SUBCHRONIC ADMINISTRATION OF VARIOUS PRETREATMENTS OF NERVE AGENT POISONING. II. COMPARED EFFICACY AGAINST SOMAN TOXICITY

OP nerve agents, such as soman, are potent irreversible inhibitors of central and peripheral acetylcholinesterases. Pretreatment of OP poisoning relies on the subchronic administration of a reversible acetylcholinesterase inhibitor. In the present study, the protective effects against soman toxicity of such compounds i.e. pyridostigmine, physostigmine (alone or associated with scopolamine) or huperzine are compared in guinea-pigs instrumented for EEG recording. Each medication is given via a subcutaneous mini-osmotic pump for 6 days at a delivery rate providing about 30% maximal inhibition of red cell acetylcholinesterase activity. The animals then receive iterative injections of soman (1/3 LD₅₀) every 10 min. With pyridostigmine, reflecting a decreased overall tolerance to the poisoning, the cumulative doses of soman producing either tremors and convulsions or seizures are lower than those found in non-pretreated intoxicated controls. On the other hand, physostigmine does not afford satisfactory protection against the early mortality after intoxication. On this specific point, physostigmine + scopolamine and huperzine, although they do not prevent the appearance of seizures, give best results. The effects of each pretreatment on acetylcholinesterase, butyrylcholinesterase and carboxylesterase (these two latter enzymes may act as endogenous scavengers of OP compounds) are also examined in vitro and in the blood of each animal during subchronic administration. Huperzine appears as a selective inhibitor of red cell acetylcholinesterase activity while pyridostigmine or physostigmine additionally inhibit plasmatic butyrylcholinesterase. Considerations about huperzine or physostigmine + scopolamine as the most appropriate candidate for the pretreatment of OP poisoning are given.     

Subchronic administration of various pretreatments of nerve agent poisoning. II. Compared efficacy against soman toxicity

OP nerve agents, such as soman, are potent irreversible inhibitors of central and peripheral acetylcholinesterases. Pretreatment of OP poisoning relies on the subchronic administration of a reversible acetylcholinesterase inhibitor. In the present study, the protective effects against soman toxicity of such compounds i.e. pyridostigmine, physostigmine (alone or associated with scopolamine) or huperzine are compared in guinea-pigs instrumented for EEG recording. Each medication is given via a subcutaneous mini-osmotic pump for 6 days at a delivery rate providing about 30% maximal inhibition of red cell acetylcholinesterase activity. The animals then receive iterative injections of soman (1/3 LD50) every 10 min. With pyridostigmine, reflecting a decreased overall tolerance to the poisoning, the cumulative doses of soman producing either tremors and convulsions or seizures are lower than those found in non-pretreated intoxicated controls. On the other hand, physostigmine does not afford satisfactory protection against the early mortality after intoxication. On this specific point, physostigmine + scopolamine and huperzine, although they do not prevent the appearance of seizures, give best results. The effects of each pretreatment on acetylcholinesterase, butyrylcholinesterase and carboxylesterase (these two latter enzymes may act as endogenous scavengers of OP compounds) are also examined in vitro and in the blood of each animal during subchronic administration. Huperzine appears as a selective inhibitor of red cell acetylcholinesterase activity while pyridostigmine or physostigmine additionally inhibit plasmatic butyrylcholinesterase. Considerations about huperzine or physostigmine + scopolamine as the most appropriate candidate for the pretreatment of OP poisoning are given.     

Huperzine A

Huperzine A, an alkaloid isolated from Huperzia serrata, is a putative nootropic agent developed by the Chinese Academy of Sciences. Huperzine A is currently in phase III trials in China for the treatment of patients with Alzheimer's disease. The mechanism of action of huperzine A is suggested to be facilitated through the slow reversible inhibition of acetylcholinesterase. Marco Hi-Tech Joint Venture has exclusive worldwide marketing and distribution rights to huperzine A. Marco Hi-Tech Joint Venture is a corporation principally owned by Hi-Tech Pharmacal and Marco International, a global trading and finance firm formed to import huperzine A from China. Marco Hi-Tech Joint Venture also has rights to synthetic analogues of huperzine A. In July 2003, Savient Pharmaceuticals acquired the exclusive rights from Marco Hi-Tech to market huperzine A in Europe and the US. Clinical trials of huperzine A in elderly patients with age-associated memory loss are underway in the US, and a phase II study funded by an NCI grant is being planned.     

Acute and chronic studies with the anticholinesterase Huperzine A: effect on central nervous system cholinergic parameters

High affinity choline transport, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were assessed in rats after acute and chronic administration of the AChE inhibitor Huperzine A. Acute treatment: Forty-five min after a single injection of Huperzine A (0.5 mg/kg i.p.) the activity of AChE was significantly decreased by 15-30% in hippocampus, striatum and septum. The activity of ChAT was not altered. In the hippocampus high affinity choline transport was attenuated by 25%, whereas no effect in the striatum was observed. After 90 min, both inhibition of AChE and attenuation of high affinity choline transport had returned to control values. A dose of 0.1 mg/kg (i.p.) did not produce significant effects. Similar results were obtained with physostigmine (0.25 mg/kg), although the duration of inhibition of AChE was shorter than that with Huperzine A. Chronic treatment: After 5 days (twice a day), at 0.5 mg/kg, the activity of AChE was significantly reduced by 20-30% in every region of the brain studied. High affinity choline transport in the hippocampus was reduced by 28%, 45 min after the last injection, but in the striatum there was no effect. The activity of ChAT was not affected in any region of the brain studied. Thus, acute or chronic treatment with Huperzine A: did not alter ChAT; reduced high affinity choline transport in the hippocampus in a transient manner; and had a longer duration of action as an AChE inhibitor than physostigmine. Moreover, tolerance to low-toxicity doses of Huperzine A was minimal, contrary to what has been observed with other inhibitors of AChE.     

Kinetic analysis of the protection afforded by reversible inhibitors against irreversible inhibition of acetylcholinesterase by highly toxic organophosphorus compounds

In organophosphate poisoning, the underlying mechanism of the therapeutic efficacy of carbamate prophylaxis, which was successfully tested in animal experiments, still awaits complete understanding. In particular, it is unclear whether survival is improved by increased acetylcholinesterase activity during the acute phase, when both carbamate and organophosphate are present. This question should be solved experimentally by means of a dynamically working in vitro model. Immobilized human erythrocytes were continuously perfused while acetylcholinesterase activity was monitored in real-time by a modified Ellman method. The concentrations of reversible inhibitors and of paraoxon were varied to assess the influence of both components on the enzyme activity under steady-state conditions. Physostigmine, pyridostigmine and huperzine A were tested for their prophylactic potential. Upon pretreatment with these reversible inhibitors the enzyme was inhibited by 20-90%. Additional perfusion with 1 microM paraoxon for 30 min resulted in a residual activity of 1-4%, at low and high pre-inhibition, respectively. The residual activity was significantly higher than in the absence of reversibly blocking agents (0.3%). After discontinuing paraoxon, the activity increased even in the presence of the reversible blockers. Stopping the reversibly blocking agents resulted in 10-35% recovery of the enzyme activity, depending on the degree of pre-inhibition. The experimental results agreed with computer simulations upon feeding with the essential reaction rate constants, showing that physostigmine was somewhat superior to pyridostigmine in enhancing residual activity in the presence of 1 microM paraoxon for 30 min. The model predicts that inhibitors with a faster dissociation rate, e.g. huperzine A, may be superior in case of a 'hit-and-run' poison such as soman.     

Effects of combined,multiple stressors on pyridostigmine-induced acute toxicity in rats

A number of studies have evaluated the possibility that stress-induced changes in blood-brain barrier permeability enhanced the central effects of the carbamate acetylcholinesterase inhibitor, pyridostigmine. We previously found relatively little evidence of stress-induced changes in the acute toxicity of pyridostigmine in rats using a variety of restraint, forced running and forced swimming stress conditions. In this study, we evaluated the effects of sequential pre-exposure to multiple stressors on the acute toxicity of pyridostigmine. Rats (n = 8 per treatment group) were either un-stressed or stressed by restraint (60 min), forced running (60 min, 15 m/min, 6 degrees incline) and forced swimming (15 min), and then given either vehicle (saline, 1 ml/kg, po) or pyridostigmine (30 mg/kg, po) immediately after the final stressor. Functional signs of cholinergic toxicity (involuntary movements, autonomic dysfunction) were recorded at 0.5, 1 and 2 h after dosing. Body temperature was measured both before stress and 2 h after dosing. Rats were sacrificed immediately after 2-h functional observations to collect tissues (whole blood, diaphragm, frontal cortex, hippocampus and cerebellum) for measurement of cholinesterase activity. Stressed rats treated with pyridostigmine exhibited higher lethality (2/8) compared to unstressed rats given pyridostigmine (0/8). Pyridostigmine elicited classical signs of cholinergic toxicity, but the rats that died did not show increased cholinergic signs and no significant differences in cholinergic signs were noted between treatment groups. Cholinesterase activity was significantly inhibited in blood (47-50%) and diaphragm (80%) following pyridostigmine exposure regardless of stress conditions. Slight but significant inhibition (11-15%) of cerebellar cholinesterase activity was observed following pyridostigmine exposure, but inhibition was not influenced by stress. We conclude that while acute lethality from pyridostigmine may be increased by combined, multiple stressors, increased lethality does not appear due to enhanced cholinergic toxicity or via increased cholinesterase inhibition in either central or peripheral tissues.     

Effects of huperzine A on acetylcholinesterase isoforms in vitro: comparison with tacrine,donepezil, rivastigmine and physostigmine

Five inhibitors of acetylcholinesterase, huperzine A, donepezil, tacrine, rivastigmine and physostigmine, were compared with regard to their effects on different molecular forms of acetylcholinesterase in cerebral cortex, hippocampus, and striatum from the rat brain. In general, huperzine A preferentially inhibited tetrameric acetylcholinesterase (G4 form), while tacrine and rivastigmine preferentially inhibited monomeric acetylcholinesterase (G1 form). Donepezil showed pronounced selectivity for G1 acetylcholinesterase in striatum and hippocampus, but not in cortex. Physostigmine showed no form-selectivity in any brain region. In cortex, the most potent inhibitors of G4 acetylcholinesterase were huperzine A (K(i) 7 x 10(-9) M) and donepezil (K(i) 4 x 10(-9) M). The potent inhibitors of cortical G1 acetylcholinesterase were donepezil (K(i) 3.5 x 10(-9) M) and tacrine (K(i) 2.3 x 10(-8) M). In hippocampus, huperzine A and physostigmine were the most potent inhibitors of G4 acetylcholinesterase, while donepezil and tacrine were most potent against G1 acetylcholinesterase. In striatum, huperzine A and donepezil were the most potent against G4 acetylcholinesterase, while again donepezil was the most potent against G1. Although the inhibition constants (K(i)) of these acetylcholinesterase inhibitors differed significantly from region to region, the nature of the inhibition did not vary. These results suggest that the use of acetylcholinesterase inhibitors in treatment of Alzheimer's disease must consider both form-specific and region-specific characteristics of acetylcholinesterase inhibition.     

Huperzine a: an acetylcholinesterase inhibitor with high pharmacological potential]

Huperzine A is an alkaloid isolated from a chinese club-moss. The molecule is a potent and selective inhibitor of acetylcholinesterase. Several pharmacological and clinical studies showed that huperzine A improves the mnesic capacity and cognitive functions. Huperzine A was also found to be a neuroprotective agent. This molecule which possesses a high pharmacological potential is under clinical evaluation for the palliative treatment of Alzheimer's disease. In addition, its use in the pretreatment of poisoning by organophosphorous nerve agents could be another indication.     

Safety and Preliminary Efficacy of the Acetylcholinesterase Inhibitor Huperzine A as a Treatment for Cocaine Use Disorder

Background:

Cholinergic transmission is altered by drugs of abuse and contributes to psychostimulant reinforcement. In particular, acetylcholinesterase inhibitors, like huperzine A, may be effective as treatments for cocaine use disorder.

Methods:

The current report describes results from a double-blind, placebo-controlled study in which participants (n=14–17/group) were randomized to huperzine A (0.4 or 0.8mg) or placebo. Participants received randomized infusions of cocaine (0 and 40mg, IV) on days 1 and 9. On day 10, participants received noncontingent, randomized infusions of cocaine (0 and 20mg, IV) before making 5 choices to receive additional infusions.

Results:

Huperzine A was safe and well-tolerated and compared with placebo, treatment with huperzine A did not cause significant changes in any cocaine pharmacokinetic parameters (all P>.05). Time-course and peak effects analyses show that treatment with 0.4mg of huperzine A significantly attenuated cocaine-induced increases of “Any Drug Effect,” “High,” “Stimulated,” “Willing to Pay,” and “Bad Effects” (all P>.05).

Conclusions:

The current study represents a significant contribution to the addiction field since it serves as the first published report on the safety and potential efficacy of huperzine A as a treatment for cocaine use disorder.     

石杉碱甲新剂型研究进展

石杉碱甲是一种高效、高选择性的中枢乙酰胆碱酯酶抑制剂,其作用时间长、易透过血脑屏障、口服生物利用度高,在国内已广泛应用于阿尔采末病的治疗。目前上市的剂型主要为口服或注射的普通制剂,需多次使用,因此对于患有认知功能缺失的老年患者存在用药不便的问题。此外,多次给药还容易引起血药浓度大幅度波动,导致机体产生不良反应,因此对其新剂型的开发势在必行。本文综述了石杉碱甲新剂型相关研究进展,主要根据药物体内外释放特征、安全性、有效性等对现有的石杉碱甲口服缓控释制剂、长效缓释注射剂、缓释贴片、鼻用原位凝胶等剂型进行了比较,同时结合脑内乙酰胆碱变化水平与学习记忆关系的理论提出了新的剂型设计思路,以期为进一步开发安全、疗效可控和依从性高的新产品提供参考。     

Toxicological scoring of Alzheimer's disease drug huperzine in a guinea pig model

Huperzine is a secondary metabolite in lycopods Huprzia and an inhibitor of acetylcholinesterase and antagonist of N-methyl-D-apartate receptor. Huperine is a suitable drug for the treatment of Alzheimer's disease as it is a part of traditional Chinese medicine. Currently, it undergoes clinical trials in the European Union and United States. The toxicological data about huperzine are missing and link between huperzine and oxidative stress has not been extensively investigated. For the above mentioned reasons, we organized experiment on a guinea pig model aimed at the investigation of adverse effects caused by huperzine. Guinea pigs were exposed to (-)-huperzine A in doses 5-625 μg/kg. Animals were sacrificed one day after exposure. Ferric reducing antioxidant power, thiobarbituric acid reactive substances, glutathione reductase, caspase 3 activity and selected biochemical markers (e.g. transaminases, blood urea nitrogen and glucose) were assayed. In frontal, parietal, temporal lobes and cerebellum, we found increase of antioxidants, glutathione reductase and oxidative stress markers in a dose dependent manner. Effects on liver, kidney and spleen were milder. We discuss ambivalent action of huperzine in the body and judge the huperzine action owing to recently reported experiments.     

Reversible inhibition of acetylcholinesterase by carbamates or huperzine A increases residual activity of the enzyme upon soman challenge

The treatment options in soman poisoning are very limited due to rapid aging of the inhibited acetylcholinesterase, which makes the enzyme essentially intractable. Hence, oxime treatment probably comes too late in realistic scenarios. As an alternative, protecting part of the enzyme by reversible inhibition prior to soman exposure has been proposed. This strategy was successfully tested in animal experiments, but its efficacy still awaits complete understanding. In particular, it is unclear whether survival is improved by a higher residual activity of acetylcholinesterase during the acute phase, when the reversible and irreversible inhibitors are present together. In previous experiments with carbamate pre-treatment and paraoxon challenge we noticed an increased residual activity of erythrocyte acetylcholinesterase compared to non-pre-treatment. This result was encouraging to also test for comparable effects when using soman. Immobilized human erythrocytes were continuously perfused for real-time measurement of acetylcholinesterase activity by a modified Ellman method using 0.45mM acetylthiocholine. After having established the inhibition rate constant of soman, we tested the prophylactic potential of physostigmine, pyridostigmine and huperzine A. Pre-treatment with the reversible inhibitors inhibited the enzyme by 20-95%. Additional perfusion with 10nM soman for 30min resulted in a residual activity of 1-5%, at low and high pre-inhibition, respectively. The residual activity was markedly higher than in the absence of reversibly blocking agents (0.1%). After discontinuation of soman and the reversible inhibitors, enzyme activity recovered up to 30% following pre-inhibition by 50%. The experimental data agreed with computer simulations when feeding the kinetic-based model with the established rate constants. The results with soman essentially agreed with those obtained previously with paraoxon.     

石杉碱甲改善阿尔采末病相关线粒体功能损伤及潜在分子机制

阿尔采末病(AD)是一种常见的神经退行性疾病,目前临床上应用最广泛的AD治疗药物是乙酰胆碱酯酶抑制剂(AChEI)。石杉碱甲是我国自主研发的AChEI,近期研究显示除了胆碱酯酶抑制作用之外,石杉碱甲在大脑中动脉栓塞大鼠、APP/PS1转基因小鼠及β-淀粉样蛋白损伤的离体线粒体等多种模型中有显著的线粒体功能保护作用。本文对线粒体在AD发生发展中的作用和石杉碱甲对AD相关线粒体功能损伤的保护作用作一综述,并探讨其潜在的分子机制。     

Prolonged effects of poly(lactic-co-glycolic acid) microsphere-containing huperzine A on mouse memory dysfunction induced by scopolamine

Huperzine A is an anticholinesterase and cognitive enhancer, which is able to alleviate the symptoms of memory dysfunction in the mouse. The fast metabolization rate and narrow therapeutic spectrum makes it unfit for clinical use. Poly(lactic-co-glycolic acid) microsphere as delivery system effectively maintains the blood concentration of huperzine A by a slow-release effect over a long time. In the present article, we investigated the prolonged protective effect of microsphere-containing huperzine A on memory dysfunction induced by scopolamine. Spectrophotometric assay was used to determine the activity of acetylcholinesterase (AChE) and passive avoidance tests to evaluate memory performance. The results show that a bolus dose of microsphere-containing huperzine A (at a dose of 300 microg/kg or 600 microg/kg) administered intramuscularly can effectively maintain drug activity and significantly decrease the activity of AChE from day 3 to 14, the strongest effect seen on day 3 and 7. Accompanying the reduction of the activity of AChE, microsphere-containing huperzine A (300 microg/kg or 600 microg/kg) remarkably increased transfer latency time and no transfer response on the second trial through mitigating the memory impairments induced by scopolamine as compared to the scopolamine model group. Microsphere-containing huperzine A showed cognitive enhancing properties and anticholinesterase activity and may thus be a candidate for treatment of memory impairment.     

石杉碱甲胶囊和片剂治疗阿尔采末病的多中心双盲研究

目的：比较石杉碱甲胶囊和片剂治疗轻中度阿尔采末病（ＡＤ）的疗效和不良反应。方法：９２例ＡＤ病人随机分２组,胶囊组５５例（男性２７例,女性 ２８例,年龄 ６９ ａ ± ｓ７ ａ）;片剂组 ３７例（男性１７例,女性 ２０例,年龄 ７１ａ± ６ａ）。给石杉碱甲胶囊或片剂 ０． １５ ｍｇ,ｐｏ, ｂｉｄ × ８ ｗｋ。结果：胶囊组和片剂组总有效率分别为 ６９％, ５７％,２组比较差异无显著意义（Ｐ＞０．０５）。结论：石杉碱甲是一个安全、有效的治疗ＡＤ药物,肢囊剂型与片剂剂型疗效相似。     

Pharmacokinetics of huperzine A following oral administration to human volunteers.

The objective of the present study was to investigate the in vivo pharmacokinetics of huperzine A in healthy human volunteers. Twelve subjects (M 6, F 6; age ranged from 20-25 years) participated in the study. Huperzine A was administered in tablet form at a single dose of 0.4 mg. Following oral administration, the presence of huperzine A started to appear in the plasma at 5-10 min, and reached the peak concentrations with a Cmax of 2.59 +/- 0.37 ng/ml at 58.33 +/- 3.89 min (time to reach peak level, Tmax. The area under plasma vs time curve (AUC(0-t)) and the area under plasma from zero to infinity (AUC(0-infinity) for huperzine A were found to be 1986.96 +/- 164.57 microg/l.min and 2450.34 +/- 233.32 microg/l.min, respectively. The results of this study indicated that the pharmacokinetics of huperzine A conformed to a two-compartmental open model. The mean values of alpha and the beta half-life were 21.13 +/- 7.28 min and 716.25 +/- 130.18 min respectively, and showed a biphasic profile with rapid distribution followed by a slower elimination rate.     

SUBCHRONIC ADMINISTRATION OF PYRIDOSTIGMINE OR HUPERZINE TO PRIMATES: COMPARED EFFICACY AGAINST SOMAN TOXICITY

ABSTRACT

Organophosphonate (OP) nerve agents, such as soman, are potent irreversible inhibitors of central and peripheral acetylcholinesterases (AChEs). Pre-treatment of OP poisoning relies on the subchronic administration of a reversible AChE inhibitor. In the present limited study, the protective effects against soman toxicity of such compounds, i.e., the current pre-treatment pyridostigmine and huperzine, a proposed pre-treatment, are compared in primates. This is the first time primates are used to study the potential of pre-treatment with huperzine. Indeed, previous studies with huperzine used nonprimate models which are not the most appropriate for pre-treatment in humans. Each medication is given via a subcutaneous mini-osmotic pump for 6 days at a delivery rate providing about 20% inhibition of red cell AChE activity. In this trial with only four primates, huperzine selectively inhibits red cell AChE activity whereas pyridostigmine also inhibits plasma butyrylcholinesterase (BuChE). This latter may act as endogenous scavenger of OP compounds helping to confer additional protection against OPs. During intoxication, the cumulative dose of soman needed to produce convulsions and epileptic activity is 1.55-fold higher in the animals pre-treated with huperzine compared to those pre-treated with pyridostigmine. Thus, replacing PYR by HUP for a subchronic pre-treatment of primates gives them better tolerance to the epileptic effects of soman.     

Preparation andin vivo evaluation of huperzine A-loaded PLGA microspheres

Huperzine A-loaded microspheres composed of poly(D,L-lactide-co-glycolide) were prepared by an ONV emulsion solvent evaporation method. The characterization of the microspheres such as drug loading, size, shape and release profile was described. The in vitro release in the initial 7 days was nearly linear with 10% released per day. Thereafter drug release rate became slow gradually and about 90% drug released at day 21. The in vitro release rate determined by dialysis bag method had a good correlation with the in vivo release rate. Huperzine A aqueous solution was intramuscularly injected (i.m.) at 0.4 mg/kg and microspheres were intramuscularly injected at 8.4 mg eq huperzine A/kg in rats. The maxium plasma concentration (Cmax ) after i.m. microspheres was only 32% of that after i.m. solution. Drug in plasma could be detected until day 14 and about 5% of administered dose was residued at the injection site at day 14. The relative bioavailability of huperzine A microspheres over a period of 14 days was 94.7%. Inhibition of acyecholinesterase activity (AchE) in rat's cortex, hippocampus and striatum could sustain for about 14 days. In conclusion, huperzine A-loaded microspheres possessed a prolonged and complete drug release with significant inhibition of AchE for 2 weeks in rats.     

Prevention of peripheral side-effects of transdermal hyoscine by adjunctive therapy with low dosage of pyridostigmine.

1. The value of low dosage of pyridostigmine (30 mg three times daily) in preventing peripheral anti-muscarinic side effects of a transdermal controlled-release formulation of hyoscine, was tested in a double-blind placebo-controlled study, involving 47 healthy subjects. 2. Salivary excretion was repeatedly measured during 48 h of combined therapy of two transdermal hyoscine patches with pyridostigmine and 14 h after its cessation. Blood acetylcholinesterase activity was also measured, serving as an index of pyridostigmine bioavailability. 3. The adjunctive therapy with pyridostigmine was highly effective in preventing the substantial impairment in salivary flow caused by the transdermal formulation. An associated 23% inhibition of blood acetylcholinesterase activity was observed. 4. Small doses of pyridostigmine may therefore have a role in increasing the tolerability of transdermal hyoscine therapy. In some patients this drug combination might also allow some increment of the hyoscine dose.     

石杉碱甲治疗重症肌无力症128例

石杉碱甲(Huperzine A)对128例重症肌无力症治疗的有效率为99.2％。该药的作用维持时间比新斯的明为长(P<0.001)。副反应中肌束颤动、头晕、出汗和视力模糊出现率也较新斯的明为低,有显著差异;仅恶心较新斯的明为高,对全身主要脏器无明显不良反应。是值得推广的一种胆碱酯酶抑制药。