Effects of Huperzine Used As Pre-Treatment Against Soman-Induced Seizures

doi:10.1016/S0161-813X(00)00015-2

NeuroToxicology

Volume 22, Issue 1, February 2001, Pages 29-37

https://doi.org/10.1016/S0161-813X(00)00015-2 Get rights and content

Abstract

Huperzine A (HUP), an alkaloid isolated from the Chinese club moss, Huperzia serrata, is a reversible inhibitor of cholinesterases which crosses the blood-brain barrier and shows high specificity for acetylcholinesterase (AChE) and a prolonged biological half-life. We tested, in vivo, its efficiency in protecting cortical AChE from soman inhibition and preventing subsequent seizures. The release of acetylcholine (ACh) was also followed in the cortex of freely moving rats using microdialysis techniques. We previously found that soman-induced seizures occurred in rodents only when the cortical AChE inhibition was over 65% and when the increase of ACh level was over 200 times the baseline level. This was verified in the present study in control animals intoxicated by 1 LD₅₀ of soman (90 μg/kg). Using the same dose of soman in rats pre-treated with 500 μg/kg of HUP, we observed that 93% of the animals survived and none of them had seizures. This dose of HUP reduced AChE inhibition to 54% and increase of ACh level to 230 times baseline value. HUP thus appears as a promising compound to protect subjects against organophosphorus intoxication.

Section snippets

INTRODUCTION

Organophosphate compounds are very potent neurotoxic agents. These agents, such as soman, irreversibly inhibit acetylcholinesterase (AChE) both centrally and peripherally. This inhibition induces a major increase of ACh level (hypercholinergy) producing muscle fasciculations, respiratory distress, epileptic fits always leading to generalised seizures. In surviving animals these seizures lead to severe incapacitation and to irreversible brain damage with lesions especially in hippocampus,

Surgery

Male Sprague–Dawley rats (300–350 g, Janvier, France) were anaesthetised with chloral hydrate (400 mg/kg; IP) and placed in a stereotaxic frame. The transcortical dialysis fibers (AN69HF HOSPAL) were implanted into the frontal cortex (AP 0.0 mm, H −2.0 mm relative to bregma) and into the somatosensory cortex (AP −3 mm, H −1.5 mm relative to bregma). Six stainless steel screws served as electrodes and were implanted into the skull on the level of the hippocampus and above the visual and frontal

Clinical Effects Induced By HUP and Soman

No clinical signs were observed in rats after the injection of HUP alone at 100 μg/kg. In the groups receiving HUP at 500 μg/kg, most of the animals had symptoms such as chewing, hypersalivation, fasciculations and tremors. These symptoms were moderate compared to those observed after the injection of soman. Moreover, they disappeared rapidly (30 min) after treatment.

After the injection of soman at 90 μg/kg, all animals had severe early symptoms of intoxication irrespective the pre-treatment

DISCUSSION

The main finding of the present work was that only a high dose of HUP (500 μg/kg) gave protection against status epilepticus due to soman. Indeed, HUP at a lower dose (100 μg/kg) did not prevent the occurrence of seizures. However, pre-treatment with HUP (irrespective of the dose) was effective in reducing lethality.

CONCLUSION

Pre-treatment with HUP, 500 μg/kg, was effective in preventing epileptic activity induced by soman. This molecule appeared to act on the three parameters necessary for an intoxicated animal to have seizures: at the enzymatic level, HUP protects the AChE; at the neurochemical level, it reduces the hypercholinergy due to soman and at the electrophysiological level, it increases the gamma index. HUP thus seems very promising to prevent soman toxicity. However, being intended to be injected as a

Acknowledgements

We wish to thank S. Nallet for correction of the English manuscript.

References (25)

Y Ashani et al.
Mechanism of inhibition of cholinesterases by HUP A
Biochem. Biophys. Res. Commun.
(1992)
J.G Clement et al.
HI-6, an oxime which is an effective antidote of soman poisoning: a structure-activity study
Toxicol. Appl. Pharmacol.
(1982)
G Damsma et al.
Basal acetylcholine release in freely moving rats detected by on-line trans-striatal dialysis: pharmacological aspects
Life Sci.
(1988)
G.L Ellman et al.
A new and rapid colorimetric determination of acetylcholinesterase activity
Biochem. Pharmacol.
(1961)
J.J Gordon et al.
The protection of animals against organophosphate poisoning by pre-treatment with a carbamate
Toxicol. Appl. Pharmacol.
(1978)
G.B Koelle et al.
Selective, near-total, irreversible inactivation of peripheral pseudocholinesterase and AChE in cats in vivo
Biochem. Pharmacol.
(1974)
G Testylier et al.
Simultaneous in vivo determination of acetylcholinesterase activity and acetylcholine release in the cortex of waking rat by microdialysis. Effect of VX
J. Neurosci. Met.
(1998)
C.A Broomfield et al.
Protection by butyrylcholinesterase against organophosphorus poisoning in nonhuman primates
Pharmacol. Exp. Ther.
(1991)
E.G Cape et al.
Differential modulation of high-frequency gamma-electroencephalogram activity and sleep-wake state by noradrenaline and serotonin microinjections into the region of cholinergic basalis neurons
J. Neurosci.
(1998)
A.Y Chang et al.
Power spectral analysis of electroencephalographic desynchronization induced by cocaine in rats: correlation with evaluation of noradrenergic neurotransmission at the medial prefrontal cortex
Synapse
(1995)

S.D Cohen

Carboxylesterase inhibition and potentiation of soman toxicity

Arch. Toxicol.

(1981)

P Filliat et al.

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

Neurotoxicology

(1999)

Cited by (50)

Study of Huperzine A derivatives with extended protection against soman intoxication
2023, Toxicology and Applied Pharmacology
Pre-administration of huperzine A (Hup A) was validated to prevent poisoning from exposure to nerve agents (NAs) by reversibly inhibiting acetylcholinesterase (AChE). However, like the currently commonly used reversible inhibitors, Hup A has a short half-life and is unable to produce a long-term preventative effect. To extend the protective time of Hup A against NAs, 42 derivatives with a CN bond were designed based on the structure of Hup A in this study. All designed derivatives showed good binding capability with AChE via molecular docking. Six compounds (H3, H4, H11, H14, H16, and H25) with representative structures were selected for synthesis by Schiff base reaction, and their structures were stable. The modified Ellman's method showed the six compounds concentration-dependently inhibited AChE, and the half maximal inhibitory concentration (IC₅₀) were higher than that of Hup A. Pretreatment of AChE with the derivatives significantly increased the IC₅₀ of soman. In vivo experiments demonstrated H3, H4, H14, H16, and H25 had longer protective capacities against 1 × LD₉₅ soman-induced death in mice than Hup A. The 12 h protective index showed that the protective ratios of H3, H4, H14 and H16 were 2.31, 1.85, 2.23 and 1.99 respectively, better than that of Hup A. The extended protection of the derivatives against soman may be explained by their transformation to Hup A in vivo. Furthermore, all six compounds showed lower acute oral toxicity than Hup A. Overall, our study provided an optional strategy to acquire pretreatment agents for NAs with extended action and low toxicity.
The reversible inhibitors of acetylcholinesterase as pretreatment options against nerve agents’ intoxications
2022, Sensing of Deadly Toxic Chemical Warfare Agents, Nerve Agent Simulants, and their Toxicological Aspects
Nerve agents rapidly inactivate acetylcholinesterase (AChE), leading to the accumulation of acetylcholine in the synaptic cleft, ultimately causing death by respiratory failure. Due to the inefficiency of the standard antidotal therapy consisting of atropine, oxime, and diazepam, the pretreatment option is introduced, given to healthy individuals when a chemical attack is expected. The pyridostigmine bromide is currently the only approved drug for the pretreatment against nerve agent exposure, but in the past few decades, several other AChE inhibitors have been proposed including carbamates (physostigmine and rivastigmine) and the reversible inhibitors AChE used for the treatment of Alzheimer’s disease (huperzine A, donepezil, and galanthamine). In this chapter, we present a comprehensive review of the newest in vitro and in vivo studies of these drugs as pretreatment options, along with the recently designed AChE reversible inhibitors, also tested in vitro for protection of the enzyme against irreversible inhibition by nerve agents. Special attention is paid to the results of the in vitro studies and kinetic behavior of AChE inhibitors and their relation to the protective effects exerted in the in vivo animal models.
Persistent brainwave disruption and cognitive impairment induced by acute sarin surrogate sub-lethal dose exposure
2021, Toxicology
Warfare neurotoxicants such as sarin, soman or VX, are organophosphorus compounds which irreversibly inhibit cholinesterase. High-dose exposure with nerve agents (NA) is known to produce seizure activity and related brain damage, while less is known about the effects of acute sub-lethal dose exposure. The aim of this study was to characterize behavioral, brain activity and neuroinflammatory modifications at different time points after exposure to 4-nitrophenyl isopropyl methylphosphonate (NIMP), a sarin surrogate. In order to decipher the impacts of sub-lethal exposure, we chose 4 different doses of NIMP each corresponding to a fraction of the median lethal dose (LD₅₀). First, we conducted a behavioral analysis of symptoms during the first hour following NIMP challenge and established a specific scoring scale for the intoxication severity. The intensity of intoxication signs was dose-dependent and proportional to the cholinesterase activity inhibition evaluated in mice brain. The lowest dose (0.3 LD₅₀) did not induce significant behavioral, electrocorticographic (ECoG) nor cholinesterase activity changes. Animals exposed to one of the other doses (0.5, 0.7 and 0.9 LD₅₀) exhibited substantial changes in behavior, significant cholinesterase activity inhibition, and a disruption of brainwave distribution that persisted in a dose-dependent manner. To evaluate long lasting changes, we conducted ECoG recording for 30 days on mice exposed to 0.5 or 0.9 LD₅₀ of NIMP. Mice in both groups showed long-lasting impairment of theta rhythms, and a lack of restoration in hippocampal ChE activity after 1-month post-exposure. In addition, an increase in neuroinflammatory markers (IBA-1, TNF-α, NF-κB) and edema were transiently observed in mice hippocampus. Furthermore, a novel object recognition test showed an alteration of short-term memory in both groups, 1-month post-NIMP intoxication. Our findings identified both transient and long-term ECoG alterations and some long term cognitive impairments following exposure to sub-lethal doses of NIMP. These may further impact morphopathological alterations in the brain.
Slow-binding inhibitors of acetylcholinesterase of medical interest
2020, Neuropharmacology
Citation Excerpt :
Huperzine A was also proposed for the pre-treatment of OP poisoning instead of pyridostigmine (Grunwald et al., 1994). In particular, it was effective against soman-induced seizures (Tonduli et al., 2001). Further studies showed that huperzine A and its derivatives present several advantages over pyridostigmine.
Certain ligands slowly bind to acetylcholinesterase. As a result, there is a slow establishment of enzyme-inhibitor equilibrium characterized by a slow onset of inhibition prior reaching steady state. Three mechanisms account for slow-binding inhibition: a) slow binding rate constant k_on, b) slow ligand induced-fit following a fast binding step, c) slow conformational selection of an enzyme form. The slow equilibrium may be followed by a chemical step. This later that can be irreversible has been observed with certain alkylating agents and substrate transition state analogs. Slow-binding inhibitors present long residence times on target. This results in prolonged pharmacological or toxicological action.
Through several well-known molecules (e.g. huperzine) and new examples (tocopherol, trifluoroacetophenone and a 6-methyluracil alkylammonium derivative), we show that slow-binding inhibitors of acetylcholinesterase are promising drugs for treatment of neurological diseases such as Alzheimer disease and myasthenia gravis. Moreover, they may be of interest for neuroprotection (prophylaxis) against organophosphorus poisoning.
This article is part of the special issue entitled ‘Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield’.
Chemical warfare agents and the nervous system
2020, Handbook of Toxicology of Chemical Warfare Agents
The use of chemicals in warfare dates back centuries, with modern chemical warfare beginning in World War I. More recent use of organophosphorus nerve agents in the Syrian civil war has highlighted the continuing worldwide concern for the misuse of chemical warfare agents (CWAs). A number of different types of CWAs can have deleterious effects on the nervous system. We briefly describe cellular function within the nervous system, factors that make the nervous system particularly sensitive to CWAs and other toxicants, and the different basic types of responses to neurotoxicants. Emphasis is placed on the acute and chronic neurological consequences of exposure to organophosphorus anticholinesterases, and both cholinergic and noncholinergic mechanisms in the expression of their toxicity and its modulation. A brief overview of the role of anticholinesterases in Gulf War illnesses is included. Additional information is provided with three other classes of CWAs, cyanides, sulfur mustard, and quinuclidinyl benzilate.
Pretreatment and prophylaxis against nerve agent poisoning: Are undesirable behavioral side effects unavoidable?
2016, Neuroscience and Biobehavioral Reviews
Citation Excerpt :
A marked protective efficacy is obtained by donepezil (2.5 mg/kg) combined with procyclidine (3 or 6 mg/kg) when given prophylactically against a lethal dose of soman (1.6x LD50) in rats (Haug et al., 2007). The combination of HI-6 (125 mg/kg) and huperzine (0.5 mg/kg) also provides an effective protection against soman (1x LD50) in rats when given 30 min before exposure (Tonduli et al., 2001). However, the combination of donepezil (2.5 mg/kg) and procyclidine (3 mg/kg) results in a cognitive deficit and radically depressed locomotor and rearing activities in the novelty test.
The threat of chemical warfare agents like nerve agents requires life saving measures of medical pretreatment combined with treatment after exposure. Pretreatment (pyridostigmine) may cause some side effects in a small number of individuals. A comprehensive research on animals has been performed to clarify effects on behavior. The results from these studies are far from unambiguous, since pyridostigmine may produce adverse effects on behavior in animals in relatively high doses, but not in a consistent way. Other animal studies have examined the potential of drugs like physostigmine, galantamine, benactyzine, trihexyphenidyl, and procyclidine, but they all produce marked behavioral impairment at doses sufficient to contribute to protection against a convulsant dose of soman. Attempts have also been made to develop a combination of drugs capable of assuring full protection (prophylaxis) against nerve agents. However, common to all combinations is that they at anticonvulsant doses cause behavioral deficits. Therefore, the use of limited pretreatment doses may be performed without marked side effects followed by post-exposure therapy with a combination of drugs.

View all citing articles on Scopus

View full text

Effects of Huperzine Used As Pre-Treatment Against Soman-Induced Seizures

Abstract

Section snippets

INTRODUCTION

Surgery

Clinical Effects Induced By HUP and Soman

DISCUSSION

CONCLUSION

Acknowledgements

Biochem. Biophys. Res. Commun.

Toxicol. Appl. Pharmacol.

Life Sci.

Biochem. Pharmacol.

Toxicol. Appl. Pharmacol.

Biochem. Pharmacol.

J. Neurosci. Met.

Protection by butyrylcholinesterase against organophosphorus poisoning in nonhuman primates

Pharmacol. Exp. Ther.

Differential modulation of high-frequency gamma-electroencephalogram activity and sleep-wake state by noradrenaline and serotonin microinjections into the region of cholinergic basalis neurons

J. Neurosci.

Power spectral analysis of electroencephalographic desynchronization induced by cocaine in rats: correlation with evaluation of noradrenergic neurotransmission at the medial prefrontal cortex

Synapse

Carboxylesterase inhibition and potentiation of soman toxicity

Arch. Toxicol.

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

Neurotoxicology