Supramolecular therapeutics to treat the side effects induced by a depolarizing neuromuscular blocking agent

Abstract

Succinylcholine (Sch) is the only depolarizing neuromuscular blocking agent widely used for rapid sequence induction in emergency rooms. Unfortunately, a variety of (sometimes lethal) adverse effects, such as hyperkalemia and cardiac arrest, are associated with its use, and currently there are no specific antidotes to reverse Sch or to treat these side-effects. Methods: The binding behaviors of Sch and several synthetic receptors, including cucurbit[7]uril, sulfo-calix[4]arene and water-soluble carboxylatopillar[6]arene (WP[6]), were first investigated. With a mouse model, a leathal dose of Sch was selected for evaluation of the antidotal effects of these synthetic receptors on Sch induced mortality. The antidotal effects of a selected synthetic receptor, WP[6], on Sch induced cardiac arrhythmias, hyperkalemia, rhabdomyolysis and paralysis were subsequently evaluated with rat and mouse models. The reversal mechanism was also investigated at a cellular level. Results: All of these macrocyclic molecules exhibited relatively high binding affinities with Sch in vitro. In a Sch-overdosed mouse model, immediate injection of these synthetic receptors right after Sch administration increased the overall survival rate, with WP[6] standing out with the most effective antidotal effects. In addition, administration of WP[6] also reversed the paralysis induced by Sch in a mouse model. Moreover, infusion of WP[6] to Sch-overdosed rats reduced the incidence of cardiac arrhythmia, inhibited the otherwise abnormally high serum potassium levels, and relieved the muscular damage. At the cellular level, WP[6] reversed the Sch induced depolarization and reduced the efflux of intracellular potassium. Conclusion: Synthetic receptors, particularly WP[6], exhibited high binding affinities towards Sch, and presented a significant potential as supramolecular therapeutics to treat the various side effects of Sch by specifically sequestering Sch in vivo.

Keywords: antidote; host-guest chemistry; neuromuscular blocking agent; pillararene; succinylcholine.

Figure 1

Chemical structures and binding behaviors of WP[6] and Sch. (A) Chemical structures of WP[6], SC[4]A, CB[7], Ch, Ach and Sch. (B) ITC titration of WP[6] and Sch in water at 25 °C. One binding site model was utilized to fit the data, affording an association constant Ka of 3.42 × 10⁶ M^-1 (ΔH: -28.2 kJ/mol, ΔG: -37.3 kJ/mol and -T ΔS: -9.16 kJ/mol). (C) Energy-optimised inclusion complexes of WP[6] and Sch. (D) ¹H-NMR spectra (400 MHz, D₂O, room temperature).Top: Sch (0.50 mM); Middle: WP[6] (0.50 mM) and Sch (0.50 mM); Bottom: WP[6] (0.50 mM). IS: internal standard (tetramethylsilane).

Figure 2

Synthetic receptors reversed Sch induced death in a mouse model. (A) Survival rates of mice that were i.v. administered with various doses of Sch. n = 6 for each group. (B) Survival rates of mice that were i.v. treated with various doses of synthetic receptors immediately after the i.v. administration of a lethal dose of Sch (0.75 mg/kg). n = 6 for each group. (C) Weight changes of mice that received the treatment of WP[6] immediately after i.v. administration of 0.75 mg/kg Sch. n = 6 for each group. (D) Hematological parameters of the blood from mice that were sacrificed at Day 14 after received the treatment of WP[6] immediately after the i.v. administration of a lethal dose of Sch (0.75 mg/kg). n = 6 for each group.

Figure 3

WP[6] reversed the Sch induced abnormal ECG pattern in a rat model. (A) Representative ECG patterns of rats that i.v. administered with or without 50 mg/kg WP[6] at 30 s after Sch administration. (B and C) Time when the abnormal ECG pattern appeared (B) and disappeared (C) after rats were i.v. administered with or without WP[6] (20 or 50 mg/kg) at 30 s after Sch administration. n = 16 for each group. (D) Incidence rate of the abnormal ECG pattern of rats that i.v. administered with or without WP[6] (20 or 50 mg/kg) at 30 s after Sch administration. n = 16 for each group.

Figure 4

WP[6] alleviated Sch induced hyperkalemia in a rat model. (A) The time dependent changes of serum potassium level of rats. Rats were i.v. administered with saline or WP[6] (20 mg/kg or 50 mg/kg) at 30s, 2 min and 5 min after Sch (0.9 mg/kg) administration. Group Ctrl and WP[6] 50 mg/kg, n = 4; other groups, n = 7. Data are presented as means ± SEM and *P < 0.05, **P < 0.01, ***P < 0.001. (B to E) Relative serum potassium level of rats at 5 min (B), 10 min (C), 15 min (D) and 25 min (E) after rats were i.v. administered with various samples. Data are presented as relative values and means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

WP[6] alleviated the Sch induced rhabdomyolysis in a rat model. (A to C) The levels of creatine kinase (A), UA (B) and Crea (C) in the serum collected from rats sacrificed at 15 min after injected (via the right anterior tibial muscle) with saline, Sch (1 mg/kg), WP[6] (20 mg/kg or 50mg/kg at 30 s after administration of Sch) and WP[6] (50 mg/kg). n = 6 for each group; Data are presented as means ± SEM and *P < 0.05, **P < 0.01. (D) H&E histopathological analysis of the right anterior tibial muscle from rats sacrificed at 15 min after injected with saline, Sch (1 mg/kg), WP[6] (20 mg/kg or 50 mg/kg at 30 s after administration of Sch) and WP[6] (50 mg/kg). Scale bar = 50 µm.

Figure 6

Reversal of paralysis in a mouse model. Mice were i.v. administered with WP[6] (20 mg/kg or 50 mg/kg) at 30 s after the administration of Sch (0.5 mg/kg). n = 8 for each group, Data are presented as the original data and means ± SEM, *P < 0.05, ***P < 0.001.

Figure 7

WP[6] reversed the changes of plasma membrane potential and reduced the loss of intracellular potassium in vitro. (A and B) Typical flow cytometry curves (A) and quantitative analysis (B) for determination of the plasma membrane potential of L6 cells that were incubated with 0, 10, 100 and 1000 µM Sch for 10 min. MFI: mean fluorescence intensity. Data are presented as means ± SEM. n = 4, *P < 0.05. (C) Quantitative analysis for determination of the plasma membrane potential of L6 cells that were incubated with 100 µM Sch, WP[6] (1 mM) or co-incubated with Sch (100 µM) and WP[6] (molar ratio 1:1, 1:2, 1:5 and 1:10) for 10 min. Data are presented as means ± SEM. n = 4, *P < 0.05, *P < 0.01 and ***P < 0.001. (D) Relative content of intracellular potassium of L6 cells that were incubated with 100 µM Sch, WP[6] (1 mM) or co-incubated with Sch (100 µM) and WP[6] (molar ratio 1:1, 1:2, 1:5 and 1:10) for 10 min. Data are presented as means ± SEM. n = 4, ***P < 0.001.