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With this, we offer you customized health insurance plans for both yourself and your entire family. At your request, you are being redirected to a third party site. The substance cytotoxicity was determined by a relative survival RS capacity of the cells, calculated as a ratio between the cloning efficiency CE of the cells plated immediately after the treatment and the normal cellular CE of non-treated cells negative controls after 7 days of culturing.
The rest of the treated CHO-k1 cells were maintained in the growth medium for 8 days to allow near-optimal phenotypic expression of any induced in hprt gene mutations. Then, to determine the frequency of the induced mutations, the cells were subcultured and maintained in the growth medium in the presence 2,, cells or in the absence cells of a selective agent 2.
After that, the number of cell colonies in both media was counted and the frequency of mutations was calculated with the formula:.
Three separate immunotoxicity tests were performed: 1 evaluation of cellular immunity in a delayed-type hypersensitivity test, 2 evaluation of animal immune response to a standard antigen, 3 evaluation of the phagocytic activity of peritoneal macrophages. For carrying out these experiments 90 male ICR mice were used, they were divided equally into three groups 30 animals per group for each study and in these groups they were subdivided into subgroups of 10 mice each and treated as follows: mice of the first subgroup were injected with normal saline im, the second subgroup animals were treated with Az 0.
The reaction index was calculated using the indicated formula:. R i —reaction index, W exp —weight of the experimental paw, W cont —weight of the control paw. After 7 days the venous mouse blood was collected from the inferior vena cava, then the blood serum was isolated and the titers of IgG antibodies to BSA were determined using a standard enzyme immunoassay. On the next day after the seven-day course of the Az administration, the mice of the third group 30 animals were injected intraperitoneally with 2 mL of ink particles suspension.
After 10 min the mice were euthanized CO 2 inhalation with a subsequent isolation of the peritoneal exudate from their abdominal cavity. Allergenicity of Az was studied in a delayed-type hypersensitivity test on male 20 animals and female 20 animals adult ICR mice. The mice were divided into four groups two control and two experimental groups of 10 animals males or females per group. Mice of the experimental groups were injected subcutaneously at the base of a tail with Az solution 0.
Five days later all animals received an injection of Az 0. To estimate the intensity of the studied allergic reaction, 6, 12 and 24 h after the second Az injection the thickness of the left and right hind mouse paws was measured with a digital caliper.
Cells were sub-cultured and plated at a density of —10, cells per well in a well black plate Corning Inc. They were grown in a CO 2 incubator for 48—72 h before testing the functional activity of natively expressed nAChRs by calcium imaging. They were sub-cultured the day before transfection and were plated at a density of 10, cells per well in a well black plate. Calcium imaging procedure was performed as published earlier [ 11 ]. Fluorescence was recorded every 2 s for three minutes following agonist addition.
Responses were measured as peak intensity minus basal fluorescence level and were expressed as a percentage of the maximal response obtained to agonist.
Az was pre-applied to an oocyte for 5 min before its co-application with agonist nicotine. Animals of the control and experimental groups were treated with normal saline and with Az at doses of 0. In addition 6 mice were treated with Az at dose of 0. The corresponding solutions were injected into the triceps muscles of the mouse forelimbs at a volume of 0. For all animals their basic forelimb grip strength was recorded before the substance administration with a grip strength meter Columbus Instruments, Columbus, OH, USA.
Further, their grip strength was measured 5, 10, 15, 20, 30, 60, 90 min after the Az or normal saline im administration. Rocuronium was administered intramuscularly as described for the measurement of acute toxicity Section 5.
Muscle strength was assessed prior to administration of the substance and then 1, 2, 3, 4, 5 min after administration. To statistically evaluate the efficacy and specificity of Az in vitro, two-tailed Mann—Whitney U test was used. The following are available online at www.
E contributed materials; I. The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript and in the decision to publish the results. National Center for Biotechnology Information , U. Journal List Toxins Basel v. Toxins Basel. Published online Jan 7. Irina V. Shelukhina , 1 Maxim N.
Zhmak , 1 Alexander V. Lobanov , 2 Igor A. Ivanov , 1 Alexandra I. Garifulina , 1 Irina N. Kravchenko , 2 Ekaterina A. Rasskazova , 2 Margarita A. Salmova , 2 Elena A. Tukhovskaya , 2 Vladimir A. Rykov , 2 Gulsara A. Slashcheva , 2 Natalya S. Egorova , 1 Inessa S. Muzyka , 1 Victor I. Tsetlin , 1 and Yuri N.
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Victor I. Find articles by Victor I. Yuri N. Find articles by Yuri N. Author information Article notes Copyright and License information Disclaimer. Received Nov 3; Accepted Jan 2. This article has been cited by other articles in PMC. Associated Data Supplementary Materials toxinss Abstract Azemiopsin Az , a linear peptide from the Azemiops feae viper venom, contains no disulfide bonds, is a high-affinity and selective inhibitor of nicotinic acetylcholine receptor nAChR of muscle type and may be considered as potentially applicable nondepolarizing muscle relaxant.
Keywords: nicotinic acetylcholine receptor, azemiopsin, preclinical studies, toxicity, pharmacokinetics, myorelaxant. Key Contribution: Investigation of the preclinical profile of azemiopsin demonstrated its high affinity and specificity for muscle type nicotinic acetylcholine receptor as well as good muscle relaxant capacity. Toxicology studies in mice indicated that azemiopsin was well tolerated during chronic dosing and showed no immunotoxicity, allergenic or mutagenic activity, which made it a good candidate for application as a local muscle relaxant.
Introduction A linear peptide azemiopsin Az isolated from the Azemiops feae viper venom contains no disulfide bonds [ 1 ] and can be easily prepared by peptide synthesis. Results 2. Az Synthesis The Az peptide was prepared by a solid phase synthesis using a general Fmoc-strategy. Open in a separate window. Figure 1. Efficacy and Specificity of Az In Vitro To study a specific activity and selectivity of Az in vitro, two different methods were used: electrophysiological method of two-electrode voltage-clamp on Xenopus oocytes and calcium imaging using the genetically encoded calcium sensor Case12 or the low-molecular weight calcium indicator Fluo Figure 2.
Figure 3. In Vivo Efficacy Tests 2. In Vivo Az Efficacy To study a specific activity of Az as an agent blocking neuromuscular transmission for the treatment of muscular dystonia, its effect on mouse muscular strength was estimated.
Figure 4. In Vivo Rocuronium Efficacy The rocuronium effect was studied at doses of 0. Figure 5. Pharmacokinetics of Az To study a pharmacokinetics of Az, its radioiodinated I-labeled analog [ I]-Az was prepared. Preparation of [ I]-Az In position 9, Az molecule contains a histidine residue which may be subjected to electrophilic iodination as published earlier [ 12 ].
Pharmacokinetics Studies Single intravenous iv and intramuscular im administrations of [ I]-Az at doses of 0. Figure 6. Acute Toxicity Tests 2. Acute Toxicity of Az The acute toxicity of Az after intraperitoneal administration to mice was determined earlier [ 1 ], the LD 50 value was 2. Acute Toxicity of Rocuronium Injection of rocuronium at a dose of 0. Subchronic Toxicity of Az For the study of the subchronic toxicity, the number of animals was increased in comparison with acute toxicity tests, because many biochemical, histological and other parameters needed to be measured.
Immunotoxicity of Az To study the possible immunotoxicity of Az, its effects on 1. Figure 7. Allergenicity of Az To test the allergenicity of Az, its ability to induce a delayed-type hypersensitivity reaction in male and female ICR mice at a dose of 0. Figure 8. Mutagenicity of Az To study Az mutagenicity in vitro, its ability to induce mutations in the hypoxanthine guanine phosphoribosyltransferase hprt gene of mammalian CHO-k1 cells was tested.
Discussion As discussed above peptide and protein drugs in many cases possess higher efficacy and better specificity as compared to low molecular weight compounds. Conclusions In summary, we investigated the preclinical profile of Az in regard to in vitro and in vivo efficacy, acute and chronic toxicity, pharmacokinetics, allergenic capacity, immunotoxicity and mutagenic potency.
Materials and Methods 5. Az Synthesis 5. Solid Phase Az Synthesis Az synthesis was performed on automatic peptide synthesizer based on Gilson automated liquid handler system according to Gilson application note Iodination of Formylated Az The iodination was carried out in general according to the published procedure [ 13 ].
Deprotection of Radioiodinated Az To remove formyl protecting groups, the radioiodinated Az derivative was treated with mM sodium hydroxide solution for 12 h. Toxicity Studies 5. Acute Toxicity Az Acute Toxicity Az acute toxicity was estimated for its intravenous iv and intramuscular im administration to male ICR mice in a stepwise procedure. Rocuronium Acute Toxicity Rocuronium was administered intramuscularly at doses of 0.
Subchronic Toxicity A repeated dose day intramuscular toxicity study was conducted on 36 SD male and 36 SD female adult rats. Pharmacokinetics The pharmacokinetic study was performed using male ICR mice. Mutagenicity The ability of Az to induce mutations in the hypoxanthine guanine phosphoribosyltransferase hprt gene of Chinese hamster ovary CHO-k1 cells was tested.
Immunotoxicity Three separate immunotoxicity tests were performed: 1 evaluation of cellular immunity in a delayed-type hypersensitivity test, 2 evaluation of animal immune response to a standard antigen, 3 evaluation of the phagocytic activity of peritoneal macrophages. Phagocytic Activity of Peritoneal Macrophages On the next day after the seven-day course of the Az administration, the mice of the third group 30 animals were injected intraperitoneally with 2 mL of ink particles suspension.
Allergenicity Allergenicity of Az was studied in a delayed-type hypersensitivity test on male 20 animals and female 20 animals adult ICR mice. Efficacy and Specificity Studies 5. Calcium Imaging Calcium imaging procedure was performed as published earlier [ 11 ]. In Vivo Muscle Relaxant Effect of Rocuronium Rocuronium was administered intramuscularly as described for the measurement of acute toxicity Section 5.
Supplementary Materials The following are available online at www. Click here for additional data file. Author Contributions I. Conflicts of Interest The authors declare no conflict of interest. References 1. Utkin Y. Azemiopsin from Azemiops feae viper venom, a novel polypeptide ligand of nicotinic acetylcholine receptor. Unwin N. Nicotinic acetylcholine receptor and the structural basis of neuromuscular transmission: Insights from Torpedo postsynaptic membranes.
Jonsson F. Pharmacological characteristics of the inhibition of nondepolarizing neuromuscular blocking agents at human adult muscle nicotinic acetylcholine receptor. Bowman W. Neuromuscular block. Habre W. The involvement of histaminic and muscarinic receptors in the bronchoconstriction induced by myorelaxant administration in sensitized rabbits.
Bornia E. Presynaptic M1, M2, and A1 receptors play roles in tetanic fade induced by pancuronium or cisatracurium. Albanese A. Phenomenology and classification of dystonia: A consensus update. Kudryavtsev D. Natural compounds interacting with nicotinic acetylcholine receptors: From low-molecular weight ones to peptides and proteins.
Russian Patent. Kasheverov I. Signal Transduct. Shelukhina I. Tsomides T. Stoichiometric labeling of peptides by iodination on tyrosyl or histidyl residues. Hall R. Clinical Pathology of Laboratory Animals. In: Gad S. Animal Models in Toxicology. Johnson G. In: Parry J. Genetic Toxicology. Matsuoka M. Chromosomal aberration tests on 29 chemicals combined with S9 mix in vitro. Legal Portal of Eurasian Economic Union. In Russian. Jonsson M. Distinct pharmacologic properties of neuromuscular blocking agents on human neuronal nicotinic acetylcholine receptors: A possible explanation for the train-of-four fade.
Grip strength in mice with joint inflammation: A rheumatology function test sensitive to pain and analgesia.
Nevins M. Quantitative grip strength assessment as a means of evaluating muscle relaxation in mice. Torii Y.
Comparison of effects of botulinum toxin subtype A1 and A2 using twitch tension assay and rat grip strength test. Wright P. Population based pharmacokinetic analysis: Why do we need it; what is it; and what has it told us about anaesthetics?
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