Interstitial muscle concentrations of rocuronium under steady-state conditions in anaesthetized dogs: actual versus predicted values

S. Ezzine and F. Varin*

Faculté de pharmacie, Université de Montréal, Montréal, Québec, Canada

* Corresponding author: Faculté de pharmacie, Université de Montréal, 2900 Edouard Montpetit, C.P. 6128, succursale centre-ville, Montréal, Québec H3C 3J7, Canada. E-mail: france.varin{at}umontreal.ca

Accepted for publication September 1, 2004.


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Introduction. The objective of this study was to compare rocuronium effect (Ce) and peripheral (C2) compartment concentrations predicted by pharmacokinetic–pharmacodynamic (PK-PD) modelling with those measured in plasma (Cp) and in the interstitial fluid of muscle tissue (CISF,u) by microdialysis in anaesthetized dogs.

Methods. After approval by the Animal Care Committee, eight adult male dogs with a body weight ranging from 7 to 18 kg were anaesthetized with pentobarbital. Each dog received a 2-min rocuronium infusion of 0.15 mg kg–1 min–1 followed by a 118-min infusion of 60 µg kg–1 min–1 via the right jugular vein. Arteriovenous gradient across the hindlimb was measured at 40, 60, 100 and 120 min. Three microdialysis samples were collected at 40-min intervals. Once the infusion stopped, arterial samples were collected every 2 min for the first 10 min and every 20 min for the next 120 min. Neuromuscular function was monitored using train-of-four stimulation until full recovery. Dogs were then killed and a biopsy of muscle tissue was performed (Cm).

Results. At steady state, the mean CISF,u value was 1353 ng ml–1. After correction for the unbound fraction in plasma, the mean Ce,corr and C2,corr were 1681 and 1481 ng ml–1, respectively. At the terminal sampling point, Cm was 10-fold higher than Cp.

Conclusion. Unbound concentration of rocuronium measured in the muscle interstitial fluid under steady-state conditions confirms that parametric PK-PD modelling gives reliable estimates of effect site concentrations. Rocuronium accumulates in muscle tissue, probably by non-specific protein binding in the interstitial space.

Keywords: pharmacokinetics, rocuronium ; pharmacology, rocuronium ; model, dog


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inferences made from pharmacokinetic–pharmacodynamic (PK-PD) studies are generally limited by the fact that plasma drug concentrations are measured instead of those within the tissue of pharmacodynamic interest. Thus, the time course of the predicted concentration in the effect compartment will depend heavily on the mathematical model while the true concentration at the effect site remains theoretical.1

In the case of neuromuscular blocking agents (NMBAs), measurement of drug concentration in muscle interstitial fluid would provide an invaluable insight into their concentration–effect relationship. In this respect, microdialysis provides a unique tool for quantitative measurement of NMBAs at their site of action. Application of microdialysis to the study of regional pharmacokinetics is more recent because quantitative determination of the drug concentration has been a limiting factor. Because rocuronium is almost exclusively eliminated unchanged by biliary and urinary routes in humans,2 it was chosen as the model drug. This study was designed specifically to compare the effect and peripheral compartment concentrations of rocuronium predicted by PK-PD modelling with those measured in plasma and interstitial fluid of the muscle tissue in anaesthetized dogs.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animal preparation
Animals
All animal experiments in this study were carried out according to the Canadian Council on Animal Care. After approval by the Animal Care Committee, eight adult male dogs (five mongrels and three Beagles) with a body weight ranging from 7 to 18 kg were obtained from Biolab (Montreal, Quebec, Canada). The animals were housed singly and maintained under a 24-h cycle of 12 h light and 12 h darkness at 21.1 (0.9)°C and 50 (10)% relative humidity. Food and water were freely available except for a 24-h period before the day of the experiment.

General anaesthesia
On the day of the experiment, each dog was anaesthetized with sodium pentobarbital (Somnotol®; Abbott Laboratories, Montreal, QC, Canada) 30 mg kg–1 i.v. The animals' lungs were ventilated mechanically using a ventilator (model 607; Harvard, South Natick, MA, USA). Monitoring of the level of anaesthesia was based on haemodynamic parameters as well as the corneal reflex. An intermittent bolus dose of 4 mg kg–1 was administered for maintenance of deep anaesthesia (approximately every 90 min). All invasive procedures were carried out before administration of rocuronium. Thereafter, blood pressure and cardiac monitoring was used to monitor depth of anaesthesia.

Surgical procedure and haemodynamic monitoring
Once a satisfactory level of anaesthesia was achieved and physiological parameters were stable, the right jugular vein was cannulated for drug infusion and the right carotid artery for blood sampling. Blank plasma collected on sodium citrate 0.129 M was used for protein binding determination. Two small incisions (2 cm) were made on the animal's skin overlying the left gastrocnemius and gracilis muscles to allow insertion of two microdialysis probes. At least one half-hour equilibration time was allowed for the tissue to recover while the probe was perfused with buffer solution. Meanwhile, a three-way stopcock was installed on the arterial line for arterial blood pressure monitoring. An electromagnetic flow probe (3 mm i.d., model FR-030T; Nihon Kohden, Tokyo, Japan) was installed on the left femoral artery and connected to a polygraph system (model RM-6000; Nihon Kohden) for muscle blood flow measurement. In the femoral vein of the same leg, an indwelling catheter (Cathelon®, Critikon, Tampa, FL, USA) was inserted to allow venous blood sampling. Heart rate was monitored via the arterial pulse, and body temperature was monitored by means of rectal probe maintained at 38°C during the experimental procedure.

Neuromuscular monitoring
The medial cutaneous antebrachial nerve was stimulated supramaximally at the right forelimb through surface electrodes with 0.2 ms, 40–70 mA impulses derived at a frequency of 2 Hz. This pattern was repeated every 12 s. The resulting force of contraction was measured using a force transducer (Grass F-10) and the signal recorded on chart paper. A stabilization period of at least 5 min was allowed before injection of rocuronium. Muscle relaxation was monitored continuously until full recovery (in all animals). The degree of neuromuscular block was expressed as the percentage of twitch height depression relative to the baseline values obtained immediately before the injection of rocuronium (onset) or after complete recovery (recovery). Using train-of-four stimulation, only the first twitch (T1) was considered, giving results comparable to those obtained with single-twitch stimulation.3

Microdialysis probe calibration
The in vivo recovery of rocuronium from the microdialysis probe was then investigated in each dog using combined retrodialysis, described in detail elsewhere.4 Briefly, rocuronium (2000 ng ml–1) and its calibrator, vecuronium (2000 ng ml–1), were delivered into the muscle before systemic administration of rocuronium (reference period). These low concentrations used for probe calibration did not produce any significant pharmacological effect as no change in twitch baseline was observed. Dialysate samples (n=3) were collected every 40 min and assayed for rocuronium and vecuronium. The relative losses of the two compounds were determined afterwards by measuring their concentrations in the perfusate and dialysate. A ratio was established between the recoveries of the two compounds. A wash-out period of 60 min was allowed to enable complete removal of rocuronium from the outflow cannula; this was confirmed later by HPLC analysis. During the steady-state period, microdialysis probe calibration was continued by delivering vecuronium (in Krebs solution) into the muscle while perfusing rocuronium systemically. The relative loss of vecuronium from the probe into the muscle, corrected for the ratio determined previously (in the reference period) was used as an estimate of the in vivo recovery of rocuronium. This method has been validated previously4 5 and has been shown to be accurate and reliable.

Drug administration
Dogs received a 2-min infusion of rocuronium 0.15 mg kg–1 min–1 followed by a 118-min infusion of 60 µg kg–1 min–1 via the right jugular vein. This infusion regimen was based on the results obtained in three dogs during preliminary studies. For dog 2, the second infusion regimen lasted only 100 min. Dog 7 inadvertently received 0.345 mg kg–1 min–1 for 2 min followed by 129 µg kg–1 min–1 of rocuronium for 90 min. Therefore, plasma and interstitial fluid concentrations of dog 7 were normalized to the dose administered in all other dogs.

Sample collection
After drug administration, arterial samples (3 ml) were collected at several time points until 100% neuromuscular block, based on twitch tension. Arterial and venous blood samples (3 ml) were collected simultaneously 40, 80, 100 and 120 min after the beginning of the rocuronium infusion. These four sampling times were used to confirm steady-state equilibrium and tissue extraction (see Data analysis). Muscle interstitial fluid (approximately 80 µl) was sampled continuously by microdialysis for 120 min (0–40, 40–80, 80–120 min) and processed as described below. The median time of each microdialysis period corresponded to the blood sampling time for the arteriovenous gradient. At the end of the steady-state period, arterial plasma (3 ml) was collected every 2 min for the first 10 min and every 20 min for the next 120 min. Plasma and dialysate samples were collected into 2 M sulphuric acid, kept on ice and stored at –70°C pending HPLC analysis. For quality control purposes, aliquots of the perfusate and i.v. solution were sampled at the end of the steady-state period for future determination of vecuronium and rocuronium concentrations, respectively.

Muscle biopsy
Euthanasia was then performed by i.v. injection of pentobarbital 130 mg followed by saturated KCl 20 ml. Muscle tissue (3 g) was excised, carefully blotted dry and homogenized for 5 min with ice-cold 0.1 M monobasic ammonium phosphate buffer, pH 3.0, at a ratio of 1:6 (1 g tissue/6 ml buffer) (PowerGen Homogenizer, model 125; Fisher Scientific, Montréal, Quebec, Canada). Samples were stored at –70°C until HPLC analysis.

Quantitative analysis
HPLC analysis
Arterial and venous plasma and microdialysis samples were assayed for rocuronium concentration by reverse-phase HPLC with electrochemical detection.6 Chromatographic separation was achieved using a CN Spherisorb (5 µm particle size) column, 15 x 4.6 mm i.d., maintained at 35°C with a column heater (CH-30; Eppendorf North America, Madison, WI, USA). The mobile phase (pH 4.75), containing bifiltered H2O, acetonitrile and methanol (55:35:10), was delivered at a flow rate of 1.8 ml min–1 by means of a ConstaMetric III pump (LDC Milton Roy; Riviera Beach, FL, USA). Detection of rocuronium and vecuronium was accomplished using an electrochemical detector linked to a 5010 analytical cell (Environmental Sciences Associates, Bedford, MA, USA). The analytical cell potential was set at 750 mV. Chromatographic data were recorded and processed with an integrator (Shimadzu, Koyoto, Japan).

Plasma samples, assayed in duplicate, were extracted on Bond Elut C1 solid-phase extraction cartridges (Varian, Harbor City, CA, USA). Microdialysis samples were diluted with blank plasma and processed in the same manner as plasma samples. Direct injection into the HPLC was impossible due to the high ionic strength of the dialysate buffer. Under the conditions described, the lower limit of quantification of rocuronium and vecuronium was 5 ng ml–1. Standard curves for rocuronium and vecuronium showed good linearity between 7.8 and 5000 ng ml–1 (r2=0.99). The coefficients of variation were below 10%.

Muscle tissue homogenates were centrifuged for 5 min at 200 g before transfer onto a SPE cartridge to prevent clogging. Recovery tests for the centrifugation step were performed at three concentrations of rocuronium and indicated a loss of ±1% trapped in the residue. Calibration curves were prepared in blank muscle tissue, ranging from 8 to 500 ng ml–1 (r2=0.9948).

In vitro protein binding
An ultrafiltration technique was performed in duplicate on spiked plasma using 30 000 µm membranes as described previously.7 The ultrafiltration devices, containing 0.5 ml of the biological matrix, were centrifuged at 1900 g for 5 min at room temperature. An aliquot (50 µl) of the ultrafiltrate was acidified with sulphuric acid 2 M and frozen. Thawed ultrafiltrates were diluted with blank plasma and processed in the same manner as plasma samples for HPLC analysis.

Arteriovenous gradient
Each plasma sample drawn during the steady-state period was assayed in duplicate. As the difference between duplicates was less than 10%, the analytical error, the mean of the two values was used. After a 40-min infusion period, steady state was assumed to be mostly achieved in plasma (based on a 20 min half-life in preliminary studies).

Data analysis
Arteriovenous gradient
Arterial concentrations measured at the 80, 100 and 120 min collection times were compared using Friedman repeated-measures analysis of variance on ranks. After statistical confirmation of steady-state conditions, arterial concentrations in each dog were averaged and corrected for the unbound fraction. The corresponding venous samples were treated similarly. The arteriovenous gradient (E) of rocuronium was then calculated by applying the mean arterial (Cpssa,u) and venous (Cpssv,u) steady-state plasma concentrations to the following formula:

Interstitial fluid
Because microdialysate concentrations are time-averaged over the collection interval, these values were translated into concentrations at a single point by assuming that the concentration obtained is the actual concentration at the middle point of the time interval. As the difference between rocuronium interstitial concentration was not statistically different using Friedman repeated-measures analysis of variance, these concentrations were also pooled and averaged to obtain a mean rocuronium interstitial concentration for each dog. Data are expressed as mean (SD) for the number of dialysate collections (n=3).

Protein binding
Rocuronium unbound fraction in plasma (fu,p) was calculated by dividing the ultrafiltrate concentration (Cp,u) by the total concentration (Cp). The in vitro ratio (fu,p) was then multiplied by the steady-state plasma concentration measured in vivo to obtain Cpss,u and as a correction factor for C2 and Ce.

Pharmacokinetic analysis
Central compartment concentration
A two-compartment mammillary model with a zero order input rate and elimination from the central compartment was fitted to the plasma concentration–time profile of rocuronium. A two-compartment model was selected after standard verification of its adequacy using the Akaike information criterion (AIC). WinNonlin 1.5 software (Pharsight, Mountain View, CA, USA) was used for pharmacokinetic analysis. Point estimates and pharmacokinetic parameters were optimized for each dog using a standard minimization method (Gauss–Newton, Levenberg and Hartley).8 9 A weighting function of 1/(predicted y)2 was applied. The following parameters were derived for each dog using standard formulae: A, B, the coefficients, {alpha}, ß, the fast distribution and elimination rate constants, V1, V2, Vss, the central, peripheral and total apparent volume of distribution at steady state, and CL, the total body clearance; and the transfer rate constants between the peripheral and central compartment, k12 and k21.

Peripheral compartment concentration
For each dog, the peripheral compartment concentration (C2) vs time curve was simulated by substituting the derived pharmacokinetic parameters into the following equation:10

where k0 is the zero-order infusion rate and T the duration of the infusion.

Pharmacokinetic–pharmacodynamic analysis
Effect compartment concentration
Using the pharmacokinetic parameters derived previously for rocuronium, a parametric link model11 coupled to a sigmoid Emax model was used to derive the equilibrium rate constant between the central and the effect compartment (keo), the effect compartment concentration at 50% block (EC50) and the slope of the sigmoid curve ({gamma}). WinNonlin software was used for all PK-PD analyses. A weighting function of 1 was applied and the goodness of fit was assessed by the AIC. For each dog, the effect compartment concentration was calculated for a two-compartment model and a zero-order input using the PK-PD estimates as described by Holford and Sheiner.12


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The eight dogs studied had a mean weight of 14 kg (range 7.4–17.5). During the calibration period, the total dose of muscle relaxants (rocuronium and vecuronium) administered locally for probe calibration was kept minimal. No systemic concentrations or twitch depression were detectable. Individual data for femoral blood flow, arterial blood pressure, unbound fraction in plasma, haematocrit and arteriovenous gradient during steady state are presented in Table 1.


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Table 1 Physiological parameters, protein binding and arteriovenous gradient in dogs under continuous infusion of rocuronium.

 
Individual pharmacokinetic estimates are presented in Table 2 as mean (SD). The mean distribution half-life t1/2{alpha} was 3.7 (1.8) min and the mean elimination half-life t1/2ß was 34 (5) min. Individual pharmacodynamic parameters are presented in Table 3. The onset time was variable among dogs and ranged from 0.6 to 6.6 min. The mean 25–75% recovery index ranged from 0.9 to 17.7 min. The in vivo time–concentration profiles of rocuronium for each dog and the percentage of neuromuscular block as a function of time are presented in Figure 1.


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Table 2 Rocuronium pharmacokinetic parameters in anaesthetized dogs.

 

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Table 3 Rocuronium pharmacodynamic parameters in anaesthetized dogs. Onset, time at 90% neuromuscular block; T25%,T50%,T75%, time to 25, 50 and 75% recovery respectively; I25–75, recovery index

 


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Fig 1 (A) Individual rocuronium plasma concentration–time curves. (B) Individual neuromuscular effect of rocuronium in individual dogs.

 
Figure 2A shows a typical plasma concentration–time profile (dog 5) with a median fit. The observed and predicted effects at different times for the same dog are shown in Figure 2B. Individual PK-PD parameters are presented in Table 4. The mean EC50 was 353 ng ml–1 and the mean t1/2ke0 was 5.54 min.



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Fig 2 (A) Observed vs predicted rocuronium plasma concentrations, predicted peripheral and effect compartment concentrations and interstitial tissue concentrations in dog 5. (B) Observed vs predicted neuromuscular effect of rocuronium in dog 5.

 

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Table 4 Rocuronium pharmacokinetic–pharmacodynamic parameters in pentobarbital anaesthetized dogs. ke0, effect compartment equilibration rate constant; EC50, effect compartment concentration at 50% block; gamma, slope factor

 
The individual effect and peripheral compartment concentrations derived from PK-PD parameter estimates are presented in Table 5. Before correction for the unbound concentration in plasma, the mean Ce and C2 values were 4029 ng ml–1 and 3615 ng ml–1, respectively.


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Table 5 Steady-state rocuronium concentrations in pentobarbital anaesthetized dogs. Ce,corr and C2,corr, effect and peripheral compartment concentrations corrected on the basis of the unbound fraction in plasma (fu); CISF,u, muscle interstitial concentration; Cpssa,u, unbound arterial plasma concentration at steady state after correction of the free fraction in plasma.

 
Unbound concentrations of rocuronium in the muscle interstitial fluid, as determined by microdialysis, are presented in Table 5. The mean CISF,u value was 1353 ng ml–1. Interstitial fluid concentrations are not given for the first three dogs because these concentrations were underestimated because of an inappropriate method of microdialysis probe calibration. This issue has been addressed in detail elsewhere.4 Interstitial fluid concentrations are not available for dog 8 because of a failure of the microdialysis probe during the experimental period.

Muscle tissue concentrations at the terminal sampling point are presented in Table 6. The mean concentration in whole muscle homogenate (Cm) was 541 ng ml–1, with a corresponding plasma concentration of 38 ng ml–1.


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Table 6 Rocuronium concentrations at terminal sampling point in pentobarbital anaesthetized dogs.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The major finding of this study is that, under steady-state conditions, the unbound concentrations of rocuronium measured in muscle interstitial fluid are equivalent to those predicted for the effect and peripheral compartments by PK-PD modelling on the basis of unbound plasma concentrations. This is important as the effect compartment concentrations remain hypothetical in the absence of in vivo confirmation of the actual concentrations at the site of action. At the beginning of the infusion, the unbound concentrations in the muscle interstitial fluid and plasma correlated well. At the terminal sampling point, rocuronium concentrations in whole muscle homogenate were several-fold higher than those in plasma.

The pharmacokinetics of rocuronium has been well characterized in patients13 but reports of studies carried out in animals are limited. To our knowledge, there is no previous study reporting the PK-PD parameters of rocuronium in animals. Our bolus dose was based on a reported ED90 of 0.18 mg kg–1 for rocuronium in Beagle dogs anaesthetized with pentobarbital.14 Rocuronium onset of action was slightly more rapid compared with that in our study (2.1 vs 3.3 min), probably as a result of the input rate (bolus vs short infusion). The infusion regimen for the steady-state period was based on the analytical sensitivity required for microdialysis. The mean equilibration rate between rocuronium plasma and effect compartment concentrations (ke0) in our dogs is comparable to that found in humans.13 15 16 However, a lower EC50 is observed in dogs. It is unclear whether this increased sensitivity is species-dependent or related to pentobarbital anaesthesia.

Microdialysis in muscle tissue has been used to describe the regional kinetics of drugs, having mostly an extracellular distribution (piperacillin17, ceftriaxone18 and gallamine19). As a bolus dose was given in anaesthetized rats, it was not possible to verify if, at distribution equilibrium, the unbound concentrations in plasma and muscle interstitial fluid were equal. The physiological models used for simulations assumed that drug transfer between plasma and tissue is driven by diffusion only. When the concentrations predicted for the tissue compartment and those measured in the muscle interstitial fluid were compared, an excellent agreement was found.

To our knowledge, there is only one study20 in which microdialysis in the muscle tissue was performed under steady-state conditions. For cefaclor, unbound arterial plasma concentrations were found to be fourfold higher than those measured in the muscle interstitial fluid. A clear explanation for the gradient could not be provided because of the well-known chemical instability of this drug. This also implies that unrestricted diffusion of the free fraction across capillaries cannot be taken for granted for all drugs.

Rocuronium was therefore chosen as the model drug for neuromuscular blocking agents because of its chemical stability and absence of significant biotransformation.2 In a previous report, we have detailed how quantitative determination of rocuronium interstitial concentrations in the muscle tissue of dogs by microdialysis were validated.4

For the collection period corresponding to the first 40 min of rocuronium infusion, muscle interstitial fluid concentrations were very close to the unbound arterial concentrations (Fig. 2). This good agreement suggests a rapid transcapillary transfer of the unbound concentration of rocuronium into the extracellular fluid. Similar findings were observed for antibiotics17 18 20 and for gallamine after an i.v. bolus dose.19 In the latter study, muscle interstitial fluid samples were collected at 15-min intervals for 8 h (equivalent to one elimination half-life). The muscle interstitial fluid and arterial plasma concentration–time profile declined in parallel during both the distribution and elimination phases. These results suggest that, for hydrophilic drugs, the unbound concentrations in muscle interstitial fluid and arterial plasma belong to the same kinetic pool.

After 2 h of infusion, rocuronium interstitial concentrations proved to be equal to unbound venous but slightly lower than arterial concentrations in all but one dog (dog 3 had no arteriovenous gradient). A mean arteriovenous gradient of 6% is usually considered as negligible as it falls within the error of the analytical method. However, this gradient subsided after an infusion period equivalent to four plasma elimination half-lives, suggesting that the equilibrium within the muscle tissue is not achieved completely (94% of steady state according to our calculations). This observation is compatible with slow transfer into muscle cells, as one would expect for a drug having a very low partition coefficient.21

When the peripheral and effect compartment concentrations are derived mathematically from plasma concentrations, one assumes that the tissue/plasma concentration ratio of the unbound fraction of drug is equal to 1 under steady-state conditions. Since the protein bound fraction is not pharmacologically active, there are no practical implications for the PK-PD relationship. This assumption was corroborated by microdialysis in our study by measuring the unbound concentration of rocuronium in the muscle interstitial fluid, which represents the biophase for neuromuscular blocking drugs. Under steady-state conditions, we found excellent agreement between the effect site concentration derived mathematically and the muscle interstitial concentration measured by microdialysis. This provides further evidence that a parametric PK-PD model can yield reliable estimates of the effect site concentrations at doses for which complete neuromuscular blockade is maintained for a long period.

Insights on the intra-tissue distribution of rocuronium were obtained shortly after sacrifice. The concentrations measured in the whole muscle tissue, although quite variable, were more than seven times higher than their corresponding concentrations in plasma, the latter being almost negligible. If one takes into account that muscle tissue represents 45% of total body weight in dogs, the amount of rocuronium in the muscle tissue would correspond to 95% of the total amount of rocuronium present in the body at the terminal sampling point. Rocuronium concentrations in whole-muscle homogenate were also more than twice those predicted for the peripheral compartment on the basis of the unbound concentration in plasma. In pharmacokinetic modelling, compartments refer only to the amount of drug in a distinct kinetic pool and do not correspond to anatomically identifiable compartments. These results therefore indicate the preferential accumulation of rocuronium in muscle tissue.

The cellular permeability of rocuronium is very low. After a 2-h infusion, the total concentration of rocuronium in the cerebrospinal fluid of anaesthetized patients was found to represent only 2–3% of the unbound concentration in plasma.22 Thus, the distribution of rocuronium is mostly confined to the extracellular fluid. In our dogs, plasma rocuronium concentrations were negligible at the time of sacrifice. It follows that the amount of rocuronium measured in the muscle tissue homogenate would originate from the interstitial space. As the volume of the latter occupies 33% of the water content of the muscle in dogs, the concentration of rocuronium in the interstitial fluid would be three-fold higher than that measured in whole muscle.

In contrast, the muscle tissue/plasma concentration ratio of gallamine was only 0.2 after a bolus dose in anaesthetized rats.19 Since gallamine does not bind to proteins, this finding supports the hypothesis that protein binding in the interstitial space may be responsible for the accumulation of rocuronium in muscle. Unfortunately, our attempts to collect muscle interstitial fluid directly with a push–pull cannula were unsuccessful. It was thus impossible to estimate the free fraction within the interstitial space. However, considering that neuromuscular function had recovered completely at that time, most of the rocuronium molecules in the muscle tissue would not be bound to acetylcholine receptors. This suggests that the high muscle/plasma concentration ratio of rocuronium at the time of sacrifice would result from non-specific protein binding in the interstitial space.

Conclusion
The major finding of this study is that quantitative measurement of the unbound fraction of rocuronium in muscle tissue provides evidence that a parametric PK-PD model can yield reliable estimates of effect site concentrations. This is the first study reporting a significant accumulation of rocuronium in muscle tissue, probably by non-specific protein binding in the interstitial space.


    Acknowledgments
 
The authors would like to thank Ms Johanne Couture and Ms Sanae Yamaguchi, Faculté de pharmacie, Université de Montréal, for their technical assistance. This research was supported by the Canadian Institutes of Health Research (Grant MA-10274). A CIHR-R&D studentship was awarded to SE.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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10 Gibaldi M, Perrier D. Pharmacokinetics. In: Gibaldi M, Perrier D, eds. Pharmacokinetics. New York: Marcel Dekker, 1982; 273–9

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12 Holford NH, Sheiner LB. Understanding the dose–effect relationship: clinical application of pharmacokinetic–pharmacodynamic models. Clin Pharmacokinet 1981; 6: 429–53[ISI][Medline]

13 Dragne A, Varin F, Plaud B, Donati F. Rocuronium pharmacokinetic–pharmacodynamic relationship under stable propofol or isoflurane anesthesia. Can J Anaesth 2002; 49: 353–60[Abstract/Free Full Text]

14 Marshall RJ, Muir AW, Sleigh T, Savage DS. An overview of the pharmacology of rocuronium bromide in experimental animals. Eur J Anaesthesiol Suppl 1994; 9: 9–15[Medline]

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16 Plaud B, Proost JH, Wierda JM et al. Pharmacokinetics and pharmacodynamics of rocuronium at the vocal cords and the adductor pollicis in humans. Clin Pharmacol Ther 1995; 58: 185–91[ISI][Medline]

17 Nolting A, Costa TD, Vistelle R, Rand KH, Derendorf H. Determination of free extracellular concentrations of piperacillin by microdialysis. J Pharm Sci 1996; 85: 369–72[CrossRef][ISI][Medline]

18 Kovar A, Dalla CT, Derendorf H. Comparison of plasma and free tissue levels of ceftriaxone in rats by microdialysis. J Pharm Sci 1997; 86: 52–6[CrossRef][ISI][Medline]

19 Sasongko L, Ramzan I, Williams KM, McLachlan AJ. Muscle distribution of the neuromuscular blocker gallamine using microdialysis. J Pharm Sci 2002; 91: 769–75[CrossRef][ISI][Medline]

20 De La Pena A, Dalla CT, Talton JD et al. Penetration of cefaclor into the interstitial space fluid of skeletal muscle and lung tissue in rats. Pharm Res 2001; 18: 1310–4[CrossRef][ISI][Medline]

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22 Fuchs-Buder T, Strowitzki M, Rentsch K et al. Concentrations of rocuronium in cerebrospinal fluid of patients undergoing cerebral aneurysm clipping. Br J Anaesth 2004; 92: 419–21[Abstract/Free Full Text]





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