An evaluation of the inhibition of human butyrylcholinesterase and acetylcholinesterase by the organophosphate chlorpyrifos oxon

Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) are enzymes that belong to the superfamily of α/β-hydrolase fold proteins. While they share many characteristics, they also possess many important differences. For example, whereas they have about 54% amino acid sequence identity, the active site gorge of acetylcholinesterase is considerably smaller than that of butyrylcholinesterase. Moreover, both have been shown to display simple and complex kinetic mechanisms, depending on the particular substrate examined, the substrate concentration, and incubation conditions. In the current study, incubation of butyrylthiocholine in a concentration range of 0.005–3.0 mM, with 317 pM human butyrylcholinesterase in vitro, resulted in rates of production of thiocholine that were accurately described by simple Michaelis–Menten kinetics, with a K_m of 0.10 mM. Similarly, the inhibition of butyrylcholinesterase in vitro by the organophosphate chlorpyrifos oxon was described by simple Michaelis–Menten kinetics, with a k_i of 3048 nM⁻¹ h⁻¹, and a K_D of 2.02 nM. In contrast to inhibition of butyrylcholinesterase, inhibition of human acetylcholinesterase by chlorpyrifos oxon in vitro followed concentration-dependent inhibition kinetics, with the k_i increasing as the inhibitor concentration decreased. Chlorpyrifos oxon concentrations of 10 and 0.3 nM gave k_is of 1.2 and 19.3 nM⁻¹ h⁻¹, respectively. Although the mechanism of concentration-dependent inhibition kinetics is not known, the much smaller, more restrictive active site gorge of acetylcholinesterase almost certainly plays a role. Similarly, the much larger active site gorge of butyrylcholinesterase likely contributes to its much greater reactivity towards chlorpyrifos oxon, compared to acetylcholinesterase.     

Evaluation of a new lead for acetylcholinesterase inhibition

Acetylcholine is the most common neurotransmitter of the parasympathetic nervous system. Several cholinergic drugs are widely used in Alzheimer’s disease, urinary retention, glaucoma, and myasthenia gravis. Acetylcholinesterase is the enzyme which destroys naturally occurring acetylcholine, and acetylcholinesterase inhibitors (AChEIs) which block the enzyme are currently used in the treatment of several diseases, most importantly Alzheimer’s disease. Previous studies from our laboratory have shown a few isoquinoline analogs to be potential acetylcholinesterase inhibitors. In the present study we have extended analysis of the structure–activity relationship (SAR) of the isoquinoline ring system as an AChEI. The corresponding reduced form of 1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline, ring-opened analog, and related analogs were evaluated for AChE inhibitory activity. Our results showed that the dihydroisoquinoline ring analog compound 6 (IC₅₀ = 7.0 ± 1.4 μM) and compound 4 (IC₅₀ = 5.5 ± 1.0 μM) displayed potent AChE inhibition. These compounds are currently the lead compounds for further studies in our laboratory.     

Acetylcholinesterase,butyrylcholinesterase, and tyrosinase inhibition studies and antioxidant activities of 33 Scutellaria L. taxa from Turkey

The members of Scutellaria L. (Lamiaceae) is known to be rich particularly in flavonoids and among them, S. baicalensis has been recorded to be used for memory-enhancing purpose. Therefore, we initiated a study to screen the methanol extracts prepared from the aerial parts of 33 Turkish Scutellaria species for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities, which are the key enzymes taking place in pathogenesis of Alzheimer’s disease. Besides, the methanol extracts were tested in vitro against another enzyme, tyrosinase, which is associated with melanin hyperpigmentation. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenger effect, ferrous ion-chelating ability, and ferric-reducing antioxidant power (FRAP) of the ethyl acetate and methanol extracts were also determined. AChE, BChE, and tyrosinase inhibition of the extracts were performed on ELISA microplate reader by spectrophotometric method. The extracts showed weak inhibition against AChE and BChE, while the best tyrosinase inhibition was caused by the methanol extract of S. brevibracteata subsp. subvelutina. The extracts had a very high DDPH radical scavenging effect and moderate antioxidant activity in ferrous ion-chelating and FRAP tests.     

Frequency and enzyme activity of the butyrylcholinesterase K-variant in a Turkish population

Objective Among variants of the butyrylcholinesterase gene (BChE), the K-variant causing Ala539Thr substitution is the most common one associated with about one-third reduction in the enzyme activity. This study aimed to detect the frequency of the K-variant allele in a Turkish population sample and also to evaluate how the plasma BChE activity was influenced by this variant.Methods Patients administered for elective surgery (n=77) were examined for the presence of the K allele. The enzyme activity was determined in plasma.Results The K-variant of BChE is a common allele with a frequency of 0.266 (CI_95% 0.196–0.336) in our sample from a Turkish population. Mean enzyme activity in subjects homozygous for the K-variant was about 40% lower than other subjects.Conclusion The frequency of the BChE K-variant was significantly higher in a Turkish population than those reported for other populations and it is associated with a diminished enzyme activity.     

Synthesis and acetylcholinesterase and butyrylcholinesterase inhibitory activities of 7-alkoxyl substituted indolizinoquinoline-5,12-dione derivatives

A series of novel 7‐alkoxyl substituted indolizinoquinoline‐5,12‐dione derivatives were synthesized. The cholinesterase inhibition assays indicated that most synthesized compounds exhibited good activity for acetylcholinesterase (AChE) and high selectivity index of AChE over butyrylcholinesterase (BuChE). Compound 12b exhibited the most potent AChE inhibitory activity with an IC₅₀ value of 0.068 µM and the highest selectivity index of 144. Kinetic study of AChE indicated that a mixed type of inhibition pattern existed for these indolizinoquinoline‐5,12‐dione derivatives. Molecular docking study indicated that compound 12b could bind to both the catalytically active site and the peripheral anionic site of AChE.     

Assessment of user safety, exposure and risk to veterinary medicinal products in the European Union

Safety is an important part of veterinary drug assessment while user safety is a critical part of the overall safety assessment. In the European Union (EU), user safety is addressed through preclinical studies and by relationships with exposure but a key part of the process is the user safety assessment. EU user safety guidelines are available and these make certain recommendations but in places they lack detail and clarity. This paper seeks to examine the relevant factors that lie behind user risk assessments for veterinary medicinal products in general while focusing on EU requirements, the determination of risk management and risk communication strategies and how this relates to user safety assessment and pharmacovigilance responsibilities.     

The characteristics of the inhibition of serum cholinesterase by metoclopramide

The inhibition of serum cholinesterase by metoclopramide has been previously characterised in vitro at high dilution of the enzyme. We examined the effect of varying enzyme dilution over a range of 1000 fold dilution, and assay temperature at 25°C and 37°C on the fractional inhibition of enzyme activity by metoclopramide. Neither enzyme concentration nor reaction temperature affected this fractional inhibition. Concentrations of metoclopramide producing 50% inhibition of enzyme activity were in the range 1.0–1.9×10^–6 M. Lineweaver-Burk analysis of the enzyme reaction suggests that the pattern of this inhibition is competitive.     

兽用抗菌药耐药性风险评估研究进展

随着我国畜牧业的快速发展,兽用抗菌药物被大量使用于动物身上,并且用量逐年上升。由于兽用抗菌药物的不合理使用,引发了耐药性的产生,从而出现了一系列治疗失败问题,给人类健康造成了潜在的危害。 因此抗菌药物的使用对人和动物健康所造成的风险受到广泛关注,目前兽用抗菌药物风险评估技术已被广泛用于预测药物引发的耐药性风险大小,揭示兽用抗菌药物的使用产生的对人和动物健康造成威胁的耐药性风险。本文主要对国内外开展的兽用抗菌药耐药性风险评估的研究进行综述,为我国开展耐药风险评估提供借鉴。     

Design,synthesis, and evaluation of novel cinnamic acid-tryptamine hybrid for inhibition of acetylcholinesterase and butyrylcholinesterase

BackgroundAcetylcholine deficiencies in hippocampus and cortex, aggregation of β-amyloid, and β-secretase over activity have been introduced as main reasons in pathogenesis of Alzheimer’s disease.MethodsColorimetric Ellman’s method was used for determination of IC₅₀ value in AChE and BChE inhibitory activity. The kinetic studies, neuroprotective and β-secretase inhibitory activities, evaluation of inhibitory potency on β-amyloid (Aβ) aggregations induced by AChE, and docking study were performed for prediction of the mechanism of action.Result and discussionA new series of cinnamic acids-tryptamine hybrid was designed, synthesized, and evaluated as dual cholinesterase inhibitors. These compounds demonstrated in-vitro inhibitory activities against acetyl cholinesterase (AChE) and butyryl cholinesterase (BChE). Among of these synthesized compounds, (E)-N-(2-(1H-indol-3-yl)ethyl)-3-(3,4-dimethoxyphenyl)acrylamide (5q) demonstrated the most potent AChE inhibitory activity (IC₅₀ = 11.51 μM) and (E)-N-(2-(1H-indol-3-yl)ethyl)-3-(2-chlorophenyl)acrylamide (5b) were the best anti-BChE (IC₅₀ = 1.95 μM) compounds. In addition, the molecular modeling and kinetic studies depicted 5q and 5b were mixed type inhibitor and bound with both the peripheral anionic site (PAS) and catalytic sites (CAS) of AChE and BChE. Moreover, compound 5q showed mild neuroprotective in PC12 cell line and weak β-secretase inhibitory activities. This compound also inhibited aggregation of β-amyloid (Aβ) in self-induced peptide aggregation test at concentration of 10 μM.ConclusionIt is worth noting that both the kinetic study and the molecular modeling of 5q and 5b depicted that these compounds simultaneously interacted with both the catalytic active site and the peripheral anionic site of AChE and BChE. These findings match with those resulted data from the enzyme inhibition assay. Graphical abstractOpen in a separate windowA new series of cinnamic-derived acids-tryptamine hybrid derivatives were designed, synthesized and evaluated as butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) inhibitors and neuroprotective agents. Compound 5b and 5q, as the more potent compounds, interacted with both the peripheral site and the choline binding site having mixed type inhibition. Results suggested that derivatives have a therapeutic potential for the treatment of AD.Electronic supplementary materialThe online version of this article (10.1007/s40199-020-00346-9) contains supplementary material, which is available to authorized users.     

Selective inhibition by fenflumizole tissue cyclooxygenase in rabbits ex vivo

A new non-steroidal anti-inflammatory drug fenflumizole when administered orally to rabbits at therapeutic doses of 10 and 30 mg/kg affects neither PGI2 generation by arterial slices nor PGE2 generation by gastric mucosa, respectively. Fenflumizole at doses of 10 and 30 mg/kg per os inhibits platelet aggregability and generation of TXA2 by platelets. It is suggested that this selective action of fenflumizole on tissue cyclooxygenase ex vivo may justify with fenflumizole in anti-thrombotic therapy.     

Malathion toxicity: In vivo inhibition of acetylcholinesterase in the fish Brachydanio rerio (Cyprinidae)

Acetylcholinesterase (AChE) activity in the nervous tissue (brain) of the fish Brachydanio rerio was significantly inhibited by exposure to the organophosphorus (OP) pesticide, malathion. The inhibition was dose- as well as time-dependent. The fish survived even when the activity of the enzyme was inhibited by 90%. There was a significant recovery in the activity of AChE when malathion stress was lifted.     

Resistance to organophosphorus agent toxicity in transgenic mice expressing the G117H mutant of human butyrylcholinesterase

Organophosphorus toxicants (OP) include chemical nerve agents and pesticides. The goal of this work was to find out whether an animal could be made resistant to OP toxicity by genetic engineering. The human butyrylcholinesterase (BChE) mutant G117H was chosen for study because it has the unusual ability to hydrolyze OP as well as acetylcholine, and it is resistant to inhibition by OP. Human G117H BChE, under the control of the ROSA26 promoter, was expressed in all tissues of transgenic mice. A stable transgenic mouse line expressed 0.5 microg/ml of human G117H BChE in plasma as well as 2 microg/ml of wild-type mouse BChE. Intestine, kidneys, stomach, lungs, heart, spleen, liver, brain, and muscle expressed 0.6-0.15 microg/g of G117H BChE. Transgenic mice were normal in behavior and fertility. The LD50 dose of echothiophate for wild-type mice was 0.1 mg/kg sc. This dose caused severe cholinergic signs of toxicity and lethality in wild-type mice, but caused no deaths and only mild toxicity in transgenic animals. The mechanism of protection was investigated by measuring acetylcholinesterase (AChE) and BChE activity. It was found that AChE and endogenous BChE were inhibited to the same extent in echothiophate-treated wild type and transgenic mice. This led to the hypothesis that protection against echothiophate toxicity was not explained by hydrolysis of echothiophate. In conclusion, the transgenic G117H BChE mouse demonstrates the factors required to achieve protection from OP toxicity in a vertebrate animal.     

Intravenous butyrylcholinesterase administration and plasma and brain levels of cocaine and metabolites in rats

Butyrylcholinesterase is a major cocaine-metabolizing enzyme in humans and other primates, catalyzing hydrolysis to ecgonine methylester. Increasing butyrylcholinesterase activity may be a treatment for cocaine addiction. We evaluated the effect of 30-min pretreatment with horse-derived butyrylcholinesterase (5-15,000 U i.v.) or with the selective butyrylcholinesterase inhibitor cymserine (10 mg/kg i.v.) on the metabolism of cocaine (17 mg/kg i.p.) in anesthetized rats. Venous blood samples were collected for two hours after cocaine administration and later assayed for cocaine and metabolites by gas chromatography/mass spectroscopy. Whole brains were collected after the last blood sample and similarly assayed. Butyrylcholinesterase significantly increased plasma and brain ecgonine methylester levels and decreased cocaine plasma half-life from 26.2 min (saline) to 16.4 min (15,000 U). Butyrylcholinesterase had no significant effect on plasma or brain cocaine or benzoylecgonine levels. Cymserine had no effect on any variable. These findings suggest that butyrylcholinesterase treatment may have benefits in enhancing cocaine metabolism and in increasing levels of ecgonine methylester, which may have a protective action against cocaine.     

In vitro sensitivity of cholinesterases and [3H]oxotremorine-M binding in heart and brain of adult and aging rats to organophosphorus anticholinesterases

Organophosphorus (OP) insecticides elicit toxicity via acetylcholinesterase inhibition, allowing acetylcholine accumulation and excessive stimulation of cholinergic receptors. Some OP insecticides bind to additional macromolecules including butyrylcholinesterase and cholinergic receptors. While neurotoxicity from OP anticholinesterases has been extensively studied, effects on cardiac function have received less attention. We compared the in vitro sensitivity of acetylcholinesterase, butyrylcholinesterase and [³H]oxotremorine-M binding to muscarinic receptors in the cortex and heart of adult (3 months) and aging (18 months) rats to chlorpyrifos, methyl parathion and their active metabolites chlorpyrifos oxon and methyl paraoxon. Using selective inhibitors, the great majority of cholinesterase in brain was defined as acetylcholinesterase, while butyrylcholinesterase was the major cholinesterase in heart, regardless of age. In the heart, butyrylcholinesterase was markedly more sensitive than acetylcholinesterase to inhibition by chlorpyrifos oxon, and butyrylcholinesterase in tissues from aging rats was more sensitive than enzyme from adults, possibly due to differences in A-esterase mediated detoxification. Relatively similar differences were noted in brain. In contrast, acetylcholinesterase was more sensitive than butyrylcholinesterase to methyl paraoxon in both heart and brain, but no age-related differences were noted. Both oxons displaced [³H]oxotremorine-M binding in heart and brain of both age groups in a concentration-dependent manner. Chlorpyrifos had no effect but methyl parathion was a potent displacer of binding in heart and brain of both age groups. Such OP and age-related differences in interactions with cholinergic macromolecules may be important because of potential for environmental exposures to insecticides as well as the use of anticholinesterases in age-related neurological disorders.     

Life without acetylcholinesterase: the implications of cholinesterase inhibitor toxicity in AChE-knockout mice

The acetylcholinesterase (AChE)-knockout mouse is a new tool for identifying physiologically relevant targets of organophosphorus toxicants (OP). If AChE were the only important target for OP toxicity, then mice with zero AChE would have been expected to be resistant to OP. The opposite was found. AChE−/− mice were more sensitive to the lethality of DFP, chlorpyrifos oxon, iso-OMPA, and the nerve agent VX. A lethal dose of OP caused the same cholinergic signs of toxicity in mice with zero AChE as in mice with normal amounts of AChE. This implied that the mechanism of toxicity of a lethal dose of OP in AChE−/− mice was the same as in mice that had AChE, namely accumulation of excess acetylcholine followed by overstimulation of receptors. OP lethality in AChE−/− mice could be due to inhibition of BChE, or to inhibition of a set of proteins. A search for additional targets used biotinylated-OP as a marker. In vitro experiments found that biotinylated-OP appeared to label as many as 55 proteins in the 100,000 × g supernatant of mouse brain. Chlorpyrifos oxon bound a set of proteins (bands 12, 41, 45) that did not completely overlap with the set of proteins bound by diazoxon (bands 9, 12, 41, 47) or dichlorvos (bands 12, 23, 24, 32, 44, 45, 51) or malaoxon (band 9). These results support the idea that a variety of proteins could be interacting with a given OP to give the neurotoxic symptoms characteristic of a particular OP.     

Selective in vivo inhibition by quinidine of methoxyphenamine oxidation in rat models of human debrisoquine polymorphism

1. Lewis and Dark Agouti (DA) rat strains (n = 4), models of human extensive and poor metabolizer phenotypes of debrisoquine/sparteine, respectively, were dosed with methoxyphenamine with and without prior administration of quinidine. Methoxyphenamine and its three metabolites, namely N-desmethylmethoxyphenamine, O-desmethylmethoxyphenamine and 5-hydroxymethoxyphenamine were quantified in 0-24 h urine. 2. The oxidative metabolic routes of methoxyphenamine which had been previously shown to involve the debrisoquine/sparteine isozyme, namely O-demethylation and aromatic 5-hydroxylation, were both significantly inhibited by quinidine in the two rat strains. 3. The oxidative metabolic route of methoxyphenamine which had been previously shown to not involve the debrisoquine/sparteine isozyme, namely N-demethylation, was not significantly inhibited by quinidine in either rat strain. 4. The Lewis strain pretreated with quinidine resembled the DA strain without such pretreatment in terms of O-desmethylmethoxyphenamine and 5-hydroxymethoxyphenamine in that the mean percentages of the dose excreted as these two metabolites and the mean O-desmethylmethoxyphenamine/methoxyphenamine and 5-hydroxymethoxyphenamine/methoxyphenamine ratios were similar to one another. 5. Ten days after quinidine administration to the Lewis strain of rat, all parameters of methoxyphenamine and its metabolites returned to normal. 6. A protocol involving substrate administration to Lewis strain rats with and without prior administration of quinidine could be developed as an attractive approach to screen substrates for metabolism in vivo by the debrisoquine/sparteine isozyme. Such an approach obviates interstrain differences.     

Inhibition of acetylcholinesterase in different parts of the brain of mice by isopropyl methylphosphonofluoridate in vitro and in vivo

AChE activity in four parts of the mouse brain, i.e. in the pons and medulla oblongata, mesencephalon, diencephalon and basal ganglia, was inhibited in vitro by isopropyl methylphosphonofluoridate. The inhibition constants, n (Hill coefficient) and I₅₀ were determined. The values of both constants, n and I₅₀, were the same for all studied parts of the brain (n=1.7 and I₅₀=5.0 × 10^–10 M). In experiments in vivo, mice were administered the poison in doses ranging from 1.4 to 28.6 mol×10^–7/kg, i.e., 0.02 to 0.40 mg/kg. AChE activity was then measured and the degree of inhibition was correlated with the dose of organophosphate given. AChE in the basal ganglia was the most resistant. The highest degree of inhibition was observed in the ponto-medullar portion. This selective inhibition lends support to a concept of this particular portion of the brain as having special importance in the toxidynamics of poisoning. The comparison of AChE inhibition in vitro and in vivo suggests that only about 1% of an injected dose is involved inhibiting AChE in the brain.     

Effect of rivastigmine in the treatment of behavioral disturbances associated with dementia: review of neuropsychiatric impairment in Alzheimer's disease

ABSTRACT

Cognitive decline is conventionally regarded as the defining clinical symptom of Alzheimer's disease (AD), but behavioral and neuropsychiatric symptoms are also present throughout the course of the disease. In fact, behavioral symptoms may appear before cognitive decline is diagnosed. The presence of these symptoms may predict an increasing need for community-based services or even nursing home placement. The characteristic behavioral and neuropsychiatric symptoms associated with AD may be related to the same pathophysiology that underlies the cognitive abnormalities. AD is characterized by a loss of cholinergic neurons as well as by the presence of neurofibrillary tangles (NFTs) and senile plaques in brain regions with cholinergic deficits, resulting in a deficiency in acetylcholine (ACh) in areas of the brain that modulate cognition, behavior, and emotion. Cholinesterase inhibitors are thought to augment or maximize the concentration of ACh in the synaptic cleft. Rivastigmine is a dual inhibitor of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), enzymes involved in hydrolysis of ACh. Literature searches using MEDLINE and EMBASE databases were performed to identify studies of rivastigmine (through August 2005) that assessed neuropsychiatric aspects of AD. Rivastigmine has been demonstrated to be safe and effective in stabilizing or improving the cognitive symptoms of AD in 3 large, well-controlled, randomized clinical trials, which also demonstrated that rivastigmine improves overall global functioning. Smaller studies and meta-analyses of pooled data from the 3 large trials have suggested that rivastigmine may improve the behavioral and neuropsychiatric symptoms associated with AD.     

Protective effect of polyurethane immobilized human butyrylcholinesterase against parathion inhalation in rat

Organophosphates (OP) are used in large quantities around the world as agricultural insecticides. Exposure to these toxic chemicals is a serious global health problem. Human plasma butyrylcholinesterase is known to be a good scavenger of organophosphorus pesticides and chemical warfare agents. In this study, purified human plasma butyrylcholinesterase (HuBChE) from pooled outdated human plasma, was immobilized onto the polyurethane foam. The immobilized enzyme showed greater stability at and above room temperature (up to 55 °C), compared to the enzyme in solution. Scavenger properties of immobilized enzyme were tested in vitro with parathion and its active metabolite paraoxon. In, in vitro experiments polyurethane foam with immobilized active enzyme removed 40% of parathion and 50% of paraoxon inhibitory effect (based on cholinesterase inhibition). In, in vivo experiments groups of rats inhaled parathion through filters with immobilized active enzyme (Group I), immobilized inactivated enzyme (Group II), and control group (Group III) inhaled solvent only without any parathion or filter. In the Group II animals, activity of plasma and red blood cells cholinesterase was significantly decreased (30 and 28%, respectively) compared to Groups I and III animals. In other tissues such as brain, skeletal muscle and lung, activity of the acetylcholinesterase (AChE) in the Group II animals, was decreased significantly (29, 28, and 22%, respectively). There was no significant differences between Groups I and III animals enzyme activities. In conclusion, immobilized butyrylcholinesterase (BChE) may be useful in scavenging and detoxifying organophosphate compounds both for medical protection and decontamination procedures.