Model of Neuromuscular Block Reversal in the Anaesthetised Rat Olivier Boucheix, David Coquard, Raphaël Coston, Stéphane Baudet Charles River Lyon - Safety Assessment, Saint Germain-Nuelles, France

1 Introduction 2 Material and Methods Test system: six male Sprague-Dawley rats (400-500g). Reversal of neuromuscular block (NMB) is of strategic relevance to prevent risks of Signal acquisition and data analyses: after a 10 to 15-minute stabilisation period, developing postoperative respiratory complications in clinical situations. Animal preparation and monitoring: after induction of anaesthesia ( 60 mg/kg square pulses (40 V, 50 µs) were delivered at a frequency of 0.1Hz (kept constant IP + 10 mg/kg/h IV), rats were tracheotomised and artificially ventilated (50 rpm, 2.3 mL over the whole experiment). The muscle force signal was captured on Notocord- For this purpose, we have developed a rat model for assessing blocking of volume, 600 ms inspiratory time). A jugular vein was catheterised to allow intravenous hem® (Notocord Systems, France) data acquisition software. neuromuscular function by two drugs, and reversal of NMB with a prototypical agent, administrations of the anaesthetic agent and test substances. ECG, heart rate and body while monitoring main safety endpoints. temperature were monitored over the whole experiment duration. The maximum twitch amplitude was calculated for each stimulation. After dosing, and twitch amplitude was expressed as % from basal (pre-NMB) value. Non-linear NMB model (adapted from Pickett et al. 2008): the rat was placed on a custom-build unit sigmoid curve fitting was performed using Graphpad Prism®. In a first step, ED90 of rocuronium and atracurium, as representative NMB agents, was determined. In a second step, reversal effect of on NMB induced by where the hindlimb was secured between blocks, such that only the tibio-tarsal joint was both agents was studied at a dose of 3xED90 for each NMB agent. mobile. An isometric force transducer was secured to the hind foot such that the tibio- Treatment design: rocuronium bromide, atracurium besylate and neostigmine tarsal angle was 90°. A pair of hypodermic monopolar needle electrodes (28G) was placed (Sigma) solutions were prepared by dilution of fresh stock solutions in water for near the sciatic nerve to deliver electrical pulses in order to promote contraction of the injection (pH 4.0 ± 0.3) and administered as an IV bolus. Neostigmine was triceps surae muscle group and extension of the foot, whose displacement was converted administered as an IV bolus, 2.5 minutes after each NMB agent administration. to force using a strain gage transducer.

3 Results Neuromuscular block reversal by neostigmine Determination of rocuronium and atracurium ED90 110% 110% 4 Conclusion 90% 90% Time to 80% recovery (s) 80 200% Rat no. 104 70% 70% Rocuronium (rat no.101) 180% Max Amplitude 70 50% Recovery (%) 50% Force (%) Force Rat # 160% (%) Force We have successfully developed a rat 60 30% 30% 140% model of neuromuscular blockade 120% 10% 10% 50 considered to be reliable and robust in 100% -5 -10% 0 5 10 15 20 25 -5 -10% 0 5 10 15 20 25 40 Rocuronium 4 mg/kg Time (min) Time (min)

Force (%) Force 80% order to allow evaluation of efficacy of

Force (g) Force Rocuronium 2 mg/kg neostigmine 30 60% Maximum Amplitude Recovery - percent Time to 80% recovery - percent change from neuromuscular blockers and/or reversal Rocuronium 1 mg/kg ED90= 1.34 ± 0.11 mg/kg 40% First rocuronium change from the initial block with atracurium 20 Rocuronium under neostigmine 30µg/kg 160% the initial block with atracurium agents, while monitoring main safety Rocuronium 0.5 mg/kg 20% Rocuronium under neostigmine 300µg/kg 140% Saline Neostigmine 30µg/Kg Neostigmine 300µg/Kg endpoints. 10 Rocuronium 0.2 mg/kg 0% 0% 120% 0 5 10 15 20 25 30 0 Time (min) 100% -10% -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 80% Since these experiments were 0.03 0.1 0.3 1 3 10 -20% Time (min) 60% conducted, this model has also been Dose (mg/kg) 40% -30% 20% shown to be as suitable under 80 -40% Atracurium (rat no. 201) 200% 0% Rat no. 204 Saline Neostigmine 30µg/Kg Neostigmine 300µg/Kg -50% anaesthesia, while blood pressure could 70 Rat # 180% be monitored in addition to other vital 160% 60 Maximum Amplitude Recovery - percent Time to 80% recovery - percent change from endpoints. 140% change from the initial block with rocuronium the initial block with rocuronium 50 120% 100% Saline Neostigmine 30µg/Kg Neostigmine 300µg/Kg Atracurium 4 mg/kg 100% 20% 40 80% Atracurium 2 mg/kg (%) Force 10% Atracurium 1.5 mg/kg 80% Force (g) Force 60% 30 neostigmine Atracurium 1 mg/kg 60% 0% 40% Atracurium 0.75 mg/kg ED = 1.21 ± 0.26 mg/kg -10% 5 References 20 90 40% First atracurium Atracurium 0.5 mg/kg Atracurium under neostigmine 30µg/kg 20% -20% Atracurium 0.2 mg/kg 20% 10 Atracurium under neostigmine 300µg/kg 0% -30% 0% -20% -40% 0 0 5 10 15 20 25 30 Pickett et al. (2008). The in vivo rat muscle Time (min) Saline Neostigmine 30µg/Kg Neostigmine 300µg/Kg -50% -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0.03 0.1 0.3 1 3 10 force model is a reliable and clinically Time (min) Dose (mg/kg) relevant test of consistency among Time course of changes in twitch amplitude after NMB with rocuronium and atracurium at 3xED . The duration of NMB preparations. Toxicon, 52, Time course of changes in twitch amplitude after NMB with rocuronium or atracurium (n=3 for each). 90 induced by either agent was shortened by neostigmine at 30 and 300 µg/kg. In addition, at 300 µg/kg, neostigmine over- 455-464. ED of either blocker was determined to be 1.34 ± 0.11 mg/kg and 1.21 ± 0.26 mg/kg, respectively. 90 reversed the neuromuscular block, as shown by the increase in twitch amplitude beyond basal level.