a complement to the available emergency therapy against organophosphate poisoning. GK-11 was injected at a dose of 0.1 mg/kg (i.v) after a 45-min latency period to heavily intoxicated (8 LD50) primates. Just after intoxication, man-equivalent doses of one autoinjector containing atropine/pralidoxime/diazepam were administered. The effects of GK-11 were examined on survival, EEG activity, signs of toxicity, recovery after challenge and central nervous system histology. The present data demonstrate that treatment with GK-11 prevents the mortality observed after early administration of classical emergency medication alone. EEG recordings and clinical observations also revealed that GK-11 prevented soman-induced seizures and motor convulsions. EEG analysis within the classical frequency bands (beta, theta, alpha, delta) demonstrated that central activity was totally restored to normal after GK-11 treatment, but remained profoundly altered in animals receiving atropine/pralidoxime/diazepam alone. GK-11 also markedly accelerated clinical recovery of soman-challenged primates. Lastly, this drug totally prevented the neuropathology observed 3 weeks after soman exposure in animals treated with classical emergency treatment alone. GK-11 represents a promising adjuvant therapy to the currently available emergency polymedication to ensure optimal management of organophosphate poisoning in man. This drug is presently being evaluated in a human clinical trial for a different neuroprotective indication.

Absorption kinetics of a 14C-labelled Escherichia coli extract after oral administration in mice.

The fate of orally administered total 14C-labelled Escherichia coli extract (TEC, OM-89) was compared to that of its major 14C-labeled high molecular weight fractions (HEC, > 30 kD) in mice. High molecular weight substances (> 30 kD) were observed in blood 1 h after oral administration of the products, TEC and HEC. This suggests absorption from the digestive tract of the total E. coli extract. Blood clearance after 24 h indicates that the product was rapidly eliminated or taken up by the tissues. Its highest organ distribution among the measured organs was found in the liver, followed by the spleen and the Peyer's patches. Low molecular weight fractions (LEC, < 30 kD) were also used as control. This pilot study provides a rationale for in vivo pharmacological investigations with oral administration of the E. coli immunomodulating extract OM-89.

GK 11: promising additional neuroprotective therapy for organophosphate poisoning.

Recent experiments with primates have demonstrated that treatment with atropine/pralidoxime/diazepam, even if administered immediately after organophosphate exposure, does not totally prevent neuronal brain damage. Using primates, we have studied, for the first time, the ability of GK-11 (gacyclidine), an antiglutamatergic drug in the process of agreement for human use, given as an additional therapy, to counteract the neuropathology due to organophosphate exposure that persists after classical treatment with oxime/atropine/benzodiazepine. We have also examined the recovery of the organophosphate-intoxicated primates. Male Cynomolgus monkeys were pretreated 1 hour before poisoning with pyridostigmine, then intoxicated with 8 LD50 of soman and immediately treated with the combination pralidoxime/atropine/diazepam. Some of the animals also received GK-11 at 0.01; 0.03 or 0.1 mg/kg (i.v.) 10 minutes after soman challenge. Recovery of the primates (reflexes, movements, feeding) and the neuropathological changes that occurred three weeks after intoxication (histological examinations and neuronal cell density measurement) were compared in GK-11-treated and control animals. At all doses tested, GK-11 prevented the neuronal rarefaction of the frontoparietal cortex that was observed in soman-intoxicated animals that received only oxime/atropine/diazepam. Moreover, the 0.01 mg/kg dose of GK-11 improved the early recovery of intoxicated primates from 1 day after intoxication. In the view of the most effective management of organophosphate intoxication that is currently available, GK-11 thus appears to be a promising additional neuroprotective therapy. This drug is presently being evaluated in a human clinical trial for a different neuroprotective indication.

Efficacy of huperzine in preventing soman-induced seizures, neuropathological changes and lethality.

Huperzine A (HUP) is a potent reversible inhibitor of acetylcholinesterase (AChE) that crosses the blood-brain barrier. Its ability to prevent seizures and subsequent hippocampal neuropathological changes induced by the organophosphate soman was studied in guinea pigs. Results were compared to guinea pigs treated with pyridostigmine (PYR, 0.2 mg/kg, subcutaneously). HUP pretreatment at 0.5 mg/kg, intraperitoneally, totally prevented seizures and ensured the survival of all animals for 24 h after intoxication. Hippocampal tissue was then free of any neuronal damage. Comparatively, all animals pretreated with PYR exhibited epileptic activity after soman poisoning and five of six animals died. Examination of the hippocampus of the only surviving guinea pig pretreated with PYR showed extensive neuropathological changes. Although HUP or PYR induced similar inhibitions of blood AChE activity, only HUP pretreatment led to a decrease in central AChE activity. In binding studies on guinea-pig brain homogenates, HUP had no affinity for muscarinic, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and gamma-aminobutyric acid (GABA)A receptors and only a very low one for N-methyl-D-aspartate (NMDA) receptors. In conclusion, HUP, unlike PYR, protects against soman-induced convulsions and neuropathological changes in the hippocampus. This efficacy seems to be related to a protection by HUP of both peripheral and central stores of AChE.

Time course and regional expression of C-FOS and HSP70 in hippocampus and piriform cortex following soman-induced seizures.

The time course of induction of the proto-oncogene c-fos and the inducible heat shock hsp70 gene was studied from 5 minutes to 24 hours at both transcriptional (c-fos and hsp70 mRNA) and translational levels (C-FOS and HSP72 proteins) in the rat hippocampus and piriform cortex (Pir) after soman-induced seizures. Induction of c-fos was noticed as early as 5 minutes after seizures onset in all fields of hippocampal formation (CA1, CA3, CA4, and dentate gyrus) and in piriform cortex. The most intense induction was observed in piriform cortex. A sustained activation of c-fos occurred in Pir and in CA1, CA3, and CA4 areas of hippocampus. Nevertheless, histological analysis showed rare affected neurons in CA4, whereas damage was severe in Pir and in CA1 and CA3 hippocampal subfields. Induction of hsp70 mRNA occurred but was delayed in all areas previously exhibiting c-fos expression. Nevertheless HSP72 protein was never expressed in the structures where injury was high (i.e., CA1 and piriform cortex) and mainly occurred in the less damaged structure (i.e., CA4 area of hippocampus). Regional expression of glial fibrillary acidic protein mRNA was also studied in order to exclude an astroglial origin of the c-fos and hsp70 gene inductions. Our results demonstrated that after soman induced-seizures 1) there was no strict correlation between time course or intensity of neuronal c-fos induction and subsequent neuropathology, and 2) the most lesioned areas did not express HSP72 protein in spite of intense mRNA induction, suggesting that transcriptional and translational events for hsp70 gene might vary according to the severity of seizure insult.

Early regional changes of GFAP mRNA in rat hippocampus and dentate gyrus during soman-induced seizures.

We investigated the time course of GFAP levels in the hippocampal formation during the first 24 h following soman intoxication in rats. GFAP mRNA expression was detected by in situ hybridization. Intense GFAP mRNA expression was present in the molecular layer of the dentate gyrus as early as 6 h after intoxication. This expression was comparatively lower in other dentate gyrus layers and hippocampal CA1, CA3 and CA4 areas and seemed to be related to excessive neuronal activity. Histopathological examination demonstrated that GFAP expression in dentate gyrus is not correlated with lesions. The high astrocytic reactivity in the molecular layer of the dentate gyrus is discussed in relation to the maintenance of the homeostasis of glutamate and of synaptic plasticity in this area during soman intoxication.

Use of digoxigenin-labeled RNA probe to test hepatitis A virus antiviral drugs.

A nucleic acid hybridization assay was used to evaluate inhibitory activity of antiviral compounds against hepatitis A virus (HAV) in cell culture and compared to radioimmunoassay by analysis of variance procedure. The 5' genomic end of the HM-175 strain was used as digoxigenin-labeled RNA probe. Dot-blot examination showed a reduction of detectable HAV RNA in infected cells when treated with amphotericin B. An antiviral dose-effect was shown by statistical analysis of densitometric measures of hybridization signals. Comparison between molecular hybridization assay and radioimmunoassay by analysis of variance procedure showed the equivalence of both methods. Data previously obtained on selected drugs by antigen and infectious titres determinations were confirmed by hybridization assay and make possible digoxigenin-labeled RNA probe use to measure an antiviral dose-effect for screening of hepatitis A antiviral compounds.

Modulation of soman-induced neuropathology with an anticonvulsant regimen.

Rat hippocampus and piriform cortex were examined for pathological changes 48 hours after exposure to a convulsant dose of soman. Animals were treated with a low dose of atropine just after soman and were then injected, after 10 or 40 minutes of seizures, with both the anticonvulsant drugs NBQX and TCP. Atropine given alone counteracted the extensive neuronal loss due to soman in both areas without prevention of neuronal suffering. Comparatively, the complete anticonvulsant regimen, given before 40 minutes of seizures, totally prevented hippocampal soman-induced neuropathology. Neurones of piriform cortex were still suffering whatever the time of injection of the drugs. This emphasizes the need for a rapid and definitive anticonvulsant treatment just after soman intoxication to block the subsequent neurotoxic effect of nerve-agent exposure.

Effects of neutron-gamma irradiation on striatal D1 and D2 receptor distribution.

The early effects of neutron irradiation on the striatal D1 and D2 dopaminergic receptor distribution were investigated by quantitative receptor autoradiography. One hour after exposure at the dose of 8.4 Gy, an increase of D1 (+21%) and D2 (+25%) receptor density was observed in the striatum, located at the most anterior levels, containing the richest plexus of dopaminergic fibers afferent from the substantia nigra. Regional differences in changes of D1 and D2 receptor density were observed. This up-regulation could contribute to the development of early radio-induced neuro-vegetative syndrome.

[Detection of hepatitis A virus by riboprobe labeled with digoxigenin: comparison of methods]. / Détection du virus de l'hépatite A par ribosonde marquée à la digoxigénine: comparaison de méthodes.

A riboprobe (RNA probe), corresponding to the 5' end of the HM175 hepatitis A virus (HAV) genome, was synthetized in vitro and was digoxigenin-labeled. Then the riboprobe was used to detect the CF53 HAV strain. Conditions of virus denaturation (with or without SDS and proteinase K, timing of assay) to release viral RNA were tested by dot-blot hybridization on a ten fold dilution of HAV suspension. Densitometric measures of dot-blot spots allowed to appreciate optimization of the method. Sensitivity of hybridization was compared with sensitivity of radioimmunoassay (RIA) and cell culture methods. Hybridization signals and scale of HAV suspension were consistent when 0.05% SDS, 0.17 micrograms/ml Proteinase K, 37 degrees C, 30 mn or 3 hours are used. 8.10(2) TCID50 HAV was detected by hybridization with digoxigenin-labeled RNA probes. Detection threshold was the same as radioimmunoassay and lower comparatively to cell culture.

Influence of a bacterial extract on antigen presentation and protection against experimental infections.

Although substances known as immunodulators are usually used to stimulate nonspecific immunity, their mode of action is not well understood. In an effort to clarify this mechanism, we investigated the effect of a lyophilized bacterial extract (Broncho-Vaxom) on experimental infections, on normal or irradiated mice, and on antigen processing.

Comparative immunogenicity of live influenza viruses and their solubilized neuraminidases: results of mouse protection experiments.

Antigenicity and immunogenicity of three influenza virus strains A/PR/8/34 (H1N1). A/Hong Kong/1/68 (H3N2) and A/Port Chalmers/1/73 (H-3H2) were assayed comparatively with their corresponding neuraminidase isolated by proteolysis, and with the recombinant virus X-42 (Heq1 N2). It was concluded that intranasal immunization of mice with live virus induced heterologous immunity. Except in homologous neuraminidase-vaccinated mice, the subunit always was shown less effective and demonstrated a significantly lower antibody response than the corresponding whole virus.

["In vitro" interactions between influenza virus and mouse lung alveolar macrophages (author's transl)]. / Interactions entre le virus grippal et le macrophage alvéolaire de souris: étude in vitro.

Interactions between influenza virus A/PR/8/34 (H0N1) and Balb/c mouse lung alveolar macrophages have been studied in vitro. One day after initiation of alveolar macrophage culture in 35 mm Falcon dishes, the virus suspension was allowed to adsorb to the cells for 1 h. Detachment of cells from the plastic substrate, morphological changes in adherent cells and decreased phagocytosis of heat-killed Candida albicans occured slowly as compared to control cultures. These facts appeared to be directly correlated to the concentration of viruses in the inoculum. Data yielded by virus titrations, electron microscopy and immunofluorescence suggest that mouse lung alveolar macrophages are able to take up a large amount of viral particles and inhibit their replication, allowing only an abortive viral cycle.

[Pinguecula and pterygium: histologic and electron microscopic study (author's transl)]. / Pinguecula et pterygion: étude histopathologique et ultrastructurale.

Two pingueculae combined with pterygia were studied by light and electron microscopy. Hyaline degeneration of the collagen, dark staining granular, von Kossa negative concretions and elastotic material were observed in both conditions together with marked changes in the fibroblasts, endothelial cells, pericytes and the basement membrane of conjunctival capillaries and small veins. The elastotic material is similar to that observed in solar elastosis, where the collagen fibers are less severely damaged. Chronic sun exposure of the pericorneal conjunctiva may damage endothelial cells primarily and disturb vascular exchanges. This would result in accelerated degeneration and regeneration of endothelial cells, in thickening of the basement membrane and, secondarily, disturbed metabolism of fibroblasts with alterations of the collagen and elastic fibers.

[Effect of trace elements on the hydroxylation of benzo(a)pyrene]. / Etude de l'influence des éléments traces sur l'hydroxylation du benzo(a)pyrène

The trace elements are surrounding factors which are able to act on the yield of benzo(a)pyrene hydroxylation reaction of the hepatic tissue. This action was studied for each element and for various concentrations. The action of these elements may occurred to arylhydrocarbon hydroxylase by activating or inhibiting it, to epoxyde hydrase or to glutathione S epoxydase, favouring or inhibiting the way epoxyde dihydrodiol, and finally to arylhydrocarbon hydroxylase induction. Our results, obtained in vitro on hepatic tissue with many metallic salts, showed that some trace elements may have a cocarcinogenic action.