Genetic Association of Butyrylcholinesterase with Major Depressive Disorder.

Major depressive disorder (MDD) is characterized as clinical depression, which primarily affects the mood and behaviour of an individual. In the present study butyrylcholinesterase (BChE), a co-regulatory cholinergic neurotransmitter enzyme implicated in several putative neuronal and non-neuronal physiological roles was investigated for its role in MDD. Eighty MDD patients and sixty-one healthy controls were recruited for the study. BChE activity was measured by Ellman's method using serum while DNA samples of the patients were genotyped for BCHE polymorphisms rs3495 (c.*189G > A) and rs1803274 (c.1699G > A) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and tetra-primer Amplification Refractory Mutation System- polymerase chain reaction (ARMS-PCR). The genotyping was further validated by Sanger Sequencing. Biochemical estimation of serum BChE levels revealed a statistically significant decrease of enzyme activity in MDD patients (69.96) as compared to healthy controls (90.97), which was independent of age and gender. BCHE single nucleotide polymorphism rs1803274 genotype GA was found to be associated with the disease under a dominant model (OR 2.32; 95% CI 1.09-4.96; p value = 0.025). Furthermore, risk allele-A frequency was higher in cases (p value = 0.013) than control. Carriers of rs1803274 GA genotype showed reduced mean BChE activity than wild-type allele GG homozygotes (p value = 0.040). Gender-based analysis revealed a protective role of rs3495 in females (χ2 = 6.87, p value = 0.032, RM: OR 0.173, CI = 0.043-0.699 (p value = 0.017). In addition, rs1803274 risk allele-A was observed to be significantly higher in males (χ2 = 4.258, p value = 0.039). In conclusion, the present study is indicative of a role of BChE in the pathophysiology of MDD where genetic polymorphisms were observed to effect BChE activity. Further replication studies in different ethnicities are recommended to validate the current observations.

Antioxidant enzymes and association of CAT SNP-21 A/T (rs7943316) with male infertility.

Infertility is a multifactorial and polygenic disease. A vast majority of infertility is still unexplained despite modern diagnostic techniques. Oxidative stress is considered a factor for male infertility but etiology in terms of functional gene polymorphism and experimental studies on human subjects is scarcely reported. The aim of the study was to investigate the status of three antioxidant enzymes; catalase, superoxide dismutase (SOD), and glutathione reduced (GSH) in clinically diagnosed infertile males and find the potential association of CAT gene variant in the promoter region -21 A/T (rs7943316). The study consisted of 55 clinically diagnosed infertile males and 50 non-infertile volunteers. The activity of antioxidant enzymes was measured through a spectrophotometer. Polymerase chain reaction-restriction fragment length polymorphism was performed for genotyping of single-nucleotide polymorphism. Catalase enzyme activity was significantly decreased while SOD and GSH were substantially increased (p ≤ 0.01) in infertile men in comparison to non-infertile. CAT gene variant rs7943316 had shown significant association in dominant, recessive model and allelic frequencies. The study concludes that rs7943316 has a substantial role in male infertility. The outcome of the study may help in resolving idiopathic infertility cases and may help in evolving novel diagnostic and therapeutic approaches. Other variants of CAT and antioxidant genes are suggested to ascertain further insight.

Experimental and Established Oximes as Pretreatment before Acute Exposure to Azinphos-Methyl.

Poisoning with organophosphorus compounds (OPCs) represents an ongoing threat to civilians and rescue personal. We have previously shown that oximes, when administered prophylactically before exposure to the OPC paraoxon, are able to protect from its toxic effects. In the present study, we have assessed to what degree experimental (K-27; K-48; K-53; K-74; K-75) or established oximes (pralidoxime, obidoxime), when given as pretreatment at an equitoxic dosage of 25% of LD01, are able to reduce mortality induced by the OPC azinphos-methyl. Their efficacy was compared with that of pyridostigmine, the only FDA-approved substance for such prophylaxis. Efficacy was quantified in rats by Cox analysis, calculating the relative risk of death (RR), with RR=1 for the reference group given only azinphos-methyl, but no prophylaxis. All tested compounds significantly (p ≤ 0.05) reduced azinphos-methyl-induced mortality. In addition, the efficacy of all tested experimental and established oximes except K-53 was significantly superior to the FDA-approved compound pyridostigmine. Best protection was observed for the oximes K-48 (RR = 0.20), K-27 (RR = 0.23), and obidoxime (RR = 0.21), which were significantly more efficacious than pralidoxime and pyridostigmine. The second-best group of prophylactic compounds consisted of K-74 (RR = 0.26), K-75 (RR = 0.35) and pralidoxime (RR = 0.37), which were significantly more efficacious than pyridostigmine. Pretreatment with K-53 (RR = 0.37) and pyridostigmine (RR = 0.52) was the least efficacious. Our present data, together with previous results on other OPCs, indicate that the experimental oximes K-27 and K-48 are very promising pretreatment compounds. When penetration into the brain is undesirable, obidoxime is the most efficacious prophylactic agent already approved for clinical use.

Combined Pre- and Posttreatment of Paraoxon Exposure.

AIMS: Organophosphates (OPCs), useful agents as pesticides, also represent a serious health hazard. Standard therapy with atropine and established oxime-type enzyme reactivators is unsatisfactory. Experimental data indicate that superior therapeutic results can be obtained when reversible cholinesterase inhibitors are administered before OPC exposure. Comparing the protective efficacy of five such cholinesterase inhibitors (physostigmine, pyridostigmine, ranitidine, tacrine, or K-27), we observed best protection for the experimental oxime K-27. The present study was undertaken in order to determine if additional administration of K-27 immediately after OPC (paraoxon) exposure can improve the outcome. METHODS: Therapeutic efficacy was assessed in rats by determining the relative risk of death (RR) by Cox survival analysis over a period of 48 h. Animals that received only pretreatment and paraoxon were compared with those that had received pretreatment and paraoxon followed by K-27 immediately after paraoxon exposure. RESULTS: Best protection from paraoxon-induced mortality was observed after pretreatment with physostigmine (RR = 0.30) and K-27 (RR = 0.34). Both substances were significantly more efficacious than tacrine (RR = 0.67), ranitidine (RR = 0.72), and pyridostigmine (RR = 0.76), which were less efficacious but still significantly reduced the RR compared to the no-treatment group (paraoxon only). Additional administration of K-27 immediately after paraoxon exposure (posttreatment) did not further reduce mortality. Statistical analysis between pretreatment before paraoxon exposure alone and pretreatment plus K-27 posttreatment did not show any significant difference for any of the pretreatment regimens. CONCLUSIONS: Best outcome is achieved if physostigmine or K-27 are administered prophylactically before exposure to sublethal paraoxon dosages. Therapeutic outcome is not further improved by additional oxime therapy immediately thereafter.

Oximes as pretreatment before acute exposure to paraoxon.

Organophosphates, useful agents as pesticides, also represent a serious danger due to their high acute toxicity. There is indication that oximes, when administered before organophosphate exposure, can protect from these toxic effects. We have tested at equitoxic dosage (25% of LD01 ) the prophylactic efficacy of five experimental (K-48, K-53, K-74, K-75, K-203) and two established oximes (pralidoxime and obidoxime) to protect from mortality induced by the organophosphate paraoxon. Mortalities were quantified by Cox analysis and compared with those observed after pretreatment with a strong acetylcholinesterase inhibitor (10-methylacridine) and after the FDA-approved pretreatment compound pyridostigmine. All nine tested substances statistically significantly reduced paraoxon-induced mortality. Best protection was conferred by the experimental oxime K-48, reducing the relative risk of death (RR) to 0.10, which was statistically significantly superior to pyridostigmine (RR = 0.31). The other oximes reduced the RR to 0.13 (obidoxime), 0.20 (K-203), 0.21 (K-74), 0.24 (K-75) and 0.26 (pralidoxime), which were significantly more efficacious than 10-methylacridine (RR = 0.65). These data support the hypothesis that protective efficacy is not primarily due to cholinesterase inhibition and indicate that the tested experimental oximes may be considered promising alternatives to the established pretreatment compound pyridostigmine.

Reversible cholinesterase inhibitors as pre-treatment for exposure to organophosphates: assessment using azinphos-methyl.

Pre-treatment with reversible acetylcholinesterase (AChE) inhibitors before organophosphorous compound (OPC) exposure can reduce OPC-induced mortality. However, pyridostigmine, the only substance employed for such prophylaxis, is merely efficacious against a limited number of OPCs. In search of more efficacious and broad-range alternatives, we have compared in vivo the ability of five reversible AChE inhibitors (pyridostigmine, physostigmine, ranitidine, tacrine and K-27) to reduce mortality induced by the OPC azinphos-methyl. Protection was quantified using Cox analysis by determining the relative risk (RR) of death in rats that were administered these AChE inhibitors in equitoxic dosage (25% of LD01) 30 min before azinphos-methyl exposure. Azinphos-methyl-induced mortality was significantly reduced by all five tested compounds as compared with the reference group that was only exposed to azinphos-methyl without prior pre-treatment (RR = 1). The most efficacious prophylactic agents were K-27 (RR = 0.15) and physostigmine (RR = 0.21), being significantly more efficacious than ranitidine (RR = 0.62) and pyridostigmine (RR = 0.37). Pre-treatment with tacrine (RR = 0.29) was significantly more efficacious than pre-treatment with ranitidine, but the difference between tacrine and pyridostigmine was not significant. Our results indicate that prophylactic administration of the oxime K-27 may be a promising alternative in cases of imminent OPC exposure.

Oxime-type acetylcholinesterase reactivators in pregnancy: an overview.

Oxime-type acetylcholinesterase reactivators (oxime-AChER) are used as an adjunct in the treatment for organophosphorus anticholinesterase poisoning. Because of the widespread usage and exposure of organophosphorus compounds (OPCs), its poisoning and fatalities is obvious in pregnant women, embryos and fetuses. OPCs irreversibly inhibit acetylcholinesterase (AChE) at nerve synapses. Furthermore, the role of AChE other than neurotransmission termination has been defined in the literature. The growing evidences show that cholinergic mechanisms are involved during growth and development of other organ systems. In contrary to the fact, the data on the use of oxime-AChER in OPC poisoning in pregnancy are scanty. The present review aimed to comprehend the status of oximes in pregnancy in lieu of the published literature. A thorough literature search was performed in January 2013, using ten popular search engines including Medline/PubMed, Google scholar, etc., using nine standard keywords. The search period was set from 1966 to present. The search did not reveal substantial data. No considerable studies were retrieved which could really demonstrate either the beneficial, harmful or even null effect of oxime-AChER usage in pregnancy. Only eighteen relevant articles were obtained for a period of about 47 years. In the literature, there is no report available to demonstrate the risk of using oxime-AChER in pregnancy for the treatment of OPC poisoning. The study reveals that the use of oxime-AChER in pregnancy is largely un-addressed, inconclusive and based on speculation albeit the incidences of OPC poisoning are quite prevalent. Well-designed studies are warranted for a tangible conclusion.

Prophylactic administration of non-organophosphate cholinesterase inhibitors before acute exposure to organophosphates: assessment using terbufos sulfone.

Poisoning with organophosphorus compounds (OPCs) poses a serious threat worldwide. OPC-induced mortality can be significantly reduced by prophylactic administration of reversible acetylcholinesterase (AChE) inhibitors. The only American Food and Drug Administration (FDA)-approved substance for such pre-treatment (to soman exposure) is presently pyridostigmine, although its efficacy is controversial. In search for more efficacious and broad-spectrum alternatives, we have assessed in vivo the mortality-reducing efficacy of a group of five compounds with known AChE inhibitory activity (pyridostigmine, physostigmine, ranitidine, tacrine and K-27), when given in equitoxic dosage (25% of LD01 ) 30 min before exposure to the OPC terbufos sulfone. Protection was quantified in rats by determining the relative risk of death (RR) using Cox analysis, with RR = 1 for animals given only terbufos sulfone, but no pre-treatment. All tested AChE inhibitors reduced terbufos sulfone-induced mortality significantly (p ≤ 0.05) as compared with the non-treatment group (RR = 1: terbufos sulfone only). Best in vivo protection from terbufos sulfone-induced mortality was achieved, when K-27 was given before terbufos sulfone exposure (RR = 0.06), which was significantly (P ≤ 0.05) superior to the pre-treatment with all other tested compounds, for example tacrine (RR = 0.21), pyridostigmine (RR = 0.28), physostigmine (RR = 0.29) and ranitidine (RR = 0.33). The differences in efficacy between tacrine, pyridostigmine, physostigmine and ranitidine were not statistically significant. Prophylactic administration of an oxime (such as K-27) in case of imminent OPC exposure may be a viable option.

Usefulness of administration of non-organophosphate cholinesterase inhibitors before acute exposure to organophosphates: assessment using paraoxon.

Reversible acetylcholinesterase (AChE) inhibitors can protect against the lethal effects of irreversible organophosphorus AChE inhibitors (OPCs), when administered before OPC exposure. We have assessed in vivo the mortality-reducing efficacy of a group of known AChE inhibitors, when given in equitoxic dosage before exposure to the OPC paraoxon. Protection was quantified in rats by determining the relative risk (RR) of death. Best in vivo protection from paraoxon-induced mortality was observed after prophylactic administration of physostigmine (RR = 0.30) or the oxime K-27 (RR = 0.34); both treatments were significantly superior to the pre-treatment with all other tested compounds, including the established substance pyridostigmine. Tacrine (RR = 0.67), ranitidine (RR = 0.72), pyridostigmine (RR = 0.76), tiapride (RR = 0.80) and 7-MEOTA (RR = 0.86) also significantly reduced the relative risk of paraoxon-induced death, but to a lesser degree. Methylene blue, amiloride and metoclopramide had an unfavorable effect (RR ≥ 1), significantly increasing mortality. When CNS penetration by prophylactic is undesirable K-27 is a promising alternative to pyridostigmine.

Sub-chronic exposure to paraoxon neither induces nor exacerbates diabetes mellitus in Wistar rat.

There is an increasing belief that organophosphorus compounds (OPCs) impair glucose homeostasis and cause hyperglycemia and diabetes mellitus. The present study was undertaken to investigate the putative diabetogenic effect of sub-lethal and sub-chronic exposure to paraoxon (POX), an extremely hazardous OPC used in pesticides. The effect of paraoxon on streptozotocin-induced diabetic rats was also examined. Each rat was injected with 100 nmol of POX 5 days per week for 6 weeks. Blood glucose levels and red blood cell acetylcholinesterase activity were measured weekly. Biochemical analysis and morphological studies were performed at the end of the experiment. The results revealed that POX neither induces nor exacerbates diabetes mellitus in experimental rats. Liver and kidney/body weight ratios revealed statistically insignificant differences when compared with controls. Biochemical analysis of urine samples showed a small but not significant increase in protein level in all groups. Urine bilirubin was significantly higher in the diabetes + POX group when compared with the control group. The number of blood cells in urine was significantly higher in the POX-treated group compared with the control group. Hyperglycemia was noted in the diabetes and diabetes + POX groups, but neither in the saline control nor in POX-treated normal rats. Electron microscopy of POX-treated pancreas did not show any morphological changes in beta cells. These results suggest that POX does not cause diabetes mellitus at sub-lethal sub-chronic exposure.

N3,N7-diaminophenothiazinium derivatives as antagonists of α7-nicotinic acetylcholine receptors expressed in Xenopus oocytes.

Derivatization of phenothiazine (PTZ, 1) has been a commonly used method to develop drugs with various pharmacological properties. In the present study, a series of PTZ derivatives 1-11 were investigated on the inhibition of the cloned α7 subunit of the human nicotinic acetylcholine receptor (α7-nAChR) expressed in Xenopus oocytes by using the two-electrode voltage-clamp technique. In the first series of experiments, the effect of unsubstituted phenothiazine 1 on α7-nAChRs was compared with that of the N3,N7-diaminophenothiazin-5-ium derivative 2, and of sequentially methylated derivatives 3-6. In the second set of experiments, the effects of N3,N7-tetra-ethyl- to n-hexylphenothiazin-5-ium derivatives 7-11 were tested. Despite the lack of activity found for 1, a reversible inhibition of α7-nAChRs, ranging from moderate to potent, was observed as a result of a sequential amine- and methylamine substitution of 1. The inhibition of ACh (100 µM)-induced currents was concentration-dependent with IC(50) values ranging from 0.4 to 16.8 µM. However, an optimal inhibitory activity was achieved by prolongation of alkyl chains up to propyl size, as found in PTZ derivative 8, whereas further lengthening of alkyl chains to n-butyl-, n-pentyl-, or n-hexyl groups resulted in inactive derivatives 9-11. The results evidently suggest the presence of a lipophilic binding pocket of narrow tolerability on the receptor protein. These results emphasize the importance of amine and/or alkylamine moieties for the inhibitory effect of PTZ derivatives and provide further insights for the development of novel antagonists targeting α7-nAChRs.

Pretreatment for acute exposure to diisopropylfluorophosphate: in vivo efficacy of various acetylcholinesterase inhibitors.

Prophylactic administration of reversible acetylcholinesterase (AChE) inhibitors before exposure to organophosphorus compounds (OPCs) can reduce OPC-induced mortality. Pyridostigmine is the only FDA-approved substance for such use. The AChE-inhibitory activity of known AChE inhibitors was quantified in vitro and their in vivo mortality-reducing efficacy was compared, when given prophylactically before the exposure to the OPC diisopropylfluorophosphate (DFP). The IC50 was measured in vitro for the known AChE inhibitors pyridostigmine, physostigmine, ranitidine, tiapride, tacrine, 7-methoxytacrine, amiloride, metoclopramide, methylene blue and the experimental oxime K-27. Their in vivo efficacy, when given as pretreatment, to protect rats from DFP-induced mortality was quantified by determining the relative risk of death (RR) by Cox analysis, with RR = 1 for animals given only DFP, but no pretreatment. Physostigmine was the strongest in vitro AChE-inhibitor (IC50 = 0.012 µ m), followed by 7-methoxytacrine, tacrine, pyridostigmine and methylene blue. Ranitidine (IC50 = 2.5 µ m), metoclopramide and amiloride were in the mid-range. Tiapride (IC50 = 256 µ m) and K-27 (IC50 = 414 µ m) only weakly inhibited RBC AChE activity. Best in vivo protection from DFP-induced mortality was achieved when physostigmine (RR = 0.02) or tacrine (RR = 0.05) was given before DFP exposure, which was significantly superior to the pretreatment with all other tested compounds, except K-27 (RR = 0.18). The mortality-reducing effect of pyridostigmine, ranitidine and 7-methoxytacrine was inferior, but still significant. Tiapride, methylene blue, metoclopramide and amiloride did not significantly improve DFP-induced mortality. K-27 may be a more efficacious alternative to pyridostigmine, when passage into the brain precludes administration of physostigmine or tacrine.

Cannabis Constituents and Acetylcholinesterase Interaction: Molecular Docking, In Vitro Studies and Association with CNR1 rs806368 and ACHE rs17228602.

The study documented here was aimed to find the molecular interactions of some of the cannabinoid constituents of cannabis with acetylcholinesterase (AChE). Molecular docking and LogP determination were performed to predict the AChE inhibitory effect and lipophilicity. AChE enzyme activity was measured in the blood of cannabis addicted human subjects. Further, genetic predisposition to cannabis addiction was investigated by association analysis of cannabinoid receptor 1 (CNR1) single nucleotide polymorphism (SNP) rs806368 and ACHE rs17228602 using restriction fragment length polymorphism (RFLP) method. All the understudied cannabis constituents showed promising binding affinities with AChE and are lipophilic in nature. The AChE activity was observed to be indifferent in cannabis addicted and non-addicted healthy controls. There was no significant association with CNR1 SNP rs806368 and ACHE rs17228602. The study concludes that in silico prediction for individual biomolecules of cannabis is different from in vivo physiological action in human subjects when all are present together. However, for a deeper mechanistic insight into these interactions and association, multi-population studies are suggested. Further studies to explore the inhibitory potential of different cannabis constituents for intended AChE inhibitor-based drug are warranted.

Molecular docking analyses of CYP450 monooxygenases of Tribolium castaneum (Herbst) reveal synergism of quercetin with paraoxon and tetraethyl pyrophosphate: in vivo and in silico studies.

Pest management in stored grain industry is a global issue due to the development of insecticide resistance in stored grain insect pests. Excessive use of insecticides at higher doses poses a serious threat of food contamination and residual toxicity for grain consumers. Since the development of new pesticide incurs heavy costs, identifying an effective synergist can provide a ready and economical tool for controlling resistant pest populations. Therefore, the synergistic property of quercetin with paraoxon and tetraethyl pyrophosphate has been evaluated against the larvae and adults of Tribolium castaneum (Herbst). Comparative molecular docking analyses were carried out to further identify the possible mechanism of synergism. It was observed that quercetin has no insecticidal when applied at the rate of 1.5 and 3.0 mg/g; however, a considerable synergism was observed when applied in combination with paraoxon. The comparative molecular docking analyses of CYP450 monooxygenase (CYP15A1, CYP6BR1, CYP6BK2, CYP6BK3) family were performed with quercetin, paraoxon and tetraethyl pyrophosphate which revealed considerable molecular interactions, predicting the inhibition of CYP450 isoenzyme by all three ligands. The study concludes that quercetin may be an effective synergist for organophosphate pesticides depending upon the dose and type of the compound. In addition, in silico analyses of the structurally diversified organophosphates can effectively differentiate the organophosphates which are synergistic with quercetin.

Biochemical Analysis and Association of Butyrylcholinesterase SNPs rs3495 and rs1803274 with Substance Abuse Disorder.

Addiction is a complex mental and behavioral disorder that changes the neurochemistry and physiology of the brain. Genetics also plays a significant role in the pathophysiology of addiction. Butyrylcholinesterase (BChE), a cholinergic enzyme, has been implicated in the metabolism of various drugs, including cocaine, and an association between single-nucleotide polymorphisms (SNPs) of the butyrylcholinesterase gene (BCHE) and neuronal disorders has been reported. We report here the first investigation to be conducted on the status of BChE activity and the potential association of two BCHE gene SNPs, rs3495 (c.*189G > A) and rs1803274 (c.1699G>A, p.Ala567Thr, K-variant), with addiction vulnerability in heroin, hashish and polydrug users. Seventy-five individuals with an addiction to heroin, hashish and/or polydrug use were recruited to this study. BChE levels in the plasma were determined by Ellman's principle. SNPs were genotyped by standard procedures, followed by Sanger sequencing. Plasma BChE levels were found to be significantly higher (p ≤ 0.05) in addicts (mean ± standard error of the mean 0.031 ± 0.004 µmol/L/min; 95% confidence interval [CI] 0.024-0.038) than in non-addicts (controls) (0.014 ± 0.001 µmol/L/min; 95% CI 0.012-0.017). Statistical significant differences were also observed between the addicted cohorts. A statistically significant association for both SNPs (rs3495 and rs1803274) was not observed in addicted subjects tested in the dominant, recessive and allele genetic models, but trends of variations of the rs3495 risk G allele were noted. The authors conclude that BChE plays significant roles in addiction pathophysiology as increased BChE activity in blood samples obtained from the cohorts with addiction was evident. Further studies in this direction may provide novel approaches for the treatment of addiction, but studies with a larger sample size and different ethnic groups are warranted for broader conclusions to be drawn.

Association of status of acetylcholinesterase and ACHE gene 3' UTR variants (rs17228602, rs17228616) with drug addiction vulnerability in pakistani population.

Substance addiction is a chronic, relapsing mental disorder Characterized by compulsive drug seeking, and loss of control over drug intake and relapse after prolonged abstinence. Genetics has been shown to contribute towards an individual's vulnerability to addiction. Acetylecholine (ACh), a cholinergic neurotransmitter hydrolyzed by acetylcholinesterase (AChE), is an essential neurotransmitter and neuromodulator in central and peripheral nervous system and has regulatory influence on numerous neuronal functions including addiction. The present study was carried out to investigate the role of acetylcholinesterase (AChE) in addiction through measurement of enzyme activity and to find potential association of ACHE gene 3'UTR variants rs17228602 and rs17228616 in heroin, hashish and poly drug addicts. Both SNPs are located within microRNA (miRNA) recognition sites with potential to affect miRNA/transcript interaction. A total of 122 addicts of heroin, hashish and polydrug were recruited from local rehabilitation centers to participate in this study. AChE activity was measured in blood by Ellman's method. SNP genotyping was performed by restriction fragment length polymorphism (PCR-RFLP) and Sanger sequencing. The AChE activity was found significantly higher (p ≤ 0.005) in addicted cohort (mean ±â€¯standard error of mean 0.020 ±â€¯0.001 µmol/L/min; 95% confidence interval (CI) 0.018-0.022) in comparison to non-addicted healthy subjects (0.011 ±â€¯0.001 µmol/L/min; 95% confidence interval CI 0.010-0.013). A statistically significant association of ACHE rs17228602 SNP with addiction vulnerability in dominant (DM: Odd's ratio OR = 2.095, 95% CI = 1.157-3.807 p = 0.009) and allelic genetic models (OR = 1.854 95% CI = 1.082-3.187, p = 0.016) was observed. However, no statistically significant association of rs17228616 SNP with substance abuse disorder was found. The data presented here shows that AChE could play significant role in substance addiction. Further studies with larger sample size and other variants of AChE are recommended to identify novel therapeutic approaches for cholinergic based treatment of addiction.

Reactivation potency of two novel oximes (K456 and K733) against paraoxon-inhibited acetyl and butyrylcholinesterase: In silico and in vitro models.

Organophosphates (OPs) irreversibly inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. The reactivation of these inhibited enzymes is paramount for their normal function. Present study evaluates reactivation potency of two newly developed oximes, K456 and K733, against paraoxon (POX)-inhibited human-RBC-AChE and human-plasma-BChE in comparison to reported reactivator, pralidoxime (2-PAM). In vitro studies showed higher intrinsic toxicities of both oximes than 2-PAM for AChE. No substantial reactivation of hBChE was noted by tested concentration. Contrary to 2-PAM, the in silico study predicted lower binding free energies for both oximes. However, the detailed interaction study revealed inability of oximes to interact with catalytic anionic site of AChE and hBChE in contrast to 2-PAM. Both in vitro and in silico studies conclude that K456 and K733 are unlikely to be used as reactivators of paraoxon-inhibited AChE or BChE.

Optimal Pre-treatment for Acute Exposure to the Organophosphate Dicrotophos.

BACKGROUND: Reversible cholinesterase inhibitors, when given prophylactically before exposure to organophosphates, are able to decrease organophosphate-induced mortality. However, the efficacy of pyridostigmine, the only pre-treatment substance approved by the US Federal Drug Administration, is unsatisfactory. METHODS: In search of a better prophylactic compound, we determined in vivo the protection conferred by five cholinesterase inhibitors (ranitidine, physostigmine, tacrine, K-27 and pyridostigmine), which were administered in equitoxic dosage (1/4 of LD01) 30 minutes before exposure to the organophosphate dicrotophos. Efficacy was measured in rats by Cox analysis calculating the relative risk of death (RR), RR being 1 for the reference group which received dicrotophos and no prophylaxis. RESULTS: K-27 (RR=0.06), physostigmine (RR=0.15), pyridostigmine (RR=0.22) and tacrine (RR=0.28) significantly (p ≤ 0.05) reduced dicrotophos-induced mortality in comparison to the reference group (dicrotophos without pre-treatment), whereas ranitidine (RR=0.86) had no significant influence. The experimental oxime K-27, when given before dicrotophos exposure, conferred the best in vivo protection. This was significantly (p ≤ 0.05) more efficacious than pre-treatment with any other tested compound. The differences in efficacy between the second best compound, physostigmine, and the less efficacious substances (tacrine and pyridostigmine) were also statistically significant. CONCLUSION: These data indicate that K-27 can be considered a very efficacious prophylactic agent for organophosphate exposure.

Phenylalanine derivatives with modulating effects on human α1-glycine receptors and anticonvulsant activity in strychnine-induced seizure model in male adult rats.

The critical role of α1-glycine receptor (α1-GLYRs) in pathological conditions such as epilepsy is well known. In the present study, structure-activity relations for a series of phenylalanine derivatives carrying selected hydrogen bond acceptors were investigated on the functional properties of human α1-GLYR expressed in Xenopus oocytes. The results indicate that one particular substitution position appeared to be of special importance for control of ligand activity. Among tested ligands (1-8), the biphenyl derivative (2) provided the most promising antagonistic effect on α1-GLYRs, while its phenylbenzyl analogue (5) exhibited the highest potentiation effect. Moreover, ligand 5 with most promising potentiating effect showed in-vivo moderate protection when tested in strychnine (STR)-induced seizure model in male adult rats, whereas ligand 2 with highest antagonistic effect failed to provide appreciable anti(pro)convulsant effect. Furthermore, ligands 2 and 5 with the most promising effects on human α1-GLYRs were examined for their toxicity and potential neuroprotective effect against neurotoxin 6-hydroxydopamine (6-OHDA). The results show that ligands 2 and 5 possessed neither significant antiproliferative effects, nor necrotic and mitochondrial toxicity (up to concentration of 50µM). Moreover, ligand 2 showed weak neuroprotective effect at the 50µM against 100µM toxic dose of 6-OHDA. Our results indicate that modulatory effects of ligands 2 and 5 on human α1-GLYRs as well as on STR-induced convulsion can provide further insights for the design of therapeutic agents in treatment of epilepsy and other pathological conditions requiring enhanced activity of inhibitory glycine receptors.

Ranitidine in acute high-dose organophosphate exposure in rats: effect of the time-point of administration and comparison with pyridostigmine.

Weak and reversible inhibitors of cholinesterase, when co-administered in large doses, can act in a protective manner against more potent inhibitors such as organophosphates. The clinically widely used histamine type 2 (H2) receptor blocker ranitidine is among H2 blockers the most potent inhibitor of acetylcholinesterase but roughly three to four orders of magnitude less potent than paraoxon (an irreversible organophosphate esterase inhibitor) or pyridostigmine (a reversible carbamate esterase inhibitor). We have previously shown that in vitro ranitidine confers some protection against inhibition of cholinesterases by paraoxon and that in vivo it both increases the number of rats surviving an acute paraoxon exposure and also protects to some degree the cholinesterases from organophosphate inhibition. The purpose of the study was to compare in a prospective non-blinded study, in a rat model of acute high-dose paraoxon exposure, ranitidine with pyridostigmine either administered simultaneously or 30 min. before exposure. There were 36 rats in each of the 5 groups. All substances were applied intraperitoneally. Additional analysis included data from a similar experiment carried out in 2005, in which 54 rats were exposed to paraoxon only (G1) and 54 to paraoxon+ranitidine simultaneously (G2). All groups (except controls; G6 & G7) received 1 micro Mol paraoxon (approximately LD75); groups 2-5 received in addition to paraoxon: G2: 50 micro Mol ranitidine within 1 min. of paraoxon, G3: 1 micro Mol pyridostigmine within 1 min. of paraoxon, G4: 50 micro Mol ranitidine 30 min. before paraoxon, G5: 1 micro Mol pyridostigmine 30 min. before paraoxon. Groups 6 & 7 received only ranitidine and pyridostigmine respectively, group G1 received only paraoxon. Mortality was recorded at 30 min., 1, 2, 3, 4, 24 and 48 hr. Mortality data were compared using Kaplan-Meier plots and logrank tests. No Bonferroni correction for multiple comparisons was applied and an alpha < or = 0.05 was considered significant. All statistical analysis was performed by using SPSS 12.0 statistical software (SPSS Inc., Chicago, IL, USA). Simultaneous administration of ranitidine or pyridostigmine with paraoxon does not significantly affect mortality. Pretreatment (30 min. before) with both ranitidine or pyridostigmine statistically and significantly reduced mortality. When administered before paraoxon, pyridostigmine is statistically significantly superior to ranitidine. Both ranitidine and pyridostigmine are protective against acute paraoxon toxicity provided they are administered before paraoxon. Pyridostigmine results are statistically significantly superior to ranitidine (< or =0.05).