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BJA Advance Access originally published online on October 3, 2006
British Journal of Anaesthesia 2006 97(5):681-686; doi:10.1093/bja/ael240
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© The Board of Management and Trustees of the British Journal of Anaesthesia 2006. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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{dagger}

H. D. de Boer1,*, J. van Egmond1, F. van de Pol1, A. Bom2, J. J. Driessen1 and L. H. D. J. Booij1

1 Department of Anaesthesiology, Radboud University Medical Centre Nijmegen The Netherlands
2 Department of Pharmacology Organon Newhouse, ML1 5SH, Scotland, UK

*Corresponding author: Department of Anaesthesiology, Martini Hospital Groningen, PO Box 30033, 9700 RM Groningen, The Netherlands. E-mail: HD.de.Boer{at}mzh.nl

Accepted for publication June 19, 2006.


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Background. Reversal of neuromuscular block can be accomplished by chemical encapsulation of rocuronium by sugammadex (Org 25969), a synthetic {gamma}-cyclodextrin derivative. The present study determined the time course of the reversal action of sugammadex on rocuronium-induced block in the anaesthetized Rhesus monkey using train-of-four stimulation.

Methods. A bolus injection of rocuronium 100 µg kg–1 (about 1xED90) was given to determine the degree of neuromuscular block reached by this dose. After complete spontaneous recovery, a rapid bolus injection of sugammadex, 1 mg kg–1, was given and at different time intervals (15, 30 or 60 min, in three different experiments) the effect of another rocuronium bolus injection of 100 µg kg–1 was determined.

Results. Injection of the first dose of rocuronium resulted in a mean neuromuscular block (depression of first twitch) of 93 (SEM=1.6)%. Fifteen minutes after injection of sugammadex the same rocuronium dose resulted in 17% (SEM=5.6) block. After 30 and 60 min these maximum blocks amounted to 49% (SEM=7.6) and 79% (SEM=4.2), respectively. The estimated half-life of sugammadex in Rhesus monkey is 30 (SEM=4.9) min.

Conclusions. The half-life of sugammadex (Org 25969), a new fast and efficient reversal agent for rocuronium-induced block, is relatively short in the Rhesus monkey, implying the possibility to perform neuromuscular block by rocuronium shortly after reversal of a prior block. In translation to the human situation differences in rocuronium sensitivity and kinetics should be taken into account.

Keywords: Sugammadex (Org 25969), reversal agent, rocuronium, neuromuscular block, time course of action


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Sugammadex (Org 25969) is a new selective relaxant binding agent (SRBA) that has been designed to reverse the steroidal blocking drug rocuronium.1–3

The mechanism, encapsulation of the rocuronium molecule, results in a rapid decrease in its free plasma concentration and its removal from the motor endplates, resulting in the reappearance of muscle activity.1 Sugammadex is highly water-soluble and has no direct or indirect action on components of cholinergic transmission (acetyl-cholinesterase, nicotinic receptors or muscarinic receptors), and therefore it is unlikely that muscarinic side effects will occur.1 Recent studies in Rhesus monkeys and humans have shown that this new method of reversing the neuromuscular effects of rocuronium is fast, efficient and lacks the well-known undesirable side effects associated with the use of a combined cholinesterase inhibitor and muscarinic antagonists.1–9 However, no reports of the time course of action of sugammadex have been published so far. This information may be important if a further dose of rocuronium is needed to re-establish neuromuscular block. The capability of re-establishment of neuromuscular block by rocuronium after reversal with sugammadex is therefore unknown.

The present study was designed to evaluate the time course of action of sugammadex in Rhesus monkeys. The reversal capacity of a single bolus injection of sugammadex (1.0 mg kg–1) as a function of time was evaluated by its blocking effect on a fixed dose of rocuronium (100 µg kg–1) before and 15, 30 and 60 min after administration. The experiments were performed with train-of-four (TOF) stimulation. A model is used, based on the equilibrium of complex formation of rocuronium and sugammadex, to solve the residual amount of sugammadex as a function of time from the observed maximum blocks.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
These in vivo experiments were performed in the experimental laboratories of the Department of Anaesthesiology at the Radboud University Medical Centre in Nijmegen, The Netherlands. The experiments were approved by the regional ethical committee on animal experiments. Four female Rhesus monkeys (CSIMS, Beijing, China) were studied. They were sedated with ketamine 10 mg kg–1 (Nimatek Eurovet) administered by i.m. injection. Two i.v. lines were sited, one for anaesthetic administration, including rocuronium, and the other for test drug administration. This was followed by i.v. injection of pentobarbital sodium (Ceva Sante Animale), 25 mg kg–1, and a subsequent continuous infusion of 5–10 mg kg–1 h–1. The monkeys were intubated endotracheally and the lungs ventilated with a mixture of oxygen and nitrous oxide in a ratio of 2:3. Heart rate and oxygen saturation were determined at the ear with a pulsoximeter (Ohmeda, Biox). Blood pressure was determined with a cuff placed around the tail (Finapres, Ohmeda). Body temperature was measured by a rectal probe and maintained at 37–38°C.

For monitoring purposes the median nerve of the right arm was supramaximally stimulated near the wrist using needle electrodes. Stimulation was performed with 2 ms square wave pulses in a TOF sequence of 2 Hz with an interval of 15 s delivered by a Grass S88 Stimulator (Grass Medical Instruments, Quincy, MA, USA). The resulting contractions of the thumb muscles were quantified with a force displacement transducer and recorded on a polygraph. After an initial i.v. bolus injection of rocuronium bromide 100 µg kg–1 (approximately ED90 value for our Rhesus monkey population) the preparation was allowed to recover spontaneously. Sixty minutes after a complete steady-state spontaneous recovery, sugammadex 1.0 mg kg–1 was given intravenously as a rapid bolus. Then another i.v. bolus injection of rocuronium 100 µg kg–1 (the same dose as the first injection) was given 15, 30 or 60 min after the administration of sugammadex (in each of the four animals the three different time intervals were applied in a series of three different experiments; a total of 12 experiments; the actual order of time intervals were 60, 15 and 30 min). To evaluate which time intervals were necessary to cover enough of the elimination of sugammadex, the first experiment was performed with a time interval of 60 min. From the experimental traces T1 and TOF-ratio recovery times were calculated at 25, 50, 75 and 90% recovery, with respect to the basic value. The maximal degree of neuromuscular block obtained, the onset time of this maximal neuromuscular block, and neuromuscular recovery times of this neuromuscular block induced by rocuronium 100 µg kg–1 before and 15, 30, or 60 min after the injection of sugammadex were calculated from the traces. Only the maximum block values were used for further analysis of the half-life of sugammadex. At the end of the experiments, the animals were allowed to recover from anaesthesia.

Model description
A model to describe the expected block as a function of rocuronium dose of Droc µg kg–1, t minutes after the injection of a dose of sugammadex Dsugammadex mg kg–1, can be obtained with the following assumptions:

  1. The dose–effect relationship of rocuronium can be described as a Hill equation: Formula.
  2. The volumes of distribution for all interacting items, rocuronium, sugammadex and the complex, are the same and amount to Vd.
  3. Sugammadex equilibrates with rocuronium to form the complex defined by the equilibrium constant, and only the free rocuronium (Croc.free) interacts in the Hill equation in assumption (i): K=Ccomplex/(Croc,free·Csugammadex,free), which together with assumption (ii), can be expressed in total amounts of material D (expressed in mol; bound+unbound; applies to rocuronium as well as sugammadex):

    Formula
    or K'=K· Vd=Dcomplex/(Droc,free·Dsugammadex,free).

  4. The amount of available sugammadex as a function of time follows a first order exponential decay: D=D0 · e(–k·t).

The procedure of estimating half-life, using the above model, is outlined: The parameters ED50 and {gamma} in the Hill equation:

Formula
are determined as follows: {gamma} follows from the dose–response relationship in our monkey population: {gamma}=5. ED50 in each individual experiment then follows by solving the Hill relationship.

The complex equilibrium constant:

K'=K·Vd=Dcomplex/(Droc,free·D25969,free),

where D expressed in mol kg–1, is calculated from the situation that a rocuronium dose of half of the ED50 only provides for a few percentage of block. This block depth is also approximately obtained by an ED90 rocuronium dose given simultaneously with sugammadex 1.0 mg kg–1. Therefore, the free rocuronium concentration must be almost equal in both situations. The free fraction of rocuronium in the latter situation then follows to be about 0.3. All three factors in K' can then be calculated from the dosages and molecular weights of sugammadex (2178) and rocuronium (609), with the result of K=6.6x106 kg mol–1.

Given the exponential decay of total sugammadex, D=D0·e(–k·t), the free fraction of injected rocuronium can be predicted from this decay and the equilibrium constant K'. The free fraction can also be calculated from the injected dose, the individually obtained ED50 and the measured T1 block because of the second rocuronium injection. The least squares fitting procedure minimizes the differences between these two results of the free fraction by adapting the time constant in the exponential decay relationship.

The same procedure is applicable to TOF-ratios. The main difference is that the parameters of the Hill equation, ED50 and {gamma}, are slightly different. However, at profound block, when both T1 and T4 are very small, TOF-ratios become very inaccurate. To avoid the bias introduced by this effect, we restricted the procedure to T1 blocks.

Measurements concerning blocks and recovery times are presented as mean (SEM). Data were analysed, using a Student's t-test, P-values <0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Twelve experiments were performed in which four different female Rhesus monkeys were used (weight 5.1–6.5 kg) three times each. In the three experiments on a single animal the time intervals between injection of sugammadex and rocuronium 100 µg kg–1 were 15, 30 and 60 min. Between experiments the Rhesus monkeys recovered for at least 6 weeks. The body temperature was 37.2–37.8°C. The tracings of three experiments, in one particular monkey, are shown in Figure 1A–C. Figure 1A depicts the overlap of the T1 twitch corresponding to the calibrating ‘initial dose’ injections. Figure 1B and C show the overlap of tracings corresponding to the T1 twitch and TOF-ratios, respectively, as a function of the second rocuronium dose. Time course of rocuronium and the effect of sugammadex are listed in Table 1. In Figure 2 the effect (Maximum T1 block) of rocuronium 100 µg kg–1 15, 30 and 60 min after the injection of sugammadex 1.0 mg kg–1 is shown and compared with the predicted effects as calculated in our model. The asymptotic value at the right of the S-shaped curve that predicts the effect of 100 µg kg–1 rocuronium is equal to the block value in the absence of sugammadex (the value reached by the calibrating first dose). The calculated half-life of sugammadex in Rhesus monkeys follows from a least squares fitting procedure: 30 min.


Figure 1
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  		Fig 1 (A) Time courses of the first twitch T1 of a ‘calibrating’ dose of rocuronium (100 µg kg–1) in three different experiments in the same monkey. This calibrating procedure always precedes sugammadex evaluation (part B of the figure). Note the reproducible response to the same dose of rocuronium in three different experiments, separated for at least six weeks. This was common in this monkey population, only few exceptions of small deviations were observed. Time t=0 min corresponds to time of injection of the ‘calibrating’ dose of rocuronium (100 µg kg–1). (B) Tracings of three experiments in the same monkey, corresponding to the first twitch T1 of the TOF sequence as the result of the second dose of rocuronium (100 µg kg–1). This second injection of rocuronium was administered 15, 30 or 60 min after injection of sugammadex 1.0 mg kg–1 in the three experiments. Time t=0 min corresponds to time of injection of sugammadex. It is clear that the effect of sugammadex (preventing block) disappears with time. (C) TOF-ratio as the result of the second dose of rocuronium (100 µg kg–1) at three different time intervals after the administration of sugammadex 1.0 mg kg–1 in three different experiments in the same monkey.

 

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  		Table 1 Time course of action of sugammadex 1.0 mg kg–1. Reversal effect of sugammadex 1.0 mg kg–1 on rocuronium 100 µg kg–1 as a function of time after injection of sugammadex. Maximal neuromuscular block measured (%) for rocuronium 100 µg kg–1, onset time to reach maximum block (min) and recovery times of the maximal neuromuscular block obtained (min) spontaneously before (pre) a bolus injection of sugammadex 1.0 mg kg –1 and 15, 30 or 60 min after sugammadex 1.0 mg kg–1. Values are means with SEM in parentheses: n=4 in each group, 12 experiments in total. BNR, this depth of block is not reached by T1 with this dose of rocuronium, 15 min after the injection of sugammadex

 

Figure 2
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  		Fig 2 Prediction of maximum block by rocuronium 100 µg kg–1 as a function of time interval after the injection of sugammadex 1 mg kg–1. The model assumes exponential decay of the concentration of sugammadex after its injection at t=0. The concentration of free rocuronium follows from the equilibrium constant for its complex formation with sugammadex. The maximum block reached by rocuronium 100 µg kg–1 can then be calculated by the Hill equation, using the free rocuronium concentration. The parameters in the Hill equation are obtained from the tracings obtained from rocuronium (100 µg kg–1) injected before sugammadex and the dose–response relation obtained in earlier experiments. Good agreement between measured and predicted maximum blocks is obtained if the elimination half-life of sugammadex is set at 30 min.

 
Injection of sugammadex did not have significant effects on blood pressure or heart rate. All animals recovered completely without complication. Signs of residual block or recurarization in the experiments with sugammadex were not observed.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Sugammadex, per-6-(2-carboxyethylthio)-per-6-deoxy-{gamma}-cyclodextrin sodium salt, is able to form a host–guest inclusion complex with rocuronium, also known as chemical encapsulation.1 Nuclear magnetic resonance spectroscopy and X-ray crystallography of this host–guest inclusion complex showed a tight 1:1 complex with the rocuronium molecule intercalated in the cyclodextrin ring.1 Encapsulating the rocuronium molecule with sugammadex results in a rapid decrease in the free concentration of rocuronium in plasma and its removal from motor endplates. Rocuronium is therefore less available to bind to nicotinic receptors in the neuromuscular junction. This will promote the liberation of free acetylcholine receptors, and muscle activity will reappear. As a result of no direct or indirect action on cholinergic transmission, it is unlikely that muscarinic side effects will occur.1 Previous studies in Rhesus monkey and humans confirmed the ability of sugammadex to reverse neuromuscular block induced by rocuronium without significant cardiovascular side effects.1–9

The present study showed that sugammadex at a dose of 1.0 mg kg–1 still has residual effects in plasma after 60 min and is still able to reverse the effects of neuromuscular block induced by rocuronium faster compared with spontaneous recovery. In the model presented, this means that after 60 min the exponentially decaying concentration is still above a non-effective level. In a least squares fitting procedure within this model the effective half-life of sugammadex in Rhesus monkeys 30 min (SEM=4.9 min, calculated from the results in individual monkeys) in this population of four monkeys. The half-life of sugammadex is relatively short. Despite this, recurarization has not been observed when sugammadex is given to reverse rocuronium induced neuromuscular block. However, present clinical experience with sugammadex is limited.

In theory re-establishment of rocuronium neuromuscular block is feasible only when most of the unbound sugammadex has disappeared (sugammadex is excreted rapidly and dose-dependently in urine).10 If this is not the case, higher amounts of rocuronium will be necessary. The presented model allows the calculation of the required amount by approximation. Another possibility to re-establish neuromuscular block is the use of atracurium or mivacurium. Sugammadex has no effect on neuromuscular block induced by the non-steroidal neuromuscular blocking agents mivacurium and atracurium because the size of the cyclodextrin cavity is too small to accommodate these bulky molecules.5 It should be emphasized that re-establishment of neuromuscular block by atracurium or mivacurium excludes the use of sugammadex for reversal of the second block.

The results of this study in Rhesus monkeys may indicate that re-establishment of rocuronium neuromuscular block is possible after reversal with sugammadex. However, further investigation is needed in humans to confirm and to extend our findings. It should be noted that when interpreting these data that the Rhesus monkey is more sensitive to rocuronium compared with humans (ED90 in humans is about 300 µg kg–1; in Rhesus monkeys ED90 is approximately 100 µg kg–1). Another obvious difference between Rhesus monkeys and humans include spontaneous recovery from 90% block: it is much faster in Rhesus monkey than humans. Taking these differences into account, the principles of the presented model will also hold in humans.

Regarding the safety of sugammadex, there were no significant effects on blood pressure or heart rate. Signs of residual or recurarization in these experiments were also not observed. In this study the half-life of sugammadex is relatively short (30 min) and the absence of recurarizations proves that the sugammadex–recoruronium complex is being eliminated just as fast or faster. As long as these time courses are relatively the same in humans, the monkey model is a good predictor for the effects of sugammadex in humans.

In conclusion, sugammadex is a promising fast and effective reversal agent for neuromuscular block induced by rocuronium, based on a new concept of chemical encapsulation, and without cardiovascular side effects.1–9 Although recurarization has not been observed, the half-life of sugammadex is relatively short in monkeys: 30 min. The mechanism of its action (encapsulation of rocuronium) and the short half-life suggest the possibility to perform rocuronium neuromuscular block after reversal of an earlier dose.


   Footnotes
 
{dagger}Declaration of interest. This study was supported by a grant from Organon. A.B. is employed by Organon and L.H.D.J.B. is a member of their scientific advisory board. H.D.B. is also working as staff anaesthesiologist at the Department of Anaesthesiology at the Martini Hospital Groningen, Groningen, The Netherlands. Back


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
1 Bom A, Bradley M, Cameron K, et al. A novel concept of reversing neuromuscular block: chemical encapsulation of rocuronium bromide by a cyclodextrin-based synthetic host. Angew Chem Int Ed Engl 2002; 41:266–70[Medline]

2 Hayes AH, Mirakhur RK, Breslin DS, et al. Post operative residual block after intermediate acting neuromuscular blocking drugs. Anesthesia 2001; 56:312–18[CrossRef][Web of Science][Medline]

3 Booij LHDJ, De Boer HD, Van Egmond J. Reversal agents for nondepolarizing neuromuscular blockade: reasons for and development of a new concept. Semin Anesth Periop Med Pain 2002; 21:92–8

4 De Boer HD, van Egmond J, van de Pol F, et al. Chemical encapsulation of rocuronium by synthetic cyclodextrin derivatives: reversal of neuromuscular block in anaesthestized Rhesus monkeys. Br J Anaesth 2006; 96:201–6[Abstract/Free Full Text]

5 De Boer HD, van Egmond J, van de Pol F, et al. Sugammadex, a new reversal agent for neuromuscular block induced by rocuronium in the anaesthetized Rhesus monkey. Br J Anaesth 2006; 96:473–9[Abstract/Free Full Text]

6 De Boer HD, van Egmond J, van de Pol F, et al. Reversal of profound rocuronium neuromuscular blockade by sugammadex in anesthetized Rhesus monkeys. Anesthesiology 2006; 104:718–23[CrossRef][Web of Science][Medline]

7 Gijsenberg F, Ramael S, De Bruyn S, et al. Preliminary assessment of Org 25969 as a reversal agent for rocuronium in healthy male volunteers. Anesthesiology 2005; 103:695–703[CrossRef][Web of Science][Medline]

8 de Boer HD, Marcus M, Schouten P, Heeringa M, et al. Reversal of rocuronium-induced (1.2 mg kg–1) neuromuscular block by Org 25969: a multi center dose finding, safety study. Anesthesiology 2005; 103:A1117

9 Shields M, Mirakhur RK, Moppett I, et al. Org 25969 (sugamadex), a selective relaxant binding agent for antagonism of prolonged rocuronium-induced neuromuscular block. Br J Anaesth 2006; 96:36–43[Abstract/Free Full Text]

10 Epemolu O, Mayer I, Hope F, et al. Liquid chromatography/mass spectrometric bioanalysis of a modified {gamma}-cyclodextrin (Org 25969), rocuronium bromide (Org 9426) in guinea pig plasma and urine: its application to determine the plasma pharmacokinetics of Org 25969. Rapid Commun Mass Spectrom 2002; 16:1946–52[CrossRef][Web of Science][Medline]


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