Subchronic physostigmine pretreatment in guinea pigs: effective against soman and without side effects.

The behavioral and neurophysiological effects of the subchronically administered cholinesterase-inhibitor physostigmine (PHY) (0.025 mg/kg/h) either with or without the muscarinergic antagonist scopolamine (SCO) (0.018 mg/kg/h) were determined in guinea pigs. In contrast to a single injection of PHY, subchronic application by osmotic minipumps of PHY, even without SCO, caused no behavioral or neurophysiological side effects. Also, the efficacy of such a pretreatment in counteracting soman-induced lethality and apparent symptoms of intoxication were determined. After subchronically administered PHY or PHY + SCO, the treated animals were protected against a 3 x LD50 dose of soman.

Toxicologic evaluation of pepper spray as a possible weapon for the Dutch police force: risk assessment and efficacy.

The efficacy and possible health risks of pepper spray were evaluated. In a number of countries, pepper spray is being used by police forces to aid in arresting aggressive individuals. Pepper spray is commercially available as a canister filled with Capsicum extract, which contains capsaicin as the active component. When applied in the form of a spray, it causes an acute inflammation, and humans involuntarily close their eyes, experience a burning feeling on the skin, and are usually rapidly incapacitated. Use by the U.S. police was successful in subduing aggressive individuals in 90% of cases, and a reduction of injury to both police and arrested individuals was noted. In general, pepper spray appeared to be a relatively safe weapon with small risk of causing acute physical harm. Despite this evidence, a number of fatalities were reported in the United States following the use of pepper spray. However, it was concluded that it was not the pepper spray but rather other factors such as drugs and hog-tying that contributed to the cause of death. In only 1 case, that of an asthmatic man, was it concluded that the use of pepper spray contributed to the cause of death. Much attention has been paid to possible genotoxic effects of Capsicum extract such as mutagenicity and carcinogenicity. It was concluded that the risk of long-term health effects is negligible. Because pepper spray may induce bronchoconstriction, people suffering from chronic obstructive lung disease may be hypersensitive to it. Although the results of one study indicate that asthmatics do not develop additional bronchoconstriction following inhalation of capsaicin, the number of experimental data are too few to draw sound conclusions.

Comparison of the efficacy of single or repeated HI-6 treatment following soman poisoning in guinea pigs and marmoset monkeys.

The therapeutic efficacy of single or repeated doses of HI-6, together with atropine, against soman poisoning were compared both in guinea pigs and in marmoset monkeys. In addition, the pharmacokinetics of HI-6 were determined after single or repeated injections. Both single and repeated HI-6 injections protected guinea pigs effectively against 2 x LD50 soman. The plasma levels of HI-6 after single HI-6 injection fitted a one-compartment elimination model. The plasma levels of HI-6 following repeated injections were in accordance with those predicted using the data obtained after single HI-6 injection. No evidence was found for any disturbance of the HI-6 elimination in guinea pigs following soman intoxication. Marmosets were intoxicated with 2 x LD50 soman (s.c.), followed after 1 min by i.m. injections of atropine and HI-6. One and 2 h later, four animals received additional HI-6 injections. The pharmacokinetics of HI-6 in plasma, after single and repeated HI-6 injections were similar to those found in the guinea pig. Furthermore, repeated HI-6 injections protected effectively against soman: four out of four animals survived, in fair condition. In contrast, only one out of four animals receiving single HI-6-treatment fully recovered within a few days. Two animals died, the fourth animal survived, but had to be euthanized 3 weeks after intoxication.

Side effects of physostigmine as a pretreatment in guinea pigs.

To prevent incapacitation following nerve agent intoxications, it is proposed to replace pyridostigmine by the centrally active carbamate physostigmine (PHY). Behavioral and neurophysiological effects of PHY were determined and whether these effects would be counteracted by scopolamine. In addition, we compared them with the effects of another reversible cholinesterase (ChE) inhibitor ethyl-p-nitrophenylphosphoramidate (PNF) At similar levels of blood AChE inhibition, PHY caused a larger shuttlebox performance decrement than PNF, which was antagonized by scopolamine (0.1 mg/kg). SCO enhanced the PHY-induced increase of the auditory startle response, whereas PNF, with or without scopolamine, had no effect. In the EEG, PHY led to a power increase at the theta 2-alpha 1 band, also found after PNF, and at the theta 1 band. SCO antagonized all EEG effects, but not the effects of PHY on visual evoked responses, in contrast to those of PNF. Based on the different effects of both inhibitors, it is suggested that at relevant doses several PHY-induced phenomena occur that are unrelated to AChE inhibition.

Pharmacological effects of oximes: how relevant are they?

The increased international concern about the threat of military and terroristic use of nerve agents, prompted us to critically consider the expected value of the currently available oxime treatment of nerve agent poisoning. Although oximes have been designed to reactivate the inhibited acetylcholinesterase (AChE), clinical experience has indicated that they are not always very effective as reactivators and at this very moment none of them can be regarded as a broad-spectrum antidote. In spite of this drawback, oximes are worth further investigating, since recent data derived from soman or tabun lethally intoxicated non-human primates suggest that the oxime HI-6 may exert a pharmacological effect that is not related to reactivation of inhibited AChE, but still leads to survival. This pharmacological effect causes recovery of neuronal transmission in the respiratory centres of the brain and recovery of neuromuscular transmission in the diaphragm. These findings have stimulated research to reveal the pharmacological basis of these effects in order to find drugs which could be more effective and less toxic than the available oximes. Since cholinergic drugs were able to exert this effect, a new concept for further treatment is suggested: maintenance of neuronal transmission in spite of continued AChE-inhibition by pharmacological manipulation of the cholinergic receptor. This should renew interest in the diverse pharmacological effects of oximes to reach a more effective treatment in the future.

Effects of low doses of cholinesterase inhibitors on behavioral performance of robot-tested marmosets.

To investigate at which dose levels undesirable effects started, behavioural performance and several physiological parameters were measured in marmosets (Callithrix jacchus) after soman (1.75 and 3.5 micrograms/kg), sarin (3 and 6 micrograms/kg), physostigmine (10 and 20 micrograms/kg), and pyridostigmine (200 and 400 micrograms/kg). Effects on performance were investigated with a discrete-trial, two-choice visual discrimination task and a hand-eye coordination task. The former test appeared more sensitive to disruption than the hand-eye coordination task. "Motor speed" was not disrupted by any of the four compounds. However, "choice time" as well as "no attempts" increased and were clearly more disturbed by soman and physostigmine than by sarin and pyridostigmine. All effects had disappeared after 24 h. Except for a small effect of sarin on heart rate and blood pressure, none of the cholinesterase (ChE) inhibitors affected a number of physiological parameters at behavioural effective does that caused a profound ChE inhibition in blood. Take together, these results strongly suggest that both soman and physostigmine may interfere with higher CNS functions at low dose levels. These effects may go undetected because physical signs are absent.

The functional role of molecular forms of acetylcholinesterase in neuromuscular transmission.

The severity of poisoning following acetylcholinesterase (AChE) inhibition correlates weakly with total AChE activity. This may be partly due to the existence of functional and non-functional pools of AChE. AChE consists of several molecular forms. The aim of the present study was to investigate which of these forms will correlate best with neuromuscular transmission (NMT) remaining after partial inhibition of this enzyme. Following sublethal intoxication of rats with the irreversible AChE inhibitor soman, diaphragms were isolated after 0.5 or 3 h. It appeared that at 3 h after soman poisoning the percentage of G1 increased, while those of G4 and A12 decreased. NMT was inhibited more strongly than in preparations obtained from the 0.5 h rats with the same level of AChE inhibition, but with a normal ratio of molecular forms. NMT correlated positively with G4 as well as with A12, but inversely with G1. In vitro inhibition with the charged inhibitors DEMP and echothiophate resulted in higher levels of total AChE, relatively less G1 and more G4 and A12 than after incubation with soman, but led to less NMT. Treatment of soman-intoxicated rats with the reactivating compound HI-6 resulted in preferential reactivation of A12, persisting low levels of G1 and concurrent recovery of NMT as compared with saline-treated soman controls with equal total AChE activity. Apparently, in rat diaphragm G4 and A12 are the functional AChE forms.

Non-reactivating effects of HI-6 on hippocampal neurotransmission.

Effects of the oxime HI-6, unrelated to reactivation of acetylcholinesterase (AChE), on field potentials in the dentate gyrus of the rat hippocampus following AChE inhibition, were investigated both in vitro and in vivo. In hippocampal slices, AChE inhibition decreased the perforant path evoked population spike amplitude (PSA). This effect could be prevented by pre-incubation of the slices with atropine (0.1-1 microM) or with the M1 muscarinic receptor antagonist pirenzepine (1 microM). A similar preventive effect was found after pre-incubation with the GABAA antagonist picrotoxin (20 microM), suggesting that the effects of AChE inhibition in vitro may be due to an enhancement of GABAergic inhibitory activity via activation of M1-muscarinic receptors. The effects of AChE inhibition in vivo were variable; both increases and decreases of the PSA were found. Following AChE inhibition, HI-6 increased the PSA dose-dependently, both in the in vivo and in the in vitro hippocampus. At higher oxime doses the perforant path stimulation elicited multiple population spikes. The effects of the oxime were presumably not mediated by an antagonism of cholinergic receptors, since they could not be mimicked with cholinergic antagonists like atropine, mecamylamine or gallamine. Further testing of the nature of the HI-6 effect in hippocampal slices in vitro, using a paired antidromic-orthodromic stimulation protocol, showed that HI-6 may interfere with GABAergic inhibition.

Search for a therapy against soman-intoxication.

This mini-review mainly describes a part of the pharmacological research carried out in our laboratory during the past decades, aimed at finding a therapy against intoxication by cholinesterase-inhibiting organophosphates, in particular against the nerve agent soman. In particular soman, because this is one of the nerve agents that consistently appears to be very resistant to treatment. Various experimental approaches are described. Yet, even after all these years of research an adequate (pre)treatment against poisoning by soman is still not available.

Comparison of the therapeutic effects and pharmacokinetics of HI-6, HLö-7, HGG-12, HGG-42 and obidoxime following non-reactivatable acetylcholinesterase inhibition in rats.

The oximes HI-6, HLö-7, HGG-12, HGG-42 and obidoxime were used in a previously developed rat model to evaluate the therapeutic effects of oximes other than acetylcholinesterase (AChE) reactivation (so-called "non-reactivating effects"). To test this anaesthetized, atropinized and artificially ventilated rats (n = 8 or 16) were poisoned with a three times LD50 dose of the potent AChE-inhibitor crotylsarin (CRS, i.v.). CRS-inhibited rat AChE dealkylates instantaneously, thereby excluding AChE reactivation by the oximes. Five minutes after poisoning the rats were treated (i.v.) with an oxime or saline and 10 min later artificial ventilation was terminated. Survival times were determined. Saline-treated animals died within 15 min. In comparison, treatment with HI-6, HLö-7, HGG-12, HGG-42 or obidoxime resulted in a significant prolongation of survival time. In the groups treated with HLö-7, HI-6 or HGG-12, 12-37% of the animals survived more than 24 h. It was investigated whether differences in therapeutic effectiveness are caused by differences in pharmacokinetics of the oximes. The plasma half-lives of HI-6, HLö-7, HGG-12, HGG-42 and obidoxime amounted to 67, 63, 27, 55 and 179 min, respectively. At doses of 75 or 150 mumol/kg, all oximes could be detected in brain and medulla oblongata in similar amounts (6-10 nmol/g tissue). In vitro, all oximes were effective in restoring failure of neuromuscular transmission (NMT) caused by CRS, albeit with varying potency. All oximes bound with affinities in the micromolar range to rat brain muscarinic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Therapeutic efficacy of HI-6 in soman-poisoned marmoset monkeys.

The therapeutic efficacy of the oxime HI-6 against intoxication with the irreversible cholinesterase (ChE) inhibitor soman was tested in marmoset monkeys. Five out of six marmosets, intoxicated with 5 x LD50 soman and treated immediately with diazepam (0.2 mg.kg-1 iv) and 15 sec later with atropine (0.5 mg.kg-1 im) and HI-6 (50 mg.kg-1 im), survived for more than 24 hr. One of these animals died after 4 days. In the HI-6-treated marmosets blood ChE activity was inhibited at a rate slower than that in three animals treated similarly but with saline instead of HI-6. The latter marmosets died within 8 min after soman. HI-6 achieved its plasma peak 5 min after injection and was eliminated with a t1/2 of about 40 min. In a second experiment similarly treated marmosets were euthanized at 5 min (three saline-treated animals) or at 10 min (three HI-6-treated animals) after the soman intoxication to enable the determination of acetylcholinesterase (AChE) activities in diaphragm and brain tissue. In addition, in these animals blood AChE and butyrylcholine esterase (BuChE) activities were determined. Low AChE activities were encountered in diaphragms and brains. These levels were not significantly different between saline- and HI-6-treated marmosets. In vitro treatment with HI-6 at 40 min after soman still led to an increase of the AChE activity, which was significant in diaphragm, suggesting that postmortem AChE inhibition had occurred. The ratio of AChE to BuChE in blood was significantly enhanced in HI-6-treated animals, indicating that HI-6 preferentially reactivated AChE. It is concluded that (i) HI-6 is an effective treatment against soman poisoning in marmosets and (ii) AChE reactivation or protection by HI-6 contributed to the survival of the animals.

Organophosphate poisoning: a method to test therapeutic effects of oximes other than acetylcholinesterase reactivation in the rat.

A method was developed to study exclusively those therapeutic effects of oximes that are not related to reactivation of organophosphate-inhibited acetylcholinesterase (AChE). The model uses the organophosphorus compound crotylsarin (CRS), which proved to be a potent, irreversible, peripherally and centrally active AChE inhibitor with a very short biological half-life. CRS-inhibited AChE appeared to age very rapidly, because in vitro addition of oximes immediately following inhibition, did not result in any AChE reactivation. Anaesthetized, atropinized and artificially ventilated rats were intoxicated with 3 x LD50 CRS and treated 5 min later with the bispyridinium oxime HI-6. Fifty percent of these animals survived more than 24 h following termination of artificial ventilation at 10 min after oxime treatment. The mean survival time of the remaining animals was 66 min, whereas all untreated animals died within 4 min. HI-6, when added in vitro to isolated intact hemidiaphragms, or to diaphragm or brain homogenates from rats which had been killed 1 min following 3 x LD50 CRS, failed to reactivate the inhibited AChE. If blood was sampled (before and) after HI-6 administration to CRS-intoxicated rats, no HI-6-induced AChE reactivation was observed. Yet, a clear improvement of the neuromuscular transmission in the hindleg muscles of these animals was found following HI-6 injection. With this model, decisive evidence is obtained that non-reactivating effects of HI-6 by themselves are therapeutically relevant.

Therapy of organophosphate poisoning in the rat by direct effects of oximes unrelated to ChE reactivation.

Isolated rat diaphragm preparations treated with soman or with the irreversible and oxime resistant cholinesterase (ChE) inhibitor S27 showed a considerable recovery of neuromuscular transmission (NMT) during incubation with the (bis)pyridinium oximes HI-6, HGG-12, P2S and obidoxime. In the soman-treated preparations this NMT recovery was predominantly caused by reactivation of acetylcholinesterase (AChE) but in the S27-treated preparations it was caused by a direct (pharmacological) effect unrelated to enzyme reactivation. Atropinized rats were artificially ventilated after injection with 3 x LD50 soman for 3 h and then treated with HI-6, i.e. at a time when oxime reactivation of soman inhibited ChE is no longer possible. Nevertheless, these rats started to breathe spontaneously and 50-60% survived more than 24 h, whereas all control animals (saline instead of HI-6) died within 10 min after artificial ventilation was terminated. In such animals no significant reactivation of ChE activity at various time intervals following HI-6 treatment was found, either in the diaphragms or in the brains. There was a significant amount of NMT (50%) in vitro in diaphragms obtained from these animals. This NMT did not improve in vitro in the presence of HI-6 and was not inhibited by soman administered to the medium. It is concluded that in this case the NMT found was based on synaptic adaptation to the continued inhibition of ChE and that the survival of the animals might be due to a combination of this synaptic adaptation and central direct effects of HI-6.

Irreversible photobinding of nitrofurantoin and of nitrofurfural to plasma proteins in vitro.

Allergic reactions form a serious problem in therapy with the urinary antiseptic nitrofurantoin (NFT). The formation of conjugates between NFT and plasma proteins is considered to be a first step in the development of such reactions. We investigated the possibility that UV-A irradiation of NFT in the presence of plasma proteins results in covalent binding. Binding to blood cell proteins was +/- 100X less. Efficient photobinding to albumin was demonstrated for NFT (up to 50 nmol mg-1 protein) and its photoproduct 5-nitrofurfural. Incubation of photodecomposition products from these two nitrofurans with plasma proteins also resulted in irreversible binding. Protein amino and, to a lesser extent, thiol groups proved to be targets for binding. Furthermore, upon photolysis both compounds induced a significant decrease in the iso-electric point of albumin. Photobinding, along with such alterations in the structure of plasma proteins, may well be able to trigger immunological responses when taking place in vivo. In this manner activation of nitrofurantoin by light may be a cause of allergic reactions by nitrofurantoin.

Formation of methemoglobin by photoactivation of nitrofurantoin or of 5-nitrofurfural in rats exposed to UV-A light.

The antibacterial drug nitrofurantoin (NFT) is notorious for causing hemolytic anemia, which may be related to the methemoglobinemia, another side-effect of NFT. As NFT is photolabile, and nitrite, well known as a MetHb generator, is an important photoproduct of NFT, it seems not unlikely that light is a cause of NFT-induced MetHb formation. When rats were irradiated with UV-A immediately after oral NFT administration, the amount of MetHb significantly increased: 0.97 +/- 0.37% n = 36 (P less than 0.001 Student's t-test, control value: 0.5%). An increase in MetHb was also observed with rats simultaneously exposed to UV-A and the major photodecomposition product of NFT, viz. 5-nitrofurfural. In addition in vitro experiments proved the formation of MetHb as a result of photoactivation of NFT. Nitrite, photochemically formed from nitrofurfural and from the metabolite nitrofuroic acid, plays an important role. A dark reaction of the other photoproduct, nitrofurfural, with hemoglobin also appeared to cause a considerable amount of MetHb in vitro. However, because of rapid deactivation of nitrofurfural by either photodecomposition or metabolism, this dark reaction is not expected to contribute to the in vivo MetHb formation.

Cytotoxicity and induction of repairable DNA damage by photoactivated 5-nitrofurfural.

5-Nitrofurfural (NFA) an important photodecomposition product and metabolite of medicinal nitrofurans is phototoxic in bacterial test systems. Its major photodecomposition product 5-hydroxymethylene-2(5H)-furanone (HMF) appears to be responsible for this. Furthermore HMF and photoactivated nitrofurfural can induce repairable DNA damage in Escherichia coli bacteria. These effects may be important with respect to skin and allergic reactions and carcinogenicity appearing in nitrofuran therapy.

Irreversible binding of the chloropromazine radical cation and of photoactivated chlorpromazine to biological macromolecules.

The irreversible binding of chlorpromazine radical cation (CPZ+.) and photoactivated chlorpromazine (CPZ) to calf thymus DNA in vitro and bacterial macromolecules in intact bacterium cells was investigated. CPZ+. may be formed in vivo metabolically and photochemically. CPZ+. and photo-activated CPZ bind covalently to double- and single-strand DNA. The conformation of the DNA appeared to be important for the CPZ+. reactivity: though CPZ+. is less stabilized by complex formation with single-strand DNA, the reaction rate and the binding capacity of DNA-complexed CPZ+. with single-strand DNA is larger than with double-strand DNA. Photoactivated CPZ binds considerably to proteins, DNA and RNA in the intact bacterium cells. In spite of the relatively short lifetime of CPZ+. in the presence of the cells CPZ+. also binds irreversibly to bacterial DNA, RNA and proteins. The consequences of covalent binding for the cytotoxicity and genotoxicity of CPZ+. and photoactivated CPZ and the possible role for CPZ+. as an intermediate in the photobinding of CPZ is discussed.