International Journal of Risk & Safety in Medicine - Volume 27, issue s1

Article Type: Other

Citation: International Journal of Risk & Safety in Medicine, vol. 27, no. s1, pp. S63-S63, 2015

Authors: Yarullina, D.R. | Damshkaln, L.G. | Bruslik, N.L. | Konovalova, O.A. | Ilinskaya, O.N. | Lozinsky, V.I.

Article Type: Abstract

Abstract: BACKGROUND: Probiotics are live microorganisms, generally either lactobacilli or bifidobacteria, which when administered in adequate amounts confer a health benefit to the host [1 ]. Due to the growing evidence of health benefits associated with their use, probiotics are of increasing interest and represent now a significant growth area in the functional foods industry [2 ]. However, to be effective, orally administered probiotics should survive preparation of dosage forms and passage through acidic environment of the gastrointestinal tract (GIT). Reaching the intestine, these microorganisms should be able to establish themselves, remain viable and perform their beneficial actions. In this context, …oral formulations have to protect probiotic bacteria from gastric acidity and delay their release in the small intestine in order to allow their complete release in the colon. OBJECTIVE: To evaluate effects of starch formulations of lactobacilli on their survival in gastric environment and probiotic properties. METHODS: Nineteen Lactobacillus strains belonging to the species L. fermentum (14 strains), L. plantarum (4 strains), and L. rhamnosus (1 strain), were isolated from dairy products and probiotics, and were used in this study. Lactobacilli were cultured in de Man, Rogosa, Sharpe (MRS) broth (Merck, Germany) under microaerobic conditions at 37°C. Amylolytic activity of lactobacilli, cultured for 3–5 days on MRS agar supplemented with 1% soluble potato starch (SPS), was determined with iodine reagent (0.01 M I2 -KI solution). Loading in starch was performed with L. plantarum 8PA3 bacteria (“Dry lactobacterin”, Perm, Russia), which were resuspended to the concentration 1010 cells/mL in 10 mL of 0.85% NaCl solution and added to 90 mL of 2.5% SPS solution. Resulting mixture was frozen at –18°C and then lyophilized (Martin Christ Alpha 1-2 LDplus, Germany). Atomic force microscopy (AFM) images of formulated L. plantarum 8PA3 cells were acquired in air by a Solver P47H atomic force microscope (NT-MDT, Moscow, Russia). Starch swelling and dissolution was studied in simulated colonic fluid (SCF), prepared according to [3 ] and in distilled water (pH = 6.0) as control. Amylase from Aspergillus oryzae (A8220, Sigma) was added to the solutions to study the influence of amylase. The formulation form was examined visually during 14 h incubation time. Fluorescence microscopy images were obtained with a Leica DM6000B (Germany) fluorescent microscope using Leica FW4000 software. L. plantarum 8PA3 loaded in SPS were placed either in HCl solution (pH 2), or in 2% oxgall bile solution, or in 0.85% NaCl solution. Viability was tested after 2, 4 and 6 h incubation at 37°C by plating diluted aliquots onto MRS agar with subsequent counting of bacterial colony forming units (CFU). In addition, viability was determined using LIVE/DEAD Bac Light bacterial viability kit L-7012 (Molecular Probes, Invitrogen) as described elsewhere [4 ]. Fluorescence in the stained samples was estimated with BD FACS Canto II (USA) flow cytometer or fluorescent microscope. Nitric oxide (NO) production was assessed with DAF-FM DA and DAA fluorescent dyes as described earlier [4 ]. Each experiment was performed in triplicate. RESULTS: In the present study we studied the probiotic composition comprising of SPS and bacteria L. plantarum 8PA3. We used AFM to confirm effective fixation of the cells to carbohydrate. The compositions were found to swell quickly (~5 min) in aqueous solutions either containing amylase, or not. Tested starch formulations disintegrated during the first 5-10 min of incubation in amylase solutions whereas in amylase-free probes dissolution was less intensive (after ~30 min). Amylolysis of starch excipients was less pronounced in aqueous amylase solution than in SCF, supplemented with amylase. None of 19 studied Lactobacillus strains hydrolyzed SPS when growing on MRS agar supplemented with it. The amount of viable L. plantarum 8PA3 cells formulated with SPS was high and did not change when stored for 6 months at 4°C. The bacterial viability tests also demonstrated that after 6 h treatment with 2% bile or HCl (pH 2) L. plantarum 8PA3 exhibited increased sensitivity (viability 14% and 0.4%, respectively). However, in similar conditions no significant differences were noticed between bacterial viability obtained for formulated with starch and non-formulated bacteria. Furthermore, we showed that loading into SPS had no effect on bacterial production of nitric oxide (NO) – a pluripotent regulatory molecule in human organism. CONCLUSIONS: Overall, the results strongly support that formulation with polymeric matrices on the basis of SPS represent an appealing technology of probiotics production. It provides slow release of bacteria in target environment and does not alter their viability and NO biosynthesis. However, SPS excipient does not preserve the bacteria from harsh conditions of upper GIT. Therefore, we conclude that for oral administration the composition should be loaded in acid-resistant capsules. Show more

Keywords: Probiotic, polymeric matrix, composition, soluble potato starch

DOI: 10.3233/JRS-150692

Citation: International Journal of Risk & Safety in Medicine, vol. 27, no. s1, pp. S65-S66, 2015

Authors: Valeeva, I.Kh. | Titarenko, A.F. | Khaziakhmetova, V.N. | Ziganshina, L.E.

Article Type: Abstract

Abstract: BACKGROUND: It is believed that the anti-inflammatory activity of medicines is closely related to their antioxidant activity. However, in clinical practice rigorous evidence-based medicine approach fails to reveal important effects of antioxidants on patient important outcomes in inflammatory disorders, as has been shown by a number of Cochrane reviews [1–3 ]. OBJECTIVE: To evaluate anti-inflammatory and antioxidant effects of newly developed pharmacological agents: dimephosphone and its structural analogues ephorane and mephoprane, and xymedon, in comparison with prednisolone and etidronate in experimental animal model of adjuvant arthritis. METHODS: Experiments were conducted in 64 white mongrel rats of …both sexes weighing 180–200 g, which were divided into 8 groups 8 rats each (4 males and 4 females each), kept under standard vivarium conditions with certified feeding ration (kombikorm). The study was approved by the local ethics committee. We induced adjuvant arthritis by administration under the plantar aponeurosis of the left hind paw of 0.1 ml of Freund’s adjuvant (Sigma) in rats of 7 study groups. The groups were as follows: 1st group - intact animals (control); 2nd group – animals to whom the solvent (distilled water) was administered with intra-gastric tube in corresponding volume (control of the model); 3rd – 8th study groups, in which animals were administered with study agents each at a dose of 1 mmol/kg body weight: dimephosphone, ephorane, mephoprane, xymedon, etindronate and prednisolone. The intensity of the modeled arthritis was determined by measurements of paw volumes with plethysmometer (UgoBasile). We calculated the difference in rat paw volume before the administration (baseline) and after administration of Freund’s adjuvant at 3, 7, 11, 15, 20, 27, 31, 38, 41 days. The development of secondary arthritis was documented by the increase in volume of both hind and fore paws and tails. On the 41st day of the experiment the animals were sacrificed under light ether anesthesia and exsanguinated. The blood was used to determine the activity of catalase and peroxidase, the content of the total, reduced and oxidized glutathione, the level of ceruloplasmin, conjugated dienes of unsaturated fatty acids (DC), TBA-interacting products (MDA), and the total antioxidant activity of serum (AOA). The results were processed statistically using the Student’s t -test. RESULTS: The primary reaction to the Freund’s adjuvant in a form of swelling of the ankle joint of the left hind paw was observed at 24 hours after its injection. External clinical manifestations of the modeled disease were more pronounced on the third day: local inflammatory reaction (redness, swelling, ulceration) was seen in all the animals at the injection site with the increase of the paw volume. On the 11th day of the experiment 20% of the animals developed secondary arthritis. The study agents dimephosphone, ephorane, and prednisone exerted anti-inflammatory effect decreasing the volume of left hind paws by 45%, 46% и 27% respectively on the 40th day of experiment. Mephoprane did not affect the primary inflammatory response to the Freund’s adjuvant (rats’ left hind paws), however it reduced the volume of the contralateral right paw (secondary arthritis) by 90% on the 20th day of the experiement. This ant-inflammatory effect was accompanied by documented antioxidant activity in case of dimephosphone, ephorane, prednisolone, but not mephoprane. Dimephosphone reduced the levels of lipid peroxidation products in rats blood by 46% (DC) and by 25% (MDA). Ephorane also reduced the levels of lipid peroxidation products in the blood by 46% (DC) and by 25% (MDA), increasing the level of glutathione by nearly half, both the total and the reduced form. Prednisolone reduced the level of lipid peroxidation products in blood by 61% (DC), but not the TBA-interacting products. Mefopran did not affect the blood level of lipid peroxidation products. Xymedon and etidronate showed no anti-inflammatory effect. Xymedon demonstrated anti-oxidant properties, decreasing the blood levels of lipid peroxidation products, while etidronate seemed to behave in pro-oxidant mode, increasing the blood levels of lipid peroxidation products. CONCLUSIONS: The effects of studied agents on the intensity of inflammation and lipid peroxidation were inconsistent. The results of the study did not show a clear link between anti-inflammatory and anti-oxidant activity. Further research in potential anti-inflammatory activity of new drugs exhibiting antioxidant properties needs to be done before recommending their use in clinical practice. Show more

Keywords: Anti-inflammatory, anti-oxidant, rat paw oedema, arthritis

DOI: 10.3233/JRS-150693

Citation: International Journal of Risk & Safety in Medicine, vol. 27, no. s1, pp. S67-S68, 2015

Authors: Zueva, I.V. | Semenov, V.E. | Mukhamedyarov, M.A. | Lushchekina, S.V. | Kharlamova, A.D. | Petukhova, E.O. | Mikhailov, A.S. | Podyachev, S.N. | Saifina, L.F. | Petrov, K.A. | Minnekhanova, O.A. | Zobov, V.V. | Nikolsky, E.E. | Masson, P. | Reznik, V.S.

Article Type: Abstract

Abstract: BACKGROUND: Alzheimer’s disease (AD) is the major age-related progressive neurodegenerative disorder. The brain of AD patients suffers from loss of cholinergic neurons and decreased number of synapses [1 ]. AD is caused by an imbalance between Aβ production and clearance, resulting in increased amount of Aβ in various forms [2 ]. Reduction of Aβ production and increasing clearance of Aβ pathogenic forms are key targets in the development of potential therapeutic agents for AD treatment. Unfortunately, only nosotropic approaches for treatment of AD are currently effective in humans. These approaches mainly focus on the inhibition of brain acetyl-cholinesterase (AChE) to …increase lifetime of cerebral acetylcholine [3 ]. It is important to emphasize that AChE itself promotes the formation of Aβ fibrils in vitro and Aβ plaques in the cerebral cortex of transgenic mouse models of AD [4 ]. This property of AChE results from interaction between Aβ and the peripheral anionic site of the enzyme (PAS) [5 ]. Dual binding site inhibitors of both catalytic active site (CAS) and PAS can simultaneously improve cognition and slow down the rate of Aβ-induced neural degeneration. Unfortunately, the assortment of AChE PAS ligands is still extremely limited. OBJECTIVE: To study putative advantages of AChE non-charged PAS inhibitors based on 6-methyluracil derivatives for the treatment of Alzheimer’s disease. METHODS: In vitro studies . Concentration of drug producing 50% of AChE/BuChE activity inhibition (IC50) was measured using the method of Ellman et al. [6 ]. Toxicological experiments were performed using IP injection of the different compounds in mice. LD50, dose (in mg/kg) causing lethal effects in 50% of animals was taken as a criterion of toxicity [7 ]. The ability of compound to block in vitro AChE-induced Aβ1–40 aggregation was studied using a thioflavin T (ThT) fluorescent probe [8 ]. In vivo biological assays . For in vivo blood–brain barrier permeation assay brains were removed 30 min after IP injection of LD50 dose of tested compound injection. The inhibitory potency was measured using the method of Ellman. Scopolamine and transgenic models of AD were used to evaluate the influence of compound 35 on spatial memory performance.Water solution of scopolamine was injected to mice (ip) 20 minutes before starting memory test during 14 days [9 ]. Mice were assigned to 7 groups, including 4 groups receiving injection (ip) of compound in different dosages, donepezil-treated mice (donepezil is conventionally used to treat Alzheimer’s disease), positive and negative control groups. Double transgenic (APP/PS1) mice expressing a chimeric mouse/human amyloid precursor protein and a mutant of human presenilin-1 [10 ] were assigned to 4 groups, including transgenic animals injected (ip) with compound 35 or donepezil solution, positive (transgenes injected with water) and negative (wild-type mice) controls. To evaluate spatial memory performance, mice were trained on a reward alternation task using a conventional T-maze [11 ]. The criterion for a mouse having learned the rewarded alternation task was 3 consecutive days of at least 5 correct responses out of the 6 free trials. For β-amyloid peptide load was evaluated quantitatively as a number and summary area of Thioflavine S fluorescent spots in cerebral cortex and hippocampal images using Image J program. Statistical analyses were performed using the Mann-Whitney test. RESULTS: We evaluated the acute toxicity of the most active compounds. The most potent AChE inhibitor compound 35 (IC50 (AChE) = 5 ± 0.5 nM) exhibited the lowest LD50 values (51 mg/kg) and inhibited brain AChE by more than 71 ± 1%. Compound 35 at 10 nM, exhibited a significant (35 ± 9%) inhibitory activity toward human AChE-induced Aβ aggregation. Scopolamine injection induced significant decrease in correct choice percentage in T-maze, as well as decrease in percentage of mice reaching criterion for learning the task by day 14. This memory deficit was relieved to some extent either by compound 35 (5 mg/kg) or donepezil (reference compound) treatment (0.75 mg/kg). Interestingly, higher doses of compound 35 (10 and 15 mg/kg) produced less therapeutic effect on spatial memory deficit. Group of APP/PS1 mice showed 3 times lower percentage of reaching behavioral criterion and lower percentage of correct choice in T-maze alternation task comparing to WT mice, whereas compound 35 (5 mg/kg) or Donepezil treatment effectively improved these parameters in APP/PS1 mice. Compound 35 treatment (5 mg/kg) during 14 days significantly reduced percentage of summary area and number of β-amyloid peptide (βAP) deposits visualized in sections of cerebral cortex, dentate gyrus, and hippocampal CA3 area in APP/PS1 mice. The most prominent reduction of βAP load by compound 35 treatment was found in CA3 area and cerebral cortex. Meanwhile, Donepezil treatment (1 mg/kg) during 14 days significantly reduced βAP load in cerebral cortex but not in dentate gyrus and CA3 area. CONCLUSIONS: Experiments showed that the most potent AChE inhibitor compound 35 (6-methyluracil derivative) permeated the blood-brain barrier, improved working memory in the APP/PS1 transgenic mice and significantly reduced the number and area of Aβ plaques in the brain. Thus, compound 35 is a promising candidate as a bi-functional inhibitor of AChE for treatment of AD. Show more

Keywords: Alzheimer’s disease, acetyl-cholinesterase inhibitors, methyluracil derivatives

DOI: 10.3233/JRS-150694

Citation: International Journal of Risk & Safety in Medicine, vol. 27, no. s1, pp. S69-S71, 2015

Authors: Petrov, K.

Article Type: Abstract

Abstract: BACKGROUND: Acetylcholinesterase (AChE) inhibitors are widely used in medicine for pharmacological correction of cholinergic neurotransmission pathologies such as myasthenia gravis (MG) and Alzheimer’s disease [1, 2 ]. The efficacy of anti-AChE drugs is based on their ability to potentiate the effects of acetylcholine (ACh) due to a decrease in the rate of AChE-catalyzed hydrolysis of ACh. Crystallographic studies showed that the active site of AChE is located at the bottom of a deep gorge [3 ]. It was shown that, in addition to its catalytic center, AChE has other sites that are crucial for the proper functioning of the enzyme. …In particular, the so-called peripheral anionic site (PAS) located at the entrance of the active site gorge is responsible for: 1) allosteric modulation of the catalytic center; 2) enzyme inhibition at high substrate concentration; 3) and non-catalytic functions such as enhancement of cell adhesion and neurite outgrowth. OBJECTIVE: Especially interesting is the relationship between the PAS and pathological beta-amyloid deposition. This led to a new hypothesis for rational design of more effective anti-Alzheimer drugs [4 ]. METHODS: Concentration of drug producing 50% of AChE activity inhibition (IC50) was measured using the method of Ellman et al. [5 ]. Toxicological experiments were performed using IP injection of the different compounds in mice. LD50, dose (in mg/kg) causing lethal effects in 50% of animals was taken as a criterion of toxicity [6 ]. Molecular docking was performed with Autodock 4.2.6 software. RESULTS: We described previously a new class of selective mammalian AChE vs. butyrylcholinesterase (BChE) inhibitors based on alkylammonium derivatives of 6-methyluracil of acyclic topology [7 ]. In the present study, taking acyclic derivatives of 6-methyluracil as a model AChE inhibitor, we attempted to develop AChE inhibitors that specifically bind to the PAS with weak binding to the active site of AChE. We attempted to increase the size of AChE ligands to restrict specific binding to the PAS of AChE. To this aim we synthesized pyrimidinophanes bearing two o-nitrobenzylethyldialkylammonium heads. Almost all of synthesized pyrimidinophanes inhibited AChE in the nanomolar range. Based on molecular docking simulations, it was suggested that compounds bind AChE to the active center as well as to the PAS or only to the PAS. Thus, we found that introduction of the spacer, flexible or rigid, between [5-(o-nitrobenzylethylammonium)pentyl] units at N atoms of the 6-methyluracil moiety allows tuning the binding of 6-methyluracil derivatives with AChE. CONCLUSIONS: In conclusion, it can be stated that pyrimidinophanes are promising lead scaffold structures for further design of specific ligands for the PAS of AChE. Also AChE inhibitors with a 6-methyluracil moiety may be considered as potential drugs for the treatment of pathological muscle weakness syndromes. Show more

Keywords: Pyrimidinophanes, beta-amyloid, peripheral anionic site, acetylcholinesterase

DOI: 10.3233/JRS-150695

Citation: International Journal of Risk & Safety in Medicine, vol. 27, no. s1, pp. S72-S73, 2015

Authors: Lushchekina, S. | Kots, E. | Kharlamova, A. | Petrov, K. | Masson, P.

Article Type: Abstract

Abstract: BACKGROUND: Myasthenia gravis (MG) is a chronic autoimmune neuromuscular disorder characterized by fluctuating weakness of voluntary skeletal muscles. The cause of autoimmune response is unknown and only symptomatic therapies for MG are currently available. Pharmacological correction of synaptic failure underlying MG, involves partial inhibition acetyl- and butyrylcholinesterase. Effectiveness of cholinesterase inhibitors in the symptomatic treatment of MG is based on their ability to potentiate the effects of acetylcholine by decreasing the rate of its enzymatic hydrolysis at neuromuscular junctions. Several new inhibitors of AChE were tested in animal model of MG and may be considered as valuable candidates for …the treatment of pathological muscle weakness syndromes. In this study, we have investigated mechanisms of ChE inhibition by one of the most active 6-methyluracil derivatives (C547), as well as the possible benefits of using this compound for MG treatment compared to traditionally used pyridostigmine bromide. It was experimentally shown that C547 is a «pseudo-irreversible» slow-binding inhibitor of human AChE. Human BChE is reversibly inhibited by C547 with an affinity about 4 orders of magnitude lower than that of human AChE. Slow-binding inhibition of AChE leads to a lasting (over 24 hours) effect on the symptoms of muscle weakness in animal model of MG after a single administration of C547. OBJECTIVE: The aim of the present molecular modeling study was to reveal mechanism of AChE inhibition by C547 and elucidate its apparent «pseudo-irreversibility». METHODS: Two principle methods used in the present study were molecular docking and molecular dynamics (MD). Molecular docking was performed with Autodock 4.2.6 software, Lamarckian Genetic Algorithm to obtain structure of protein inhibitor complexes and Local Search for MD snapshots to compare relative binding affinity. For MD simulations NAMD 2.10 software with Charrm 36 force field was used, for the ligand C547 Charmm General Force Field was used, and missing parameters were obtained with quantum mechanical calculations. Unconstrained MD, steered MD (SMD) and free energy calculations with adaptive biasing force were performed. RESULTS: During unconstrained MD, C547 very rapidly binded to the peripheral anionic site (PAS) of AChE. To pass the bottleneck, application of the external force was required (SMD). Both SMD modelling and free energy calculation revealed that after crossing the AChE bottleneck, C547 falls into very favorable position. At the same time the rupture of interactions as well as overcoming the bottleneck gates in the course of pulling out procedure requires application of much higher force than during the pulling-in process. This difference between binding and dissociating processes explains apparent «pseudo-irreversibility» of the inhibitor. CONCLUSIONS: These findings are in good agreement with kinetics study showing that C-547 is a slow-binding inhibitor of type B, i.e. after rapid initial binding of inhibitor, the enzyme-inhibitor complex undergoes an isomerization step. Position obtained by SMD is in good agreement with X-ray data obtained by F. Nachon, IBS, France. Show more

Keywords: Molecular modeling, myasthenia gravis, slow-binding, inhibitor, acetylcholinesterase

DOI: 10.3233/JRS-150696

Citation: International Journal of Risk & Safety in Medicine, vol. 27, no. s1, pp. S74-S75, 2015

Authors: Cong, H.H. | Khaziakhmetova, V.N. | Zigashina, L.E.

Article Type: Abstract

Abstract: BACKGROUND: Non-steroidal anti-inflammatory agents (NSAIDs), steroids and representatives of other pharmacological groups [1, 2 ] are widely used for pharmacological regulation of inflammation. However, their anti-inflammatory effects are accompanied by serious adverse reactions [3, 4 ]. There was a hope that newer NSAIDs, selective inhibitors of COX-2, would be safer, but their longer-term use appeared to cause an increased risk of heart attacks and stroke [5 ]. Carrageenan rat paw oedema model is traditionally used for search and development of new NSAIDs with assessment of effects after 3 to 5 hours after oedema induction [6, 7 ], neglecting longer-term effects …[8 ]. OBJECTIVE: To compare effects of traditional NSAIDs (indomethacin, naproxen) on the development, duration and intensity of carrageenan rat paw oedema. METHODS: Carrageenan paw oedema was induced in 18 rats by sub-plantar injection into the right hind paw of the animals of 0.1 ml of 1% aqueous gel of carrageenan-λ (22049 SIGMA λ-Carrageenan plant-mucopolysaccharide, Sigma-Aldrich). We assessed the intensity of the oedema development and its duration by measurements of rat paw volume using plethysmometer 37140 (UgoBasile, Italy). Measurements were made prior to induction of oedema (base-line volume) and at 1, 2, 3, 4, 5, 24, 48, 72, 96, 120, 144, 168 and 192 hours after sub-plantar carrageenan injection. Calculating the percentage of increase in paw volume assessed the intensity of the oedema. The base-line paw volume was taken for 100%. Animals were divided into 3 groups of 6 rats each; group 1: control (solvent); group 2: naproxen 15 mg/kg and group 3: indomethacin 10 mg/kg. These doses are known as ED50 (effective doses 50) on carrageenan rat paw oedema with single-dose NSAIDs administration [9 ]. To get the most accurate estimate of the intensity of the simulated by carrageenan inflammatory response and the potential effects of some NSAIDs with their longer-term use we calculated areas under the curve «increase in paw volume – time» using standard method of numerical integration - trapezoidal method. Statistical analysis was performed using Microsoft Office Excel 2007 with the calculation of arithmetic means M, their standard deviations (δ) and standard errors (m). We applied Student’s t -test and accepted as significant the differences with P values equal to or less than 0.05. RESULTS: The inflammatory reaction induced by carrageenan, developed in a form of swelling/oedema with an increase in the rat paw volume up to 55% of the baseline volume. The maximum volume of oedema was observed in the control group at 3 h after the injection of carrageenan, which is in accordance with the literature data on the development of carrageenan paw edema in rats [10, 11 ]. Naproxen at a dose of 15 mg/kg showed anti-inflammatory activity at 1, 2, 3, 4 and 5 hours after administration of carrageenan with suppression of oedema development by 59, 81, 73, 60 and 39% (p = 0.03; 0.001; 0.001; 0.001 and 0.01), respectively. There was no oedema inhibition by naproxen at later time-points. Indomethacin at a dose of 10 mg/kg showed anti-inflammatory effect at 2, 3, 4, and 5 hours after carrageenan oedema induction with inhibition of oedema development by 54, 54, 54 and 33% (p = 0.01, 0.004, 0.001 and 0.01) respectively. Again there was no oedema inhibition by indomethacin at the later time-points. When comparing the calculated areas under the curve «increase in paw volume – time» we found no differences between the values of control and study groups: naproxen (15 mg/kg) and indomethacin (10 mg/kg). We think that these values of areas under the curve «increase in paw volume – time» represent the total inflammatory reaction induced by carrageenan and need to be used for the assessment of future potential anti-inflammatory agents which should not only produce short-term symptomatic oedema suppression, but change the nature of the oedema response, potentially with alternative mechanisms of action. Our experimental findings are in accordance with the well-known lack of effects of NSAIDs on the outcomes of chronic inflammatory diseases [12 ]. This may be due to the fact that they suppress the development and symptoms of inflammation at the early stages, but the reaction to inflammatory stimuli develops fully over the longer period of time and takes its full course nonetheless. This proves that traditional modeling approaches to future potential anti-inflammatory agents development needs re-assessment. CONCLUSIONS: Single-dose administration of naproxen (15 mg/kg) or indomethacin (10 mg/kg) exerts decrease in rat paw oedema volume at no later than 5 hours after oedema induction by carrageenan. Evaluating anti-inflammatory activity by the areas under the curve «increase in paw volume – time» proves that a single-dose NSAID’s administration has no effect on the inflammatory response when evaluated not by single time-point index (at 3 or 5 hours), but by assessing the oedema development and duration over 192 hours (8 days). Show more

Keywords: Rat paw oedema, NSAIDs, timing of effects, anti-inflammatory

DOI: 10.3233/JRS-150697

Citation: International Journal of Risk & Safety in Medicine, vol. 27, no. s1, pp. S76-S77, 2015

Authors: Vyshtakalyuk, A.B. | Nazarov, N.G. | Zobov, V.V. | Semenov, V.E. | Galyametdinova, I.V. | Tcherepnev, G.V. | Reznic, V.S.

Article Type: Abstract

Abstract: BACKGROUND: Research and development of effective hepatoprotective medicines is one of the priority areas of research in Russia. Literature data shows that active research and development of hepatoprotectors is carried out both in Russian and other countries [1–6 ]. Pirimidines are used as hepatoprotective medicines stimulating protein synthesis and reparation of hepatocytes in toxical and infectious liver disorders [7 ]. In our previous work ee have shown hepatoprotective properties of pyrimidine derivative, named Xymedon [8 ]. This research, funded by the Russian Science Foundation, is aimed at identifying the most effective hepatoprotectors among pirimidine derivatives. OBJECTIVE: To test …hepatoprotective properties of one of the new Xymedon (Xym) derivative - L-ascorbate 1-(2-hydroxyethyl)-4,6-dimethyl-1,2-dihydro-pirimidine-2-one (Asc-Xym) on the toxic liver damage model induce by carbon tetrachloride (CTC, CCl4 ). METHODS: The toxic liver damage in rats was modeled by subcutaneous injection of CTC (CCl4 ) in vegetable oil (mixed at 1:1 ratio) at a dose of 2 ml per kg. The experiments were carried out under two schemes: 1) oral administration of Xym or Asc-Xym preparations by gavage at the doses of 10 and 20 mg/kg followed by subcutaneous injection of CTC 1 hour after pyrimidine oral administration and continued for 3-4 days; - this was the design of preventive pyrimidine use, 2) liver damage modeling by CTC subcutaneous injections for 3 days followed by oral administration of Xym, Asc-Xym or Thiotriazolin (Thi) preparations at the doses of 20 mg/kg for 5 days; - this was the design of therapeutic scheme. The rats of control groups were injected with CTC according to the same schemes, but did not get any preparations. We looked at some biochemical parameters of blood serum: alanine aminotran-sferase (AlAT), aspartate aminotransferase (AsAT), their ratio (de Rytis coefficient), and the total protein level as the markers of toxic liver damage. We performed statistical data analysis by rank nonparametric Mann-Whitney U-criterion for comparison of two independent groups. We evaluated pathomorphologic characteristics of liver damage on the histological slices colored with hematoxylin and eosin. RESULTS: Carbon tetrachloride (CTC) caused profound changes in the studied biochemical parameters of rats’ blood serum. The AlAT activity level in the serum of control animals in the preventive scheme was 116,23 (the median) with the lower quartile and the upper quartile of 76,96 and 211,71 U/l respectively; the AsAT level was 230,08/201,49-290,03 U/l; this was the increase in comparison with the reference values. De Rytis coefficient was 1,76 /1,47-2,67. This was the decrease in comparison with the reference values of intact group (36,37/28,18-43,3 U/l; 132,95 /118,24-164,00 U/l and 4,26/3,03-5,23 respectively). The differences were statistically significant at P < 0,001. In the experimental groups the changes of the biochemical parameters with respect to the reference values were less marked than in Control. The AlAT level was 89,86/87,06-165,15; 103,23/38,19-270,87 U/l; 80,28/6,12-141,82 and 100,33/62,24-144,64 U/l in the groups of rats treated with Xym at the doses of 10 and 20 mg/kg or Asc-Xym at the doses of 10 and 20 mg/kg respectively. Similarly, in the same groups the AsAT level was 211,19/170,20-250,16; 193,61 /181,57-274,69 U/l; 190,91 /65,21-198,65 and 173,25/135,50-210,70 U/l respectively. The differences of the AsAT level were statistically significant at P < 0,05 in comparison with Control in the both groups treated with Asc-Xym. Nnearly 2 times increase of the AlAT level (67,60/1,22-94,60 U/l) (P = 0,00002) was shown in comparison with the reference values in the rats of Control group in the therapeutic scheme. However the AsAT level (163,80/130,1-178,8 U/l) was only slightly higher than reference values. De Rytis coefficient (2,07/1,78-3,48) was significantly lower than the reference values (P = 0,001). The total protein level (59,36/55,17-60,10 g/l) was lower than the reference values (65,06/62,06-68,98 g/l) by 8,4%. The differences of biochemical parameters as compared with the reference values in rats of experimental groups treated with Xym, Thi and Asc-Xym at the doses 20 mg/kg were less than those in the Control groups. They were: AlAT 52,49/44,64-62,30 and 61,42/53,20-96,66 U/l, AsAT 105,00/94,7-142,3 and 235,35/111,7-335,6 U/l, de Rytis coefficient 2,09/1,87-2,28 and 3,24/1,86-4,53, total protein 63,10/62,46-64,27 and 62,46/58,70-64,43 g/l respectively in the groups treated with Xym and Thi. The values of the studied biochemical parameters AlAT (39,04/32,46-44,24 U/l), AsAT (111,9/105,27-155 U/l), de Rytis coefficient (2,87/2,72-3,30), total protein (62,89/61,46-68,14 g/l) of the rats, treated with Asc-Xym, were the most close to the reference values in comparison with other experimental groups. The analysis of histological slices revealed large areas of steatosis and necrosis of hepatocytes in Control groups in both schemes. These were less pronounced in experimental groups than in Control groups and particularly in rats, treated with Asc-Xym. CONCLUSIONS: Hepatoprtotective properties of the new compound L-ascorbate 1-(2-hydroxyethyl)-4,6-dimethyl-1,2-dihydropirimidine-2-one were established. The hepatoprotective efficacy of the compound is higher than that of Xymedon and Thiotriazolin. Show more

Keywords: Rats, pyrimidine, liver damage, carbon tetrachloride, biochemical markers

DOI: 10.3233/JRS-150698

Citation: International Journal of Risk & Safety in Medicine, vol. 27, no. s1, pp. S78-S79, 2015

Authors: Lushchekina, S. | Delacour, H. | Lockridge, O. | Masson, P.

Article Type: Abstract

Abstract: BACKGROUND: Prolonged apnoea following injection of ester-containing myoralaxants was first described in 1953. Because a large part of administered succinylcholine is shortly hydrolyzed by plasma butyrylcholinesterase (BChE) under normal conditions, prolonged apnoea was attributed to deficiency in BChE. It was found that BChE deficiency was due to genetic variations. Human BChE gene shows a large polyallelism. About 75 natural mutations of the BCHE gene have been documented so far [1 ]. Most of them cause alteration in BChE activity through point mutation effect on catalytic activity. Frame shifts and stop codons may also affect expression, or cause truncations in …the sequence. OBJECTIVE: Recently, two novel BChE “silent” variants, Val204Asp [2 ] and Ala34Val [3 ], causing prolonged neuromuscular block after administration of mivacurium, were discovered. Mutations were genetically and kinetically characterized. The aim of the current study was to understand how these mutations determine “silent” phenotype. METHODS: Molecular dynamics studies were carried out with NAMD 2.9 software at the Lomonosov supercomputer. Charmm 36 force field was used, periodical boundary conditions, 1 atm pressure, 298 K. 100 ns molecular dynamics runs were performed for the wild-type BChE and its mutants Val204Asp and Ala34Val. RESULTS: Unlike wild-type BChE, which retained its operative catalytic triad through the whole MD simulation, the catalytic triad of mutants was disrupted, making chemical step impossible. Val204Asp mutation leads to reorganization of hydrogen bonding network around the catalytic triad, which in turn increases the distance between catalytic residue main chains. Mutation Ala34Val, located on the protein surface, leads to increased fluctuations in the Ω-loop and subsequent disruption of the gorge structure, including disruption of the catalytic triad and formation of new hydrogen bonds involving catalytic center residues. CONCLUSIONS: Comparative study of the “silent” Ala328Asp mutant and the catalytically active mutant Ala328Cys shows that MD approach can discriminate between the differential effects of point mutations at a same position. Show more

Keywords: Genetic, polymorphism, butyrylcholinesterase, molecular modeling, allelozymes

DOI: 10.3233/JRS-150699

Citation: International Journal of Risk & Safety in Medicine, vol. 27, no. s1, pp. S80-S81, 2015

Authors: Gabdrakhmanov, A.I. | Khayrullin, A.E. | Grishin, C.H. | Ziganshin, A.U.

Article Type: Abstract

Abstract: BACKGROUND: Extracellular purine compounds, adenosine triphosphate (ATP) and adenosine, are involved in regulation of many cell functions, engaging in rapid and long-term cellular processes. The nucleotides, including ATP, exert their extracellular effects by influencing membrane P2 receptors. ATP outside of the cell rapidly is metabolized by the ecto-enzyme system to produce adenosine, which acts on separate adenosine (P1) receptors. Since adenosine and ATP often are functional antagonists, ATP degradation not only limits its effect, but also brings new ligand with different, often opposing, properties. Great variety and widespread of P2 and adenosine receptors in the body emphasize the important physiological …and pathophysiological significance of these receptors, and make them very attractive as targets for potential drug action. The existence of several subtypes of P2 and adenosine receptors has been shown in the skeletal muscles. ATP as a co-transmitter is densely packed together with classical neurotransmitters in the presynaptic vesicles of vertebral motor units but until recently ATP was refused to have its own functional role there and was recognized only as a source of adenosine. However, on the eve of the third millennium there appeared data that ATP, released from the nerve ending and acting on presynaptic P2 receptors, suppresses subsequent quantum release of acetylcholine. The final product of its degradation, adenosine, performs a similar inhibitory effect acting on presynaptic adenosine receptors. Despite the fact that the mechanisms of presynaptic inhibitory action of ATP and other purines were studied earlier, the object of those studies was usually neuromuscular synapse of cold-blooded animals. The few studies, in which experiments were carried out on preparations of warm-blooded animals, described the basic effects of purines. These often were guided by the convenience of preparation of the synapses of the diaphragm. We think that those results cannot be considered as typical effects of ATP and other purines on skeletal muscles and could not be extrapolated to all warm-blooded animals. Furthermore the role of ATP and its derivatives in the accumulation of vertebrate muscular effort has not been investigated. It is known that in physiological conditions vertebrates may mobilize only up to a third of the maximum muscle force. Why the two-thirds of muscular strength are not used normally but may be used at stress, remains unknown. It is known that the body’s adaptive response to stress is a change in the activity of the endocrine system. The leading role in this is given to catechol amines and glucocorticoids, mobilized in significant quantities in blood under stress. We have found previously that incubation of frog sartorius muscle with hydrocortisone resulted in a decrease of contraction amplitude. However, when hydrocortisone was used in combination with ATP, its inhibitory effect on contractile responses disappeared. It is interesting that hydrocortisone had no effect on the inhibitory effect of adenosine. In the following experiments, assessing the effect of hydrocortisone on rat soleus muscle, it was established that hydrocortisone and purines had similar inhibitory effect. When ATP and hydrocortisone were given together the same oppression occurred. OBJECTIVE: To study the effects of ATP and adenosine on contraction parameters of rat skeletal muscle and assess the impact of the catechol amines on these processes. METHODS: Contractions of rat soleus muscles were recorded isometrically by mechanical sensor Linton FSG-01 (UK) according to standard procedures. The average of muscle parameters received within 30 seconds (30 responses) was treated as one result. Amplitude and time characteristics of the curve reductions were estimated. During all experiments standard Krebs solution flowed through the bath continuously to which agents were added at necessary concentrations. All experimental animals were maintained and prepared for dissection under the European Convention for the Protection of Vertebrate Animals used in scientific experiments. All agents used in the study were supplied by Sigma Chemical Company Ltd. (UK), Tocris Cookson and Research Biochemicals International (USA). RESULTS: The concentration of 100 μM for adenosine is close to saturation [1 ], and for its predecessor ATP this concentration is created after the passage of a pulse through the synapse [2 ]. We used this concentration of purines to study the mechanism of action of adenosine and ATP on neuromuscular synapse. The effect of adenosine was partially inhibited in the presence of 100 μM 8-SPT, an antagonist of adenosine receptors. The contraction force of "fast" and "slow" rat skeletal muscles was raised by half in the presence of norepinephrine. In the presence of norepinephrine adenosine exerted its effect fully, but ATP by half reduced its depressor effect on the contraction force of both muscles. CONCLUSIONS: 1. Norepinephrine increases half times of the reduction of “fast” and “slow” skeletal muscle. 2. In the presence of norepinephrine, inhibitory effect of adenosine on contraction force is maintained. 3. Inhibitory effect of ATP on contraction force of studied skeletal muscles becomes twice less pronounced in the presence of norepinephrine. We think that reduction of ATP depressive effect on the skeletal muscle by norepinephrine may be an adaptive response to acute stress. Show more

Keywords: Norepinephrine, effect of ATP, rat, skeletal muscle, neuromuscular synapse

DOI: 10.3233/JRS-150700

Citation: International Journal of Risk & Safety in Medicine, vol. 27, no. s1, pp. S82-S83, 2015