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.

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.

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.

Efficacy of HI-6 and HLö-7 in preventing incapacitation following nerve agent poisoning.

The therapeutic efficacy of the oximes HI-6 and HLö-7 (132.5 mumol/kg), in combination with atropine, in soman- or tabun-intoxicated guinea pigs was compared, particularly with respect to recovery of shuttlebox performance and electroencephalograms (EEGs). After 1.5 x LD50 soman SC, therapy with HI-6 or HLö-7 resulted in survival of 87.5% of the animals in each group. In both groups postintoxication performance decrements and EEG abnormalities lasted approximately 2 weeks after intoxication. After 3 x LD50 soman all HLö-7-treated animals died within 5 h; 70% of the HI-6-treated animals were still alive after 8 h; however, only 10% survived more than 24 h. After 2 x LD50 tabun 36% of the HI-6-treated animals died; HLö-7 prevented lethality and led to faster recovery of performance and EEG than after HI-6. Even after 7.5 x LD50 tabun, followed by HLö-7, full recovery was reached within 1 week in the surviving animals (82%). In soman-intoxicated guinea pigs HI-6 is therapeutically slightly more effective than HLö-7. HLö-7 is far more effective, under similar conditions, against tabun intoxication than HI-6.

A simple automated test to measure exploratory and motor activity of marmosets.

An automated device is described to test the exploratory and motor activity of common marmosets (Callithrix jacchus). The device consists of four boxes interconnected by PVC tubes. The presence of an animal in a box is detected by a photocell. Calibration takes place with an electric model train. Movements of the animal from one box to another are detected by disappearance from one and appearance in another box. The apparatus is linked to a PC. The effects of two doses of methamphetamine and of pentobarbital are shown.

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.

Behavioral performance, brain histology, and EEG sequela after immediate combined atropine/diazepam treatment of soman-intoxicated rats.

It is known that rats poisoned with near-lethal doses of pinacolyl methylphosphonofluoridate (soman) develop brain lesions, particularly when convulsions are induced. When rats were intoxicated with a LD50 of soman and treated immediately thereafter with a combination of low doses of atropine and diazepam (LOW AS/DZ treatment), large decrements in performance of an earlier acquired shuttle-box task were found 6 days after intoxication. In contrast, no such decrements were found in soman-intoxicated animals treated similarly with a combination of high doses of these drugs (HIGH AS/DZ treatment). Surprisingly, surviving LOW AS/DZ animals acquired the same task again at a speed that was almost as fast as before intoxication. Similarly treated animals were examined light-microscopically 24 h after intoxication; in LOW-AS/DZ-treated animals, neuropathology was only observed in animals that had exhibited convulsions, whereas in HIGH AS/DZ animals neither convulsions nor brain damage were observed. Power spectra, obtained from electroencephalograms (EEGs) 6 days after intoxication, revealed significant differences between both treatment groups, particularly in the delta-, theta-, and beta-frequencies. After the HIGH AS/DZ treatment, a significant increase in delta activity was found compared to control values, suggestive of neuropathology. It is concluded that, in contrast with the LOW AS/DZ combination, HIGH AS/DZ prevents active avoidance deficits, convulsions, and light-microscopically detectable neuropathology after soman intoxication. However, the results of EEG measurements suggest that some aberrations may still remain even after the HIGH AS/DZ treatment.

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.

Active avoidance behavior in guinea pigs: effects of physostigmine and scopolamine.

Behavioral training of guinea pigs by conventional methods, such as used for rats and mice, appears difficult. Hence, only a few behavioral experiments with guinea pigs have been described in the literature. An active avoidance technique in an automated two-way shuttlebox is described using sound as a conditioned (CS) and a tactile stimulus (a stream of air ruffling their fur) as an unconditioned (UCS) stimulus. Acquisition is fairly rapid and reproducible. Doses of physostigmine that caused moderate blood acetylcholinesterase inhibition induced dose-dependent performance decrements. These decrements were counteracted by a sign-free dose of scopolamine.

The isolated blood-perfused pig ear: an inexpensive and animal-saving model for skin penetration studies.

To overcome most of the disadvantages of current models to investigate percutaneous penetration of drugs or toxic substances, a model is proposed here based on the isolated pig ear, which is obtained at the slaughterhouse, and perfused with oxygenated blood from the same pig. To determine the viability of the preparations, we measured glucose consumption and lactate production as metabolic parameters, Na+ and K+ ions, as well as lactate dehydrogenase activity in blood as markers for cell damage, whereas vasomotor reactivity was assessed by administering noradrenaline and isoxsuprine. After 60 min of equilibration, only insignificant changes in these parameters were observed during the subsequent 3-hr test period (longer periods were not tested). A slight weight increase was noted during the total period 4 hr, presumably due to slight edema formation. On the basis of several types of measurements, such as in vivo blood flow and ear temperature and in vitro glucose metabolism, standard procedures were developed. It is concluded that this technique offers an easy to handle, cost-efficient, and animal-saving model for skin penetration studies that lacks most of the disadvantages of existing models.

On the development of behavioral tolerance to organophosphates. IV: EEGand visual evoked responses.

Several earlier studies showed that, in contrast with DFP, repeated injections with soman did not lead to behavioral tolerance in rats. The reason for the difference between the effects of these two organophosphate cholinesterase inhibitors was not clear and a neurophysiological approach was undertaken. Four experiments (A, B, C and D) were carried out, each consisting of three groups of rats, SC injected with saline, DFP (600 micrograms/kg) or soman (60 micrograms/kg) respectively. In Experiment B and D the rats were trained to criterion in a two-way shuttlebox. Thereafter, the animals of Experiment B were fitted with suitable electrodes and two days later their EEGs and visual evoked responses (VERs) were recorded, 1 and 24 h after a single dose of the above-mentioned compounds. In Experiment D the trained animals were subsequently injected 3 times per week for 4 weeks with the same doses and their performance was tested 5 days per week, 1 and 24 h after injection. After those 4 weeks, when the DFP-treated animals had developed behavioral tolerance, electrodes were fitted and EEGs and VERs were recorded after two days, again 1 and 24 h after injection, as in Experiment B. The difference with Experiments A and C was that these animals were not trained. Otherwise, treatment schedules and recording procedures of Experiment A were identical to those of Experiments B and of Experiment C to those of Experiment D. In all cases the EEGs and VERs were recorded from animals slowly walking in a rotating hollow transparent wheel. The results show a similar pattern in all four experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Some animal models and their probability of extrapolation to man.

A number of examples of experimental models are presented which are subdivided into models that provide results with a "high" or a "moderate" probability for (qualitative) extrapolation to man. Models with a high predictive value for man are those that produce results that can be directly verified in man or human organs, in contrast with models that have a moderate predictive value, where one has to rely on similarities or analogies of signs and symptoms between man and the animal model. Models with low probability should be rejected.

On the development of behavioral tolerance to organophosphates. III: Behavioral aspects.

As part of a study on the mechanisms underlying behavioral tolerance to cholinesterase-inhibiting organophosphates (OP's) the present investigation was focussed on behavioral procedures affecting the development of tolerance. The effects of chronic administration of the OP's DFP (600 micrograms/kg SC) and soman (60 micrograms/kg SC) were compared in rats. These doses do not cause detectable effects upon close observation of the animals. As was found before, behavioral tolerance developed following DFP, but not following soman. Repeated behavioral testing affected the development of tolerance. Cross-tolerance between these two inhibitors was not found. Surprisingly, when DFP was administered 48 hr after soman, all animals were observationally normal, and when soman was given 48 hr after DFP the majority of the animals died. This indicates that the sequence in which these inhibitors were administered was of major importance. It is concluded that practice-related and/or state-dependent factors are important for the development of behavioral tolerance and that one should be careful in making generalizing statements about tolerance to cholinesterase-inhibiting OP's.

On the development of behavioral tolerance to organophosphates. I: Behavioral and biochemical aspects.

The development of tolerance to organophosphates (OPs) was investigated by SC injections of saline and sublethal doses of DFP or soman three times per week or every other day for at least 4 weeks. Shuttlebox performance was tested 1 hr and 24 hr after the injections. Notwithstanding a progressive inhibition of AChE to very low values in various organs, shuttlebox performance was virtually normal 24 hr after the OP injections. However, whereas the performance decrements measured 1 h after the injection of DFP practically disappeared in the course of weeks, the decrements 1 hr after soman remained approximately the same. These differences between the effects of DFP and soman cannot be explained: 1) by differences in inhibition or de novo synthesis of AChE in various regions of the CNS, the striated muscle or blood, 2) by differences in the reductions of the muscarinic receptors in various regions of the CNS, 3) by differences in the number of nicotinic receptors in the diaphragm muscle, or 4) by differences in phosphorylphosphatase (DFP-ase or somanase) activity in blood plasma or liver.

Side effects of therapeutic drugs against organophosphate poisoning.

The possible side effects of therapeutic drugs against organophosphate poisoning were investigated. First, dose-effect curves were obtained with atropine sulphate (AS), P2S, obidoxime, aprophen, N-methylatropine nitrate and HI-6. The first three drugs are currently used in the therapy of organophosphate poisoning, the others are potentially useful candidates. Automated tests measuring open field behavior, motor coordination and shuttlebox performance, as well as neurophysiological techniques such as the quantified EEG (qEEG) and visual evoked responses were used. The sign-free doses of these compounds were determined; it appeared that open field behavior and the qEEG were the most sensitive methods for these drugs. Subsequently, these two methods were used to investigate the effects of the combinations of AS and P2S, AS and obidoxime or AS and HI-6, each compound given in a sign-free dose. Synergistic or additive effects were found with the combination of AS and P2S, which were smaller with the combination of AS and obidoxime and absent with the combination of AS and HI-6. These results indicate that the untimely use (false alarm, panic) of the current drug combinations may cause undesirable side effects.

Retention of soman in rats, guinea-pigs and marmosets: species-dependent effects of the soman simulator, pinacolyl dimethylphosphinate (PDP).

Whether the temporary retention of intact soman in the rat and its subsequent delivery from tissues into the circulation of the blood is also demonstrable in guinea-pigs and marmosets has been investigated as was whether the soman simulator PDP (pinacolyl dimethylphosphinate) prevented this retention. Electric eel AChE, intravenously injected 1.5 h after an intravenous soman intoxication into anaesthetized, atropinized and artificially ventilated guinea-pigs (150 micrograms kg-1 soman), marmoset monkeys (100 micrograms kg-1 soman) and rats (330 and 172.5 micrograms kg-1 soman) lost its activity faster than enzyme injected in non-intoxicated animals. Electric eel AChE incubated in the presence of pectoralis or diaphragm muscle isolated from soman-intoxicated rats, guinea-pigs and marmosets 0.5 or 1.5 h after the intoxication, was progressively inhibited, indicating that those muscles still delivered soman into the incubation medium. In rats, PDP (6.4 mg kg-1 i.v.) pretreatment was effective in preventing inhibition of intravenously injected electric eel AChE 1.5 h after intoxication with a high dose of soman (330 micrograms kg-1). But after intoxication with a low dose (172.5 micrograms kg-1), PDP pretreatment was ineffective in this action, however, it did lead to less soman delivery from muscle tissue isolated 30 min following the 172.5 micrograms kg-1 soman intoxication, suggesting that there was less soman in the tissue. In PDP (6.4 mg kg-1 i.v.)-pretreated marmosets (100 micrograms kg-1 soman) and guinea-pigs (150 micrograms kg-1 soman), to the contrary, the trend was for the injected AChE to be more inhibited, whereas only slightly less soman was delivered from isolated muscle tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of topical foscarnet in a new model of herpes simplex skin infection.

Because of the lack of agreement about the effects of topically applied antiviral agents on herpes simplex virus (HSV) skin infections in humans and in animals, an in-vivo human skin model of infection was developed. Human skin was grafted on to congenitally athymic nude mice and the therapeutic effects of topically applied viral DNA polymerase inhibitor phosphonoformate (foscarnet) on the course of the disease were studied. Following infection with HSV, the human skin grafts developed herpes vesicles similar to those seen in human skin in situ. Vesicles developed within three days of inoculation, and coalesced and crusted over by the fifth day post-inoculation. Healing of the wound did not occur and non-treated animals died approximately 13 days after inoculation. Treatment with topically applied foscarnet starting 24 h after inoculation suppressed both the development of the clinical signs of the disease and the replication of HSV in the grafted human skin. However, when therapy was withdrawn the symptoms of the disease proceeded to develop. Late onset (day two post-inoculation) of the foscarnet treatment was without effect on the course of the disease. Because foscarnet showed an antiviral effect when applied to infected human skin, the lack of effect of foscarnet in clinical studies on recurrent genital or labial herpes may be due to differences in the pathogenesis of the primary and recurrent infections.

Stereoisomers of soman (pinacolyl methylphosphonofluoridate): inhibition of serum carboxylic ester hydrolase and potentiation of their toxicity by CBDP (2-(2-methylphenoxy)-4H-1,3,2-benzodioxaphosphorin-2-oxide) in mice.

The interaction of C(+/-)P(+/-)-soman (pinacolyl methylphosphonofluoridate) and its individual stereoisomers with serum carboxylic-ester hydrolase and potentiation of their toxicity by a carboxylic-ester hydrolase inhibitor CBDP (2-(2-methylphenoxy)-4H-1,3,2-benzodioxaphosphorin-2-oxide) was investigated. C(+/-)P(+/-)-Soman and the individual stereoisomers all inhibited purified mouse serum carboxylic-ester hydrolase to different degrees. C(+/-)P(+/-)-Soman and the C(-)P(-)- and C(+)P(-)-isomers had Ki values of 30.6, 18.7, and 35.7 nM, respectively, and C(-)P(+)- and C(+)P(+)-isomers had Ki values of 1412 and 2523 nM, respectively. In toxicity experiments CBDP (0.5 mg/kg; iv 1 hr prior to soman) pretreatment potentiated the toxicity of C(+/-)P(+/-)-, C(+)P(-)-, and C(-)P(-)-soman to a similar degree. Thus, it appears that the toxic stereoisomers of soman have a similar affinity for mouse serum carboxylic-ester hydrolase, and CBDP pretreatment does not enhance selectively the toxicity of one stereoisomer over the other.

Further evidence for the effect of pinacolyl dimethylphosphinate on soman storage in muscle tissue.

Diaphragms isolated from rats 60 or 120 min after the intravenous injection of 6 X LD50 soman were incubated with electric eel acetylcholinesterase. As calculated from the enzyme inhibition, detectable amounts of P(-)-soman (1,2-dimethylpropyl methylphosphonofluoridate) were released from the diaphragm into the medium even 120 min post-injection. This release was reduced by additional pretreatment of the rats with pinacolyl dimethylphosphinate, providing further evidence that this compound prevents the storage of soman in diaphragm tissue.

Prophylactic and therapeutic efficacy of the soman-simulator, pinacolyl dimethylphosphinate.

Atropinized rats, intoxicated with 6 or 8 X LD50 soman and subsequently treated with the oxime HI-6 died several hours after intoxication. Oral or intravenous administration of the soman-simulator, pinacolyl dimethylphosphinate (PDP), given at progressively increasing time intervals before soman, appeared to be very active in preventing death and secondary failure of neuromuscular transmission that followed HI-6-induced recovery. Therapeutically, PDP was only effective when given immediately after soman intoxication and oxime treatment. In animals that received no treatment otherwise, intravenous administration of PDP 10 min before intoxication with 1 X LD50 soman did not enhance lethality.