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Toxins (Basel)
2018 Jan 07;101:. doi: 10.3390/toxins10010034.
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Azemiopsin, a Selective Peptide Antagonist of Muscle Nicotinic Acetylcholine Receptor: Preclinical Evaluation as a Local Muscle Relaxant.
Shelukhina IV
,
Zhmak MN
,
Lobanov AV
,
Ivanov IA
,
Garifulina AI
,
Kravchenko IN
,
Rasskazova EA
,
Salmova MA
,
Tukhovskaya EA
,
Rykov VA
,
Slashcheva GA
,
Egorova NS
,
Muzyka IS
,
Tsetlin VI
,
Utkin YN
.
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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. In this study, we investigated its preclinical profile in regard to in vitro and in vivo efficacy, acute and chronic toxicity, pharmacokinetics, allergenic capacity, immunotoxicity and mutagenic potency. The peptide effectively inhibited (IC50 ~ 19 nM) calcium response of muscle nAChR evoked by 30 μM (EC100) acetylcholine but was less potent (IC50 ~ 3 μM) at α7 nAChR activated by 10 μM (EC50) acetylcholine and had a low affinity to α4β2 and α3-containing nAChR, as well as to GABAA or 5HT₃ receptors. Its muscle relaxant effect was demonstrated at intramuscular injection to mice at doses of 30-300 µg/kg, 30 µg/kg being the initial effective dose and 90 µg/kg-the average effective dose. The maximal muscle relaxant effect of Az was achieved in 10 min after the administration and elimination half-life of Az in mice was calculated as 20-40 min. The longest period of Az action observed at a dose of 300 µg/kg was 55 min. The highest acute toxicity (LD50 510 μg/kg) was observed at intravenous injection of Az, at intramuscular or intraperitoneal administration it was less toxic. The peptide showed practically no immunotoxic, allergenic or mutagenic capacity. Overall, the results demonstrate that Az has good drug-like properties for the application as local muscle relaxant and in its parameters, is not inferior to the relaxants currently used. However, some Az modification might be effective to extend its narrow therapeutic window, a typical characteristic and a weak point of all nondepolarizing myorelaxants.
Figure 1. Analytical UPLC-MS on Phenomenex Aeris PEPTIDE XB-C18 column (1.7 µm, 2.1 × 150 mm) using a linear acetonitrile gradient from 10 to 35%. Inset. Deconvoluted mass-spectrum of Az sample obtained after the final purification step. a.u., arbitrary unit.
Figure 2. Interaction of Az with muscle and neuronal nAChRs. Inhibitory curves of Az action on ACh (30 and 10 μM)-evoked intracellular calcium concentration ([Ca2+]i) rises in neuroblastoma Neuro2a cells expressing (a) muscle α1β1εδ and (b) α7 nAChRs, respectively. (c) Representative nicotine (Nic)-induced current traces through α4β2 nAChR and (d) dose-response curve of [Ca2+]i amplitude rise in neuroblastoma SH-SY5Y cells expressing α3-containing nAChRs in response to different concentrations of Nic. There are no inhibitory effects of Az on Nic-evoked (c) ion currents and (f) calcium responses mediated by α4β2 and α3-containing nAChRs, respectively (p > 0.05, Mann–Whitney U test). (e) Inhibitory curve of α-conotoxin MII action on Nic (100 μM)-evoked [Ca2+]i rise in SH-SY5Y cells expressing α3-containing nAChRs. Each point represents data obtained from 4 independent experiments (mean ± SEM).
Figure 3. Interaction of rocuronium with muscle and neuronal nAChRs. Inhibitory curves of rocuronium action on ACh (30 and 10 μM)-evoked intracellular calcium concentration ([Ca2+]i) rises in neuroblastoma Neuro2a cells expressing (a) muscle α1β1εδ and (b) α7 nAChRs, respectively. Inhibition of [Ca2+]i amplitude rise induced by Nic (100 μM) in neuroblastoma SH-SY5Y cells expressing α3-containing nAChRs by different concentrations of rocuronium (c). Each point represents data obtained from 4 independent experiments (mean ± SEM).
Figure 4. Muscle relaxant effect of Az. The time courses of a grip strength of mouse (ICR males) forelimbs at 0–90 min after Az (0.03, 0.1 and 0.3 mg/kg) or normal saline (control) intramuscular administration. The results are presented as mean values ± SEM, n = 10. Significant differences in the forelimb strength were revealed between control and experimental groups (one-way repeated measures ANOVA, * p < 0.05).
Figure 5. Muscle relaxant effect of rocuronium. The time courses of a grip strength of mouse (ICR males) forelimbs at 0–5 min after rocuronium (0.08, 0.1 mg/kg) or normal saline (control) intramuscular administration. The results are presented as mean values ± SEM, n = 4. Significant differences in the forelimb strength were revealed between control and experimental groups (one-way repeated measures ANOVA, * p < 0.05).
Figure 6. Pharmacokinetic curves of [125I]-Az concentration in blood of male ICR mice 0–24 h after its (a) intravenous and (b) intramuscular administration at doses of 0.25 and 0.5 mg/kg. The results are presented as mean values ± SD, n = 5 (each plot point represents mean results for five animals).
Figure 7. The influence of Az on immune system. The effect of 7-day intramuscular Az (0.15 and 0.5 mg/kg) administration to male ICR mice (a) on their delayed-type hypersensitivity (DTH) to a specific antigen (10 mM trinitrobenzenesulfonic acid) manifested by paw edema, (b) on their immune response to a bovine serum albumin (BSA) and (c) on phagocytic activity of their peritoneal macrophages. (a) The presented indexes of DTH reactions reflect the normalized difference in the weights of treated and control mouse hind paws. (b) The titers of IgG in blood serum of mice from experimental and control groups after their standard immunization with BSA are presented. (c) The number of total and phagocytic (engulfing ink particles) macrophages in 1 μL of peritoneal exudate isolated from experimental and control animals. In all three tests, there was no significant difference between control and experimental animals (p > 0.05, Kruskall-Wallis ANOVA on ranks) in the parameters studied. The results are presented as mean values ± SEM, n = 5–10.
Figure 8. The capacity of Az (0.15 mg/kg subcutaneously) to provoke allergic (a delayed-type hypersensitivity) reaction in male and female ICR mice. The differences in the thickness of the experimental left (injected with Az) and the control right hind paws are presented for animal groups with preliminary Az sensitization and without it 6, 12 and 24 h after the second peptide administration. There was no significant difference in the increase in the thickness of left hind paws between all animal groups (p > 0.05, Kruskall-Wallis ANOVA on ranks). The results are presented as mean values ± SEM, n = 10.
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