In vivo animal studies with sugammadex

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1 In vivo animal studies with sugammadex L. H. D. J. Booij, 1 J. van Egmond, 2 J. J. Driessen 3 and H. D. de Boer 4 1 Professor of Anaesthesiology, 2 Physicist, 3 Associate Professor of Paediatric Anaesthesiology, Department of Anaesthesiology, Radboud University Medical Centre, Nijmegen, The Netherlands 4 Consultant in Anaesthesiology, Martini Ziekenhuis, Groningen, The Netherlands Summary A review is presented of animal studies of the selective steroidal neuromuscular blocking drug binding agent sugammadex. These studies demonstrate that sugammadex is faster in onset than the currently used acetylcholinesterase inhibitors, has no muscarinic effects, and is characterised by lack of adverse effects on other organs. These results offer support for the further development of sugammadex for clinical use in humans.... Correspondence to: Prof. Dr L. H. D. J. Booij l.booij@owi.umcn.nl Accepted: 15 December 2008 The first reported clinical use of a neuromuscular blocking drug (NMB) dates back to 1912, when in Germany the surgeon Läwen administered 0.8 mg curarine to a patient under ether anaesthesia to facilitate intraperitoneal surgery [1]. However, lack of supplies prevented its further development. In 1928 de Caux used curare in seven patients in London [2]. However, it was only in 1942 that Griffith and Johnson introduced curare for routine clinical use [3]. The initial enthusiasm of the introduction of NMBs was tempered by the report by Beecher and Todd in 1955 [4] in almost surgical patients that the use of NMBs was associated with a sixfold increase in anaesthesia-related deaths, the most common causes being a result of respiratory depression (63%) and cardiovascular collapse (37%). Sixteen leading anaesthetists who were already regularly using NMBs responded to this report [5] to argue that whenever NMBs were used, patients lung ventilation should be assisted or controlled, and hypokalaemia, dehydration, respiratory and metabolic acidosis should be corrected. Furthermore, they advised that incomplete recovery from NMBs at the end of anaesthesia should be reversed. In later years it was shown that the use of NMBs resulted in a trebling of postoperative pulmonary complications [6]. Even now the clinical use of NMBs appears associated with an increased morbidity and even mortality. With the development of new agents in both the steroidal and benzylisoquinoline groups of NMBs, a marked improvement has been seen in the time course of action and safety profiles of NMBs. However, an ultrashort duration of action NMB has not been developed that has minimal variability in duration of effect, and thus the risk of residual paralysis at the end of anaesthesia remains. A large number of the patients in the recovery room still show residual paralysis [7]. Routine monitoring of neuromuscular transmission is one of the tools that can be used to improve the safety of the use of NMBs. Appropriate quantitative monitoring in the UK is only performed by 10% of anaesthetists, while many others use inappropriate or non-validated clinical testing [8]. It is assumed that similar figures hold true for the rest of the Western world. Thus residual paralysis remains a problem [9]. Development of ultra-short acting NMBs with only a small variability in time course of action might solve the problem. Many compounds have been synthesised and studied [10 14]. However, none of them has fulfilled the desired profile, and many have caused histamine release or adverse cardiovascular effects, or have even been toxic. Another approach to improving safety is the routine administration of reversal agents to all patients who have been given a non-depolarising NMB. Non-depolarising NMBs are competitive antagonists of the nicotinic acetylcholine receptor. Acetylcholinesterase inhibitors have been available for 85 years and their use includes reversal of neuromuscular blockade, treatment of myasthenia gravis, treatment of organophosphate poisoning, glaucoma and Alzheimer s disease. The competition with acetylcholine can be influenced by administration of acetylcholinesterase inhibitors like neostigmine, pyrido- 38 Journal compilation Ó 2009 The Association of Anaesthetists of Great Britain and Ireland

2 L. H. D. J. Booij et al. Æ In vivo animal studies with sugammadex stigmine and edrophonium, which increase the postsynaptic acetylcholine concentration by slowing down its breakdown. However, even with routine administration, residual paralysis remains a concern [15, 16]. Anticholinesterases also cause muscarinic side-effects such as bradycardia, hypersalivation, bronchoconstriction, nausea and vomiting. Therefore they are administered in combination with the muscarinic antagonists atropine and glycopyrrolate, which themselves can also exert sideeffects such as tachycardia, blurred vision, dry mouth, etc. The desirable solution was the development of more reliable, specific and safe reversal methods for neuromuscular blockade. Initially this approach involved a search for more specific acetylcholinesterase inhibitors [17 19]. Early investigations in animals demonstrated that with most compounds the adverse muscarinic effects were retained, and that with the others the potency was too low, and solubility and pharmacodynamic profile unsatisfactory. They also lacked the ability to reverse profound neuromuscular blockade. Therefore research into other possibilities was initiated. Encapsulation as a method for reversal It had been suggested in the past that removal of the NMB molecules in the plasma is a theoretical option for reversal of blockade, because it will promote the return of the NMB into the central compartment and stimulate the dissociation of the compound from the acetylcholine receptor [20]. Such an approach, for instance by chelating or encapsulating the NMB molecule, will not interfere with the breakdown of acetylcholine, and thus will not be accompanied by muscarinic side effects such as those that are seen with anticholinesterases. It is also likely that deep neuromuscular blockade, corresponding to higher NMB concentrations, can be reversed by increasing the concentration of the binding agent. Encapsulation of NMBs by exogenous compounds was therefore considered a realistic possibility. There are various types of molecules that are able to encapsulate other molecules. One of them is the family of the cyclophanes that are known to form complexes with quaternary ammonium and steroidal guest molecules. A cyclophane was successfully synthesised that chelated the steroidal compound pancuronium, and gallamine [21]. In an attempt to develop a new solvent for rocuronium it was noticed that cyclodextrins bind rocuronium. The binding is based on a 1 : 1 ratio. The cyclodextrin molecules are well tolerated in humans, are water soluble, and have a lipophilic cavity. A large number of insoluble drugs are administered encapsulated in cyclodextrins, which proved to have a safe toxicological profile [22]. Cyclodextrins were previously used safely for binding steroids [23]. This led to the development of cyclodextrins that selectively bind steroidal neuromuscular blocking agents as reversal agents [24]. A series of cyclodextrins that were theoretically able to encapsulate steroidal NMBs were synthesised in the Organon laboratories (now part of Schering-Plough) in Newhouse, Scotland [25, 26]. In vivo studies in anaesthetised guinea pigs at Newhouse showed that the c-cyclodextrins (c-cd) with a cavity size of Å had the greatest potency to reverse neuromuscular blockade induced by rocuronium. None of the compounds showed cardiovascular sideeffects in experiments in anaesthetised cats. Extension of the cavity of the c-cd by building side chains with negatively charged ions onto each glucose molecule, increased the binding force. A number of the most potent compounds were further tested in our Laboratory of Experimental Anaesthesiology at Radboud University in Nijmegen. We thus investigated the capacity of nine synthetic cyclodextrin derivatives (Org 25288, Org 25289, Org 25467, Org 25168, Org 25169, Org 25555, Org 25166, Org 26142, and Org 25969) in Rhesus monkeys. From these studies it was evident that Org had the greatest potency for binding rocuronium [27, 28]. The encapsulation of rocuronium by Org was confirmed by X-ray crystallography and found to reverse a rocuronium-induced neuromuscular block much faster than neostigmine. Adverse effects were not observed. This compound thus was selected for further evaluation and received the name sugammadex. A recording of the effect of sugammadex on a rocuronium-induced neuromuscular block in the Rhesus monkey in comparison to that of spontaneous recovery and reversal by neostigmine is shown in Fig. 1. Figure 1 Recovery of rocuronium-induced neuromuscular block: spontaneous in comparison to after neostigmine + atropine, Org 25288, Org (Sugammadex). Journal compilation Ó 2009 The Association of Anaesthetists of Great Britain and Ireland 39

3 L. H. D. J. Booij et al. Æ In vivo animal studies with sugammadex Anaesthesia, 2009, 64 (Suppl. 1), pages Justification of animal studies In drug development governments require data on the mechanism of action, the pharmacodynamic and pharmacokinetic profiles, and the safety of drugs. Despite the enormous developments in the field of molecular biology and computer technology, and the development of nonanimal tests, such integral data can only be obtained in whole animal studies. Integrated reactions from all biological systems are present in animals and they mimic the clinical situation. This makes in vivo pharmacology indispensable. Also, intact animal studies are required to determine whether the in vitro effects actually translate to the in vivo situation. Molecular biology and in vitro pharmacology are therefore complementary methods in the development of new drugs. Extrapolation from animal data to expected effects in humans is one of the basic problems in drug development. Discordance between animal and human results are in most cases to be expected. Various species differ in affinity and distribution of receptors, and in pharmacokinetic distribution, metabolism and elimination of drugs. The pathway of neuromuscular transmission and its blockade, however, does not differ much between animal species. The differences that exist in their effects relate mainly to pharmacokinetic distribution, metabolism and elimination of the NMBs. Therefore animal studies give much insight into the pharmacological profile of NMB reversal agents. However, sugammadex has a mechanism of action completely different as it does not interfere with receptors at all, but only has a physico chemical interaction with the NMB, called encapsulation. This mechanism is not different between species, enabling easier translation of the results in animal experiments to the human situation. Animal studies in the development of sugammadex were therefore even more valuable. Cyclodextrins must possess some specific properties in order to be an acceptable alternative to acetylcholinesterase inhibitors for the reversal of neuromuscular block. The existence of these properties in the cyclodextrins have been investigated in animal studies. Proof of complex formation of rocuronium and cyclodextrin in vivo and selection of sugammadex (Org 25969) Nine, c-cyclodextrins were selected from a series synthesised in the Organon laboratories as being potential reversal agents. These were tested in anaesthetised Rhesus monkeys in Nijmegen [28]. In these animals a steady state 90% neuromuscular blockade was maintained for at least 10 min by infusion of rocuronium and was then allowed to recover spontaneously after stopping the infusion. Then the same procedure was repeated but now after stopping the infusion one of the test compounds or a combination of neostigmine and atropine was administered. Time intervals from injection to 25%, 50%, 75% and 90% recovery were measured. Blood pressure and heart rate were monitored continuously. Two compounds, Org (in doses of 0.59 and 1.18 mg.kg )1 ) and Org (in doses of 0.5 and 1.0 mg.kg )1 ), had a faster recovery then neostigmine atropine (40 lg.kg )1 plus 15 lg.kg )1 ). The other cyclodextrins were less effective and required much higher dosages or produced a slower recovery than the neostigmine atropine combination. None of the cyclodextrins caused significant changes in heart rate or blood pressure. Residual block or recurarisation were not observed. Based on the fact that Org showed the strongest effects, this compound was chosen for further development. A continuous 30 min infusion of rocuronium was administered until a constant 90% depression of muscle contraction was obtained in a study in guinea pigs [29]. Then either sugammadex or saline was administered by infusion for 30 min while the rocuronium infusion continued. During the whole procedure, arterial blood samples were taken at 10 min intervals to determine the rocuronium concentration. In the sugammadex group the evoked twitch height decreased while the plasma concentration of rocuronium increased. In the saline group these variables did not change. This increase of rocuronium in the plasma is explained by an increase in the sugammadex-bound rocuronium fraction, with a simultaneous decrease in the free rocuronium fraction, observed as an increase in twitch height. Unfortunately bound and free rocuronium fractions could not be determined separately at this stage. These studies confirmed the concept that sugammadex binds rocuronium, resulting in its removal from plasma and the effect compartment, thus resulting in recovery from neuromuscular block. Efficacy of sugammadex in vivo Acetylcholinesterase inhibitors are unable to reverse deep neuromuscular blockade, for example immediately after administration of a NMB in the case of failed tracheal intubation. It is therefore suggested that acetylcholinesterase agents are given only when some recovery of blockade has occurred. Moreover, the onset time and the time to peak effect of the acetylcholinesterase inhibitors are considerably longer. A new drug that can replace the acetytlcholinesetrase inhibitors should have a fast onset with short time to peak effect and should preferably be able to reverse even profound neuromuscular blockade. 40 Journal compilation Ó 2009 The Association of Anaesthetists of Great Britain and Ireland

4 L. H. D. J. Booij et al. Æ In vivo animal studies with sugammadex It was theoretically anticipated that sugammadex would also be effective in reversing profound neuromuscular blockade and thus might be used immediately after administration of rocuronium as a rescue drug in cannot intubate, cannot ventilate situations, in which acetylcholinesterase inhibitors are usually ineffective. In a study in anaesthetised Rhesus monkeys, 100 lg.kg )1 was given and neuromuscular function was allowed to recover spontaneously [30]. The resulting peak blockade was approximately 90% in all animals (mean 93%). After recovery for 1 h, each animal was given rocuronium 500 lg.kg )1 and 1 min thereafter either saline or one of two doses of sugammadex (1.0 or 2.5 mg.kg )1 ). Sugammadex proved to be able to reverse even the profound neuromuscular blockades without showing cardiovascular effects. These studies demonstrate that sugammadex is an effective agent for the rapid reversal of residual and profound steroidal agent-induced neuromuscular blockade. Selectivity of sugammadex In guinea pigs it was demonstrated that sugammadex reverses blockade induced by rocuronium, pancuronium and vecuronium. However, blockade induced with suxamethonium, d-tubocurarine, atracurium and mivacurium were not reversed [31]. It was anticipated that this is because the size of the cavity and the NMB molecule do not match, neither for the small suxamethonium molecule, nor for the other bulky molecules. This selectivity of sugammadex for steroidal NMBs was also studied in primates [32]. In eight anaesthetised Rhesus monkeys a steady state neuromuscular block of 90% from baseline was induced with a rocuronium infusion. After 10 min steady-state the infusion was stopped and spontaneous recovery was allowed to occur. A steady state blockade was instituted again and the infusion was stopped, but now sugammadex (0.5 or 1.0 mg.kg )1 ) was given as a single dose. Neuromuscular blockade was monitored. Sugammadex rapidly reversed the blockade caused by rocuronium. In the second part of the study, instead of rocuronium, either atracurium or mivacurium was used to induce blockade. The study showed that sugammadex had no effect on the time-course of mivacurium or atracurium. A summary of this experiment is shown in Fig. 2. Sugammadex is a reversal agent specific for steroidal NMBs, and is not effective against blockade induced by other agents. Urinary excretion of sugammadex Cyclodextrin-rocuronium complexes are highly hydrophilic and thus are expected to be excreted via the kidneys easily. Epemolu et al. [33] demonstrated in a study that sugammadex is indeed excreted via the kidneys. In (a) (b) Figure 2 Effect of sugammadex 1 mg.kg )1 on neuromuscular blockade from (a) steroidal relaxants, (b) benzylisoquinoline relaxants. anaesthetised guinea pigs it was demonstrated that sugammadex increased the renal excretion of rocuronium several fold compared to excretion after saline administration. In anaesthetised cats a 2 ED 90 (ED 90 being the effective dose required for 90% effect) dose of rocuronium was allowed to recover spontaneously; after 90 min the renal arteries were ligated and 30 min later the same dose of rocuronium was administered. In one group the rocuronium-induced neuromuscular block was allowed to recover spontaneously, and in other group sugammadex 5 mg.kg )1 was given. Spontaneous recovery of rocuronium block before and after ligation of the arteries was not different from each other. In the group given sugammadex the recovery was significantly shorter than in the spontaneous recovery group and recurarisation did not occur [34]. It is clear that whereas rocuronium is mainly excreted via the liver, the rocuronium-sugammadex complex is mainly excreted via the kidneys. Nevertheless, in animals without renal function sugammadex administration still results in complete and sustained reversal of rocuroniuminduced neuromuscular block. Side-effects of sugammadex in in vivo animal studies Sugammadex has neither a direct nor an indirect action on components of cholinergic transmission (acetyl-cholinesterase, nicotinic receptors or muscarinic receptors), Journal compilation Ó 2009 The Association of Anaesthetists of Great Britain and Ireland 41

5 L. H. D. J. Booij et al. Æ In vivo animal studies with sugammadex Anaesthesia, 2009, 64 (Suppl. 1), pages and therefore it is unlikely that muscarinic side effects will occur. In none of the studies mentioned above did sugammadex caused any change in heart rate or blood pressure, nor were there other side-effects such as regurgitation, retching, changes in airway pressure, wheezing, etc. In studies in cats no cardiovascular effects were observed after administration of cyclodextrins while reversing rocuronium-induced neuromuscular blockade [25]. Because of the lack of muscarinic side-effects of sugammadex, administration in combination with atropine or glycopyrrolate is not needed as is the case with reversal by cholinesterase inhibitors. As sugammadex in the wide dose ranges used in the studies, did not produce adverse effects in animal studies, it is anticipated that it will not produce adverse effects in humans either. Re-induction of neuromuscular blockade after sugammadex administration In some relatively rare situations, it is necessary to re-establish neuromuscular blockade after reversal of a previous block. In those circumstances some free sugammadex may still be present and will encapsulate newly administered NMBs. The duration of action or half-life of sugammadex is important in this situation. This duration of action was studied in anaesthetised Rhesus monkeys [35]. Firstly, the effect of a single dose of rocuronium 100 lg.kg )1 was determined. After full recovery, sugammadex was administered in a single dose of 1.0 mg.kg )1 and then respectively after a delay of 15, 30 and 60 min, the effect of the very same dose of rocuronium was studied. The effect of this second dose of rocuronium increased with the time delay after the sugammadex administration and almost reached the original effect after a delay of 60 min. From the obtained data the half-life of sugammadex in Rhesus monkeys was calculated to be 30 min. This experiment indicates that after reversal of neuromuscular blockade with sugammadex, it is possible to reinstate blockade with rocuronium, although, depending on the elapsed time, a higher dose of rocuronium will be required. Since non-steroidal NMBs are not reversed by sugammadex, is it also possible to re-establish neuromuscular block with these NMBs. However, such a blockade cannot then be reversed with sugammadex. Acid base balance and reversal by sugammadex In a state of acidosis, acetylcholinesterase inhibitors are poor at reversing neuromuscular blockade. It has been demonstrated in anaesthetised Guinea pigs that acid-base balance disturbances have no effect on the reversal potency of sugammadex [36]. Discussion and conclusion In animal studies sugammadex proved to be a safe, rapid, and effective reversal agent for normal and profound neuromuscular block induced by steroidal neuromuscular blocking agents, and in particular rocuronium. Because of its ability to reverse profound blockade, it can be used not only to prevent or treat steroidal-induced residual curarisation, but it can also be used in situations of cannot intubate, cannot ventilate. However, sugammadex is not effective against non-steroidal neuromuscular blocking agents. Its mechanism of action is encapsulation of rocuronium. Because of this mechanism it can be anticipated that drug interactions between NMBs and other drugs, as far as neuromuscular transmission is concerned, can also be reversed by administration of sugammadex. Sugammadex itself has no adverse effects in animals. The onset of sugammadex was rapid in all in vivo animal experiments and indeed a faster recovery will occur after sugammadex than after neostigmine, pyridostigmine or edrophonium administration. Rocuronium dose calculations for re-establishing neuromuscular block with rocuronium after sugammadex reversal, showed that re-establishment of neuromuscular blockade is possible by increasing the total amount of rocuronium present. The sugammadex-rocuronium complex is excreted via the kidneys but, even in animals without renal function, the administration of sugammadex resulted in fast, complete and sustained reversal of blockade. Thus encapsulation by sugammadex proved in animals to be a potentially effective method for reversal of neuromuscular blockade. Since this encapsulation is a pure physicochemical process, in which neither neurotransmitters nor receptors are involved, the successful extrapolation of animal study results to the human situation was highly likely. Conflicts of interest LHDJB is on the scientific advisory board on anaesthesia for Schering-Plough (formerly Organon), and has received honoraria for lectures. The remaining authors have received grants to perform studies. References 1 Läwen A. Über die Verbindung Lokalanästhesie mit der Narkose, über hohe Extraduraal Anästhesie und peridurale Injektionen anästhesierende Lösungen bei tabetische Magenkriesen. Beitrage der Klinische Chiruirgie 1912; 80: Wilkinson DJ. Dr F.P. de Caux the first user of curare for anesthesia in England. Anaesthesia 1991; 46: Journal compilation Ó 2009 The Association of Anaesthetists of Great Britain and Ireland

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7 L. H. D. J. Booij et al. Æ In vivo animal studies with sugammadex Anaesthesia, 2009, 64 (Suppl. 1), pages De Boer HD, van Egmond J, van de Pol F, Bom A, Booij LHDJ. Sugammadex, a new reversal agent for neuromuscular block induced by rocuronium in the anaesthetized Rhesus monkey. British Journal of Anaesthesia 2006; 96: Epemolu O, Mayer I, Hope F, Scullion P, Desmond P. Liquid chromatography mass spectrometric bioanalysis of a modified-cyclodextrin (Org 25969) and Rocuronium bromide (Org 9426) in guinea pig plasma and urine: its application to determine the plasma pharmacokinetics of Org Rapid Communications in Mass Spectrometry 2002; 16: Bom A, van Egmond J, Hope F, van de Pol F. Rapid reversal of rocuronium-induced neuromuscular block by Org is independent of renal perfusion. Anesthesiology 2003; 99: A De Boer HD, van Egmond J, van de Pol F, et al. Time course of action of sugammadex (Org 25969) on rocuronium-induced block in the Rhesus monkey, using a simple model of equilibration of complex formation. British Journal of Anaesthesia 2006; 97: Bom AH, Mason R, McIndewar I. Org causes rapid reversal of rocuronium-induced neuromuscular block, independent of Acid-base status. Anesthesiology 2002; 96: A Journal compilation Ó 2009 The Association of Anaesthetists of Great Britain and Ireland