Effects of subchronic pyridostigmine pretreatment on the toxicity of soman

The effect of subchronic pyridostigmine pretreatment on the toxicity of soman, in the absence of supporting therapy (atropine, oxime, and (or) anticonvulsant), as well as its effect on muscarinic cholinoceptor binding characteristics was assessed in the rat. Pretreatment with pyridostigmine by means of an implanted Alzet osmotic minipump for a 5-day total exposure dose of 12 mg/kg inhibited whole blood acetylcholinesterase activity by 73%. This pyridostigmine pretreatment lowered the soman LD50 from 104 micrograms/kg in control animals to 82 micrograms/kg. In addition, the time to onset of soman-induced convulsions in pyridostigmine pretreated animals was significantly (p less than 0.001) reduced. Pyridostigmine pretreatment produced no significant effect on muscarinic cholinoceptor binding in brain or ileum. Lower doses of pyridostigmine pretreatment inhibited acetylcholinesterase activity (65 and 25%); however, LD50 and time to onset of convulsions following soman (140 micrograms/kg) were not significantly different from controls.     

Protection of red blood cell acetylcholinesterase by oral huperzine A against ex vivo soman exposure: next generation prophylaxis and sequestering of acetylcholinesterase over butyrylcholinesterase

As part of a phase Ib clinical trial to determine the tolerability and safety of the highly specific acetylcholinesterase (AChE) inhibitor huperzine A, twelve (12) healthy elderly individuals received an escalating dose regimen of huperzine A (100, 200, 300, and 400 microg doses, twice daily for a week at each dose), with three (3) individuals as controls receiving a placebo. Using the WRAIR whole blood cholinesterase assay, red blood cell AChE and plasma butyrylcholinesterase (BChE) were measured in unprocessed whole blood samples from the volunteers following each dose, and then for up to 48h following the final and highest (400 microg) dose to monitor the profile of inhibition and recovery of AChE. Significant inhibition of AChE was observed, ranging from 30-40% after 100 microg to >50% at 400 microg, and peaking 1.5h after the last dose. Gradual recovery of AChE activity then occurs, but even 48 h after the last dose red blood cell AChE was about 10% below control (pre-dose) values. Huperzine A levels in plasma peaked 1.5h after the final 400 microg dose (5.47+/-2.15 ng/mL). Plasma BChE was unaffected by huperzine A treatment (as expected). Aliquots of huperzine A-containing (from three individuals) and placebo blood samples were exposed ex vivo to the irreversible nerve agent soman (GD) for 10 min, followed by removal of unbound huperzine and soman from the blood by passing through a small C(18) reverse phase spin column. Eluted blood was diluted in buffer, and aliquots taken at various time intervals for AChE and BChE activity measurement to determine the time taken to achieve full return in activity of the free enzyme (dissociation from the active site of AChE by huperzine A), and thus the proportion of AChE that can be protected from soman exposure. Huperzine A-inhibited red blood cell (RBC) AChE activity was restored almost to the level that was initially inhibited by the drug. The increased doses of huperzine A used were well tolerated by these patients and in this ex vivo study sequestered more red blood cell AChE than has been previously demonstrated for pyridostigmine bromide (PB), indicating the potential improved prophylaxis against organophosphate (OP) poisoning.     

Protection of red blood cell acetylcholinesterase by oral huperzine A against ex vivo soman exposure: Next generation prophylaxis and sequestering of acetylcholinesterase over butyrylcholinesterase

As part of a phase Ib clinical trial to determine the tolerability and safety of the highly specific acetylcholinesterase (AChE) inhibitor huperzine A, twelve (12) healthy elderly individuals received an escalating dose regimen of huperzine A (100, 200, 300, and 400 μg doses, twice daily for a week at each dose), with three (3) individuals as controls receiving a placebo. Using the WRAIR whole blood cholinesterase assay, red blood cell AChE and plasma butyrylcholinesterase (BChE) were measured in unprocessed whole blood samples from the volunteers following each dose, and then for up to 48 h following the final and highest (400 μg) dose to monitor the profile of inhibition and recovery of AChE. Significant inhibition of AChE was observed, ranging from 30–40% after 100 μg to >50% at 400 μg, and peaking 1.5 h after the last dose. Gradual recovery of AChE activity then occurs, but even 48 h after the last dose red blood cell AChE was about 10% below control (pre-dose) values. Huperzine A levels in plasma peaked 1.5 h after the final 400 μg dose (5.47 ± 2.15 ng/mL). Plasma BChE was unaffected by huperzine A treatment (as expected).Aliquots of huperzine A-containing (from three individuals) and placebo blood samples were exposed ex vivo to the irreversible nerve agent soman (GD) for 10 min, followed by removal of unbound huperzine and soman from the blood by passing through a small C₁₈ reverse phase spin column. Eluted blood was diluted in buffer, and aliquots taken at various time intervals for AChE and BChE activity measurement to determine the time taken to achieve full return in activity of the free enzyme (dissociation from the active site of AChE by huperzine A), and thus the proportion of AChE that can be protected from soman exposure. Huperzine A-inhibited red blood cell (RBC) AChE activity was restored almost to the level that was initially inhibited by the drug. The increased doses of huperzine A used were well tolerated by these patients and in this ex vivo study sequestered more red blood cell AChE than has been previously demonstrated for pyridostigmine bromide (PB), indicating the potential improved prophylaxis against organophosphate (OP) poisoning.     

Relationship between reversible acetylcholinesterase inhibition and efficacy against soman lethality

Carbamate pretreatment (45% inhibition, reversible), combined with therapy, protected rats from soman-induced lethality [The Pharmacologist 23, 224 (1981)]. The present study was done to see if less than 45% inhibition protects and to see if reversible acetylcholinesterase (AChE) inhibition and efficacy against soman lethality are correlated. At 30 min pre-soman, guinea pigs and rats received (im) either pyridostigmine (Py) or physostigmine (Ph) to inhibit whole blood AChE from 10 to 70%; at 1 min post-soman (sc), they received (im) atropine (16 mg/kg)/2-PAMCl (50 mg/kg) and mecamylamine (0.8 mg/kg)/atropine (16 mg/kg), respectively. Protective ratios (PRs) were computed and they ranged from 3.1 to 7.7 for guinea pigs and from 1.8 to 2.4 for rats. In guinea pigs the PRs for Py + therapy were roughly similar to those of Ph + therapy. In both species at 30 min after im injection of Py and Ph, a linear relationship was found between percentage of whole blood AChE inhibition and ln dosage of carbamate. Positive correlation (p less than 0.05) was found between the degree of reversible AChE inhibition by pretreatment, coupled with therapy, and efficacy against soman lethality. The present data indicate that inhibition levels as low as 10% may provide some protection.     

Carbamate Protection of AChE Against Inhibition by Agricultural Chemicals

The carbamate pyridostigmine bromide has been used as a pretreatment to protect individuals from the nerve agent soman. Previous research showed that pyridostigmine significantly protected human muscle acetylcholinesterase in vitro from soman and bovine red blood cell acetylcholinesterase from some organophosphorous pesticides. Research presented here demonstrates that pretreatment with other carbamates also protects acetylcholinesterase from inhibition by the pesticides chlorpyrifos‐oxon and diazinon‐oxon, but not from malaoxon. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:506‐509, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21456     

+]-Huperzine A protects against soman toxicity in guinea pigs

The chemical warfare nerve agent (CWNA) soman irreversibly inhibits acetylcholinesterase (AChE) causing seizure, neuropathology and neurobehavioral deficits. Pyridostigmine bromide (PB), the currently approved pretreatment for soman, is a reversible AChE inhibitor that does not cross the blood–brain barrier (BBB) to protect against central nervous system damage. [−]-Huperzine A, a natural reversible AChE inhibitor, rapidly passes through the BBB and has numerous neuroprotective properties that are beneficial for protection against soman. However, [−]-Huperzine A is toxic at higher doses due to potent AChE inhibition which limits the utilization of its neuroprotective properties. [+]-Huperzine A, a synthetic stereoisomer of [−]-Huperzine A and a weak inhibitor of AChE, is non-toxic. In this study, we evaluated the efficacy of [+]-Huperzine A for protection against soman toxicity in guinea pigs. Pretreatments with [+]-Huperzine A, i.m., significantly increased the survival rate in a dose-dependent manner against 1.2× LD₅₀ soman exposures. Behavioral signs of soman toxicity were significantly reduced in 20 and 40 mg/kg [+]-Huperzine A treated animals at 4 and 24 h compared to vehicle and PB controls. Electroencephalogram (EEG) power spectral analysis showed that [+]-Huperzine A significantly reduces soman-induced seizure compared to PB. [+]-Huperzine A (40 mg/kg) preserved higher blood and brain AChE activity compared to PB in soman exposed animals. These data suggest that [+]-Huperzine A protects against soman toxicity stronger than PB and warrant further development as a potent medical countermeasure against CWNA poisoning.     

石杉碱甲结构改造的研究进展

石杉碱甲是高效、高选择性的可逆性乙酰胆碱酯酶抑制剂,是治疗早老性痴呆症的一个有前景的药物.本文概述了石杉碱甲的性质、结构和构效关系,从结构简化、C10、吡啶酮环、脂桥环、环外双键和桥头氨基等方面综述了石杉碱甲结构改造的研究进展,并描述了所得的新型石杉碱甲类似物的抗乙酰胆碱酯酶的生物活性.在已合成的大量的石杉碱甲类似物中,部分类似物的活性优于天然石杉碱甲,石杉碱甲结构改造的研究取得了可喜的进展.     

Protection by physostigmine against the pressor effect of soman in the rat

Intravenous injection of soman, 30 ug/kg, increased mean arterial blood pressure by 57 mmHg in urethane-anesthetized rats. The response declined slightly after a few minutes and then remained stable at about 39 mmHg for the next 20 minutes. The increase in pressure was accompanied by marked inhibition of brain acetylcholinesterase (AChE). In rats pretreated with a threshold pressor dose of physostigmine (50 ug/kg, i.v.), the peak pressor response to soman rose to the same level as that in the control group, but showed a more rapid recovery, reaching 17 mmHg at 20 minutes. The recovery of blood pressure was accompanied by partial recovery of brain AChE.     

Non-cholinergic Effects of Huperzine A: Beyond Inhibition of Acetylcholinesterase

The use of acetylcholinesterase inhibitors to decrease the breakdown of the neurotransmitter acetylcholine has been the main symptomatic therapy for mild to moderate Alzheimer’s patients, though the etiology of Alzheimer’s disease remains unclear and seems to involve multiple factors. Further evidence has indicated that some of these acetylcholinesterase inhibitors also have non-cholinergic functions on the pathogenesis of Alzheimer’s disease including the formation and deposition of β-amyloid. Huperzine A, a potent and reversible inhibitor of acetylcholinesterase that was initially isolated from a Chinese herb, has been found to improve cognitive deficits in a broad range of animal models and has been used for Alzheimer’s disease treatment in China. The novel neuroprotective effects of huperzine A might yield beneficial effects in Alzheimer’s disease therapy and provide a potential template for the design of new selective and powerful anti-Alzheimer’s drugs. The present paper gives an overview on the neuroprotective effects of huperzine A beyond its acetylcholinesterase inhibition. These effects include regulating β-amyloid precursor protein metabolism, protecting against β-amyloid-mediated oxidative stress and apoptosis. The structure–function relationship of huperzine A is also discussed.     

ORIGINAL ARTICLE: Evaluation of miosis,behavior and cholinesterase inhibition from low‐level,whole‐body vapor exposure to soman in African green monkeys (Chlorocebus sabeus)*

Background Relatively little is known about the effects of very low‐level exposures to nerve agents where few signs or symptoms are present. Methods African green monkeys (Chlorocebus sabeus) (n = 8) were exposed for 10 min, whole‐body, to a single concentration of soman (0.028–0.891 mg/m³). Results EC₅₀ values for miosis were determined to be 0.055 mg/m³ and 0.132 mg/m³ when defined as a 50 percent reduction in pupil area and diameter, respectively. In general, performance on a serial probe recognition task remained unchanged at lower concentrations, but responding was suppressed at the largest concentration tested. Soman produced concentration‐dependent inhibition of acetylcholinesterase activity and, to a lesser extent, butyrylcholinesterase activity. Conclusions These results characterize threshold soman exposure concentrations that produce miosis in the absence of other overt signs of toxicity and extend previous studies indicating that miosis is a valuable early indicator for the detection of soman vapor exposure.     

Evidence that Alterations in γ-Aminobutyric Acid and Acetylcholine in Rat Striata and Cerebella Are Not Related to Soman-Induced Convulsions

Many reports have suggested that gamma-aminobutyric acid (GABA) may play a role in organophosphate-induced convulsions. The balance between GABA and acetylcholine (ACh) in the brain also has been suggested by some investigators to be related to brain excitability. We examined these questions by studying the levels of GABA and ACh and the ratios of GABA to ACh in rat striata and cerebella (two major motor control areas in the CNS) after the administration of soman, an organophosphate acetylcholinesterase inhibitor also known as nerve gas. Male Sprague-Dawley rats weighing 250-300 g were injected subcutaneously with three different doses of soman: a subconvulsive dose of 40 micrograms/kg (approximately 30% of the ED50 for convulsions in rats), a convulsive dose of 120 micrograms/kg (approximately one ED50 for convulsions), and a higher convulsive dose of 150 micrograms/kg (approximately 120% of the ED50 for convulsions). The incidence and severity of convulsions were monitored in individual rats until they were sacrificed by focused microwave irradiation of the head at the following time points after soman administration: 4 min, a time prior to the onset of convulsions; 10 min, the time of onset of convulsions; 1 h, the time of peak convulsive activity; and 6 h, a time at which rats were recovering from convulsions. Results showed that in rat striata and cerebella, neither changes in levels of GABA and ACh nor changes in ratios of GABA to ACh were related to soman-induced convulsions, i.e., none of the changes in either levels or ratios of these two neurotransmitters were related to the initiation of, maintenance of, or recovery from soman-induced convulsions.     

Kinetics for the inhibition of acetylcholinesterase from the electric eel by some organophosphates and carbamates

The inhibition kinetics for some organophosphates (paroxon, diisopropylfluorophosphate, sarin, VX, soman and soman isomers) and carbamates (physostigmine, neostigmine, pyridostigmine and carbaryl) in the reaction with acetylcholinesterase from electric eel have been studied. Dissociation constants and rate constants for the irreversible step were determined. The great differences in inhibitory power of the organophosphates were almost entirely due to differences in affinity. A possible correlation between affinity and bonding rate is discussed.     

Huperzine A as Potential Treatment of Alzheimer's Disease: An Assessment on Chemistry,Pharmacology, and Clinical Studies

Alzheimer's disease (AD) is the fourth leading cause of death in adults, characterized by hallmark neuritic plaques and neurofibrillary tangles. Current treatments focus only on symptom relief. As a possible new treatment option for AD, huperzine A's chemistry, pharmacology, and clinical effectiveness are assessed. The chemical synthesis of huperzine A has been optimized, while an in vitro technique has provided a renewable plant source. Pharmacological studies showed that the drug inhibits the enzyme acetylcholinesterase reversibly and selectively. Huperzine A also displayed good pharmacokinetics with a rapid absorption and a wide distribution in the body at a low to moderate rate of elimination. Presently, inadequate toxicity data in human have been reported, yet animal studies demonstrated mild to moderate cholinergic side effects at therapeutic doses. Previous clinical trials have shown improvement in memory function using MMSE, MQ, ADAS‐COG, and ADL tests. In an unpublished phase II clinical trial, the ADAS‐COG and MMSE tests indicated cognitive enhancement at a dose of 0.4 mg, yet no improvement was observed at a dose of 0.2 mg. The MMSE scores indicated cognitive enhancement at 0.4 mg. Promising data suggested that huperzine A is well tolerated at doses up to 0.4 mg for 24 weeks. Therefore, huperzine A seems to be a potential treatment option for AD.     

Identification of a novel endophytic fungus from Huperzia serrata which produces huperzine A

Huperzine A is isolated from Huperzia serrata and is used for treatment of Alzheimer’s disease, due to its low toxicity and long effective period. The decrease in H. serrata sources means that natural huperzine A cannot meet the needs of clinical therapy. In this study, >200 endophytic fungal strains were isolated from H. serrata, and screened using high-performance liquid chromatography. Strain ES026 produced huperzine A. Production was identified and quantified by liquid chromatography–tandem mass spectrometry, and the yield of huperzine A was 1 μg/g dried mycelium. ES026 strain was identified as Colletotrichum gloeosporioides by morphology, polymerase chain reaction with species-specific primers and rDNA internal transcribed spacer sequence. ES026 contributes to the breeding of cultivated strains with high yield of huperzine A. Meanwhile, the strain was suitable for the study of biosynthesis of huperzine A.     

Protection and induced reactivation of cholinesterase by HS-6 in rabbits exposed to soman

Rabbits intoxicated with soman were treated with various doses of HS-6 at 3 min following administration of soman to establish whether the antidotal efficacy reported for HS-6 against soman can be attributed in part to reactivation of the inhibited cholinesterase (ChE) enzymes. Within 5 min after treating animals intoxicated with soman with 15 or 30 mg/kg of HS-6 (iv) the whole blood ChE activity increased from 6.0 to 30.5 and 44.2% of control activity, respectively. Because HS-6 apparently is able to reactivate completely the unaged inhibited enzyme, HS-6, 60 mg/kg (iv) was used to measure for the first time the $\text{in vivo}$ rate of aging of whole blood ChE in soman-intoxicated rabbits. The half time for aging was determined to be 7.6 (5.8 ? 9.4) min, P = 0.05. HS-6 in combination with atropine and pyridostigmine was tested as a pretreatment against soman. When only atropine + pyridostigmine was used in the pretreatment regimen, none of the rabbits survived a 10 LD50 dose of soman (iv). However, when HS-6 (30 mg/kg, iv) was used together with atropine + pyridostigmine in the pretreatment regimen, 87% of the animals survived this high dose of soman. Since HS-6 is a powerful reactivator of unaged, soman-inhibited ChE, the antidotal effectiveness of HS-6 against soman can be attributed in part to the restoration of vital enzyme activity.     

Synergism of toxicity of N,N-diethyl-m-toluamide to German cockroaches (Orthoptera: Blattellidae) by hydrolytic enzyme inhibitors

Various compounds were tested for effects on the toxicity of the insect repellent N, N-diethyl-m-toluamide (DEET) in German cockroaches, Blattella germanica (L.). Organophosphate and carbamate acetylcholinesterase inhibitors carbaryl, DEF, eserine (physostigmine, malathion and pyridostigmine bromide synergized DEET toxicity also synergized the toxicity of the formamidine pesticides. Amitraz and chlordimeform. Results suggest that DEET may have some toxic actions that are similar to those of formamidine pesticides. DEET synergized the toxicity of some acetylcholinesterase inhibitors but not others. Results further suggest that some mechanism other than acetylcholinesterase inhibition was responsible for the toxic interactions observed between DEET and the acetylcholinesterase inhibitors.     

Soman intoxication in hypothyroid rats: alterations in brain neuronal RNA,acetylcholinesterase and survival

Studies were conducted to investigate relationships among soman (pinacolyl methylphosphonofluoridate) induced seizure activity, central metabolic impairments and lethality in normal vs thyroid-deficient rats. Quantitative cytophotometric measurements of individual cerebrocortical (layer V) and striatal neuron RNA contents were made following dosages of 0.5, 0.9 and 1.5 LD₅₀ soman (LD₅₀ = 135 μg/kg, sc). Hypothyroidism was associated with a marked diminution of overt convulsive activity and reduced susceptibility to lethal actions of soman as indicated by enhanced 24- and 48-h survival rates at 0.9, 1.2 and 1.5 LD₅₀. Hypothyroidism per se produced RNA depletion in both cortical and striatal neurons. Soman treatment diminished cortical RNA to essentially the same extent in thyroid-deficient rats as in euthyroids, whereas there was no further reduction of striatal neuron RNA. It was found that amelioration of convulsive activity and lethal- ity in hypothyroid rats was accompanied by reduced cerebral acetylcholinesterase (AChE, EC 3.1.1.7) inactivation, and that plasma cholinesterase (EC 3.1.1.8) and aliesterase (EC 3.1.1.1) levels were significantly higher in hypothyroid than in euthyroid saline-control rats. The overall data indicate that soman- induced central metabolic impairments can occur independent of paroxysmal neural activity and lethal actions of the agent. Resistance to soman observed with thyroid deficiency may be due in large part to increased binding to plasma enzymes and diminished delivery of soman to AChE in vital cholinergic sites.     

Galantamine and carbon monoxide protect brain microvascular endothelial cells by heme oxygenase-1 induction

Galantamine, a reversible inhibitor of acetylcholine esterase (AChE), is a novel drug treatment for mild to moderate Alzheimer’s disease and vascular dementia. Interestingly, it has been suggested that galantamine treatment is associated with more clinical benefit in patients with mild-to-moderate Alzheimer disease compared to other AChE inhibitors. We hypothesized that the protective effects of galantamine would involve induction of the protective gene, heme oxygenase-1 (HO-1), in addition to enhancement of the cholinergic system. Brain microvascular endothelial cells (mvECs) were isolated from spontaneous hypertensive rats. Galantamine significantly reduced H₂O₂-induced cell death of mvECs in association with HO-1 induction. These protective effects were completely reversed by nuclear factor-κB (NF-κB) inhibition or HO inhibition. Furthermore, galantamine failed to induce HO-1 in mvECs which lack inducible nitric oxide synthase (iNOS), supplementation of a nitric oxide (NO) donor or iNOS gene transfection on iNOS-deficient mvECs resulted in HO-1 induction with galantamine. These data suggest that the protective effects of galantamine require NF-κB activation and iNOS expression, in addition to HO-1. Likewise, carbon monoxide (CO), one of the byproducts of HO, up-regulated HO-1 and protected mvECs from oxidative stress in a similar manner. Our data demonstrate that galantamine mediates cytoprotective effects on mvECs through induction HO-1. This pharmacological action of galantamine may, at least in part, account for the superior clinical efficacy of galantamine in vascular dementia and Alzheimer disease.