Skip Navigation


BJA Advance Access originally published online on December 23, 2005
British Journal of Anaesthesia 2006 96(2):201-206; doi:10.1093/bja/aei306
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
96/2/201    most recent
aei306v1
Right arrow E-Letters: Submit a response to the article
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (17)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by de Boer, H. D.
Right arrow Articles by Booij, L. H. D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by de Boer, H. D.
Right arrow Articles by Booij, L. H. D. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Board of Management and Trustees of the British Journal of Anaesthesia 2005. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Chemical encapsulation of rocuronium by synthetic cyclodextrin derivatives: reversal of neuromuscular block in anaesthetized Rhesus monkeys{dagger}

H. D. de Boer1,*, J. van Egmond1, F. van de Pol1, A. Bom2 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, Radboud University Medical Centre Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: HD.de.Boer{at}mzh.nl

Accepted for publication October 12, 2005.


   Abstract
Top
 Footnotes
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Background. At present, reversal of neuromuscular block induced by steroidal neuromuscular blocking agents (NMBAs) is achieved by administration of cholinesterase inhibitors. Chemical encapsulation of steroidal NMBAs, such as rocuronium, by a cyclodextrin is a new concept in neuromuscular block reversal. The present study evaluates 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) to reverse constant neuromuscular block of ~90%, induced by rocuronium infusion in the Rhesus monkey, using single twitch stimulation. The ability of these cyclodextrin derivatives to reverse neuromuscular block was compared with the reversal of the same neuromuscular block by the commonly used combination of neostigmine and atropine.

Methods. After a bolus injection of rocuronium, continuous infusion was started to reduce twitch contractions to ~10% of baseline values. After a steady state block of at least 10 min the infusion was stopped and the preparation was allowed to recover spontaneously. This process was repeated, but at the time the infusion was stopped, either one of the nine cyclodextrin derivatives or a combination of neostigmine and atropine was given.

Results. Recovery with cyclodextrin derivatives Org 26142 and Org 25969 was faster than after a combination of neostigmine and atropine (P<0.05). Injection of these cyclodextrin derivatives did not affect blood pressure or heart rate. Signs of residual block or recurarization were not observed in any of these experiments. In the experiments in which a combination of neostigmine and atropine was given, two animals showed signs of abdominal discomfort as frequently seen after the administration of neostigmine and significant changes in circulatory variables.

Conclusions. Chemical encapsulation or chelation of rocuronium is a new concept in reversing neuromuscular block induced by rocuronium.

Keywords: chemical encapsulation; neuromuscular block, rocuronium; reversal agent, cyclodextrins


   Introduction
Top
 Footnotes
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Steroidal neuromuscular blocking drugs, such as rocuronium, are widely used in clinical anaesthesia to facilitate tracheal intubation and to allow surgical access to body cavities.1 Residual neuromuscular block and subsequent respiratory insufficiency are associated with substantial morbidity and mortality, although the quality of tracheal intubation has significantly improved because of the clinical use of neuromuscular blocking agents (NMBAs), resulting in a reduction of pharyngo-laryngeal lesions.2–4 Therefore, many anaesthetists routinely reverse neuromuscular block to facilitate rapid and complete recovery after surgery to prevent residual block.5 At present, the reversal of neuromuscular block is associated with well known undesirable side effects.

The emphasis for the development of a new reversal agent should focus on agents with minimal side effects and reliable efficacy. One of the so far unexplored possibilities is encapsulation of the NMBAs by other agents. Cyclodextrins, a group of oligosaccharides, are well known for their ability to encapsulate lipophilic guest molecules such as steroids.6 7 Cyclodextrins have mainly been used as carriers for insoluble drugs or as stabilizers of unstable drugs and are currently used as carriers for transdermal nasal drug administration.8 Applying cyclodextrin derivatives was therefore considered for the reversal of neuromuscular block by chemical encapsulation of the steroidal rocuronium molecule. This mechanism has been confirmed by calorimetry and X-ray crystallography.7

The present study was undertaken to evaluate the effect of nine different synthetic cyclodextrin derivatives on constant neuromuscular block of ~90%, induced by rocuronium infusion in the anaesthetized Rhesus monkey, using single twitch stimulation. With one exception these compounds are all derivatives of {gamma}-cyclodextrin (listed in Table 1 in the order of increasing molecular weight) and were designed to improve the encapsulation of the rocuronium molecule. The capacity of each of these cyclodextrin derivatives to reverse neuromuscular block was compared with reversal of the same neuromuscular block by a combination of neostigmine and atropine.


View this table:
[in this window]
[in a new window]
  		Table 1 Chemical description of the nine different synthetic cyclodextrin derivatives, with increasing molecular weight, used in this study. Cd, cavity diameter; CD, cyclodextrin; Ch, cavity height; DS, degree of substitution of OH-group; MW, molecular weight

 

   Methods
Top
 Footnotes
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
In vivo experiments were performed in the experimental laboratories of the Department of Anaesthesiology at the University Medical Centre in Nijmegen, The Netherlands. The experiments were approved by the regional ethics committee on animal experiments.

Thirty-eight experiments were performed on 13 different female Rhesus monkeys (CSIMS, Beijing, China), weighing between 4.2 and 6.5 kg. The number of experiments on the individual animals varied between one and six, averaging three experiments. Experiments on the same animal were separated by at least 6 weeks of rest.

The animals were sedated in the cage with ketamine 10 mg kg–1 (Nimatek Eurovet), administered i.m. In the experimental laboratory two i.v. lines were mounted (one for the anaesthetics including rocuronium, the second line for the test drug), anaesthesia was induced by i.v. injection of pentobarbital sodium (Ceva Sante Animale, 25 mg kg–1), the monkey was intubated tracheally and the lungs ventilated with a mixture of oxygen and nitrous oxide at a ratio of 2:3. Anaesthesia was maintained by pentobarbital infusion at a rate of 5–10 mg kg–1 h–1. Heart rate (HR) and oxygen saturation were determined with a pulse oximeter (Ohmeda, Biox, Finland) at the ear. Blood pressure was determined with a cuff placed around the tail (Ohmeda, Finapres). Body temperature was maintained at 37–38°C. For monitoring purposes the median nerve of the right arm was stimulated supramaximally near the wrist using needle electrodes. Stimulation was performed with single twitch 2 ms square wave pulses (0.1 Hz) delivered by a Grass S88 Stimulator (Grass Medical Instruments, Quincy, MA, USA). The resulting contractions of the thumb were quantified with a force displacement transducer and recorded on a polygraph. After a bolus injection of rocuronium bromide (dose 100 µg kg–1), continuous infusion (infusion rate varied between 266 and 615 µg kg–1 h–1) was started to steadily reduce the twitch contractions to ~10% of their baseline value. After a steady block of ~90% had developed for at least 10 min, the infusion was stopped and the animal was allowed to recover from neuromuscular block spontaneously. This procedure was repeated, but at the time the infusion was stopped, either one of the nine different cyclodextrin derivatives or a combination of neostigmine 40 µg kg–1 and atropine 15 µg kg–1 (the effective dose for neostigmine and atropine was determined in separate series of monkey experiments) was given i.v. as a rapid bolus (for doses see Table 2). Using the experimental traces twitch recovery times were measured at 25, 50, 75, and 90% recovery, with respect to basal values. Residual block and recurarization were assessed by continuing neuromuscular monitoring for another 60 min after twitch restoration. At the end of the experiment, the animals were allowed to recover from anaesthesia.


View this table:
[in this window]
[in a new window]
  		Table 2 Recovery times with each cyclodextrin derivative and with neostigmine/atropine, as a function of increasing molecular weight. Mean (SEM) values are listed.

 
Experimental time course is illustrated in the tracing of one particular experiment and presented in Figure 1A. Various periods can be recognized: equilibration (A), constant 90% block (B), spontaneous recovery (C and D), constant 90% block (E), recovery in the presence of test drug (F), and period for the evaluation of any recurarization (G). Evaluation of the effect of the test drug is performed by comparison of the two recovery periods within the same experiment (C and F) as depicted in Figure 1B.


Figure 1
View larger version (19K):
[in this window]
[in a new window]
  		Fig 1 (A) Time course of the experiments (A) equilibration, (B) steady 90% block, (C, D) spontaneous recovery, (E) steady 90% block, (F) recovery with one of the test drugs, and (G) finally a period of evaluation of recurarization. (B) Comparison of spontaneous recovery of rocuronium-induced neuromuscular block with recovery in the presence of the test drug (in this case 0.5 mg kg–1 of Org 25969). Here the two recovery traces are depicted on top of one another.

 
Cyclodextrin derivatives were excluded from further investigation in the case of lack of potency or if any side effects were observed.

Statistics
To evaluate the effect of a particular cyclodextrin derivative, recovery with the reversal agent was compared with the recovery without reversal agent in the same experimental session. First, the ratios (r) of recovery times were calculated at 25, 50, and 75% recovery: r25=t25 with/t25 without, with similar equation for r50 and r75. Then, the average ratio of these three variables was calculated: r=;(r25+r50+r75)/3. Because of the susceptibility of t90 to any instability in the experiment, this was avoided in the calculation. The statistical significance of the reversal effect of a test drug was tested by comparing this averaged ratio r with 1 in a Student's t-test for paired observations. For comparison of the reversal effect of a test drug with that of neostigmine the non-parametric median test was used. In statistical procedures P-values <0.05 were considered statistically significant. Measurements concerning the recovery times and calculated ratios are presented as mean (SEM). Relative changes in circulatory variables are presented as mean (SD).


   Results
Top
 Footnotes
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Details of the nine synthetic cyclodextrin derivatives used in this study are presented in Table 1 and Table 2 presents the number of the experiments performed, the dose of the cyclodextrin derivatives and the neostigmine/atropine combination and recovery times. The number of experiments on a single cyclodextrin derivative varied between 1 and 4. Each experiment, however, starts with a spontaneous recovery from 90% block. In Table 2 the average spontaneous recovery times of all these experiments (n=38) are presented. In Figure 2 spontaneous and cyclodextrin-induced recoveries are depicted. It is important to note that the effect of the various test drugs is determined in a paired fashion as described above (see also Fig. 1B).


Figure 2
View larger version (25K):
[in this window]
[in a new window]
  		Fig 2 Course of neuromuscular recovery for spontaneous vs either one of the nine cyclodextrin derivatives or the neostigmine/atropine combination (neo/atro). Note: The values for spontaneous recovery depicted are means from all 38 experiments. Note that only Org 25969 and Org 26142 (at either of two dosages) provide faster recovery than the neostigmine/atropine combination.

 
All animals in which a cyclodextrin derivative was injected recovered completely with no complications. Recovery after the cyclodextrin derivatives Org 26142 and Org 25969 was faster than after the neostigmine/atropine combination. All the other cyclodextrin derivatives yielded a slower recovery time than that for neostigmine–atropine. With the exception of Org 25288, Org 26142, and Org 25969 only the highest doses are reported in Table 1. No cyclodextrin derivative was excluded from further investigation because of side effects.

In all experiments with the nine cyclodextrin derivatives, changes in mean arterial pressure (MAP) and HR were <10% of baseline values. This is illustrated in Figure 3, in which circulation variables are displayed for 1 h after injection of the most efficient reversing cyclodextrin, Org 25969, here at the highest dose, 1 mg kg–1. The relative changes in HR and MAP are reported in Table 3. Only the two cyclodextrin derivatives that reverse neuromuscular block better than neostigmine are reported, both at two doses.


Figure 3
View larger version (14K):
[in this window]
[in a new window]
  		Fig 3 Typical example of the absence of effect of cyclodextrin on circulatory variables. Here, the most efficiently reversing cyclodextrin Org 25969 is injected at time=0 at the highest dose (1 mg kg–1). Spontaneous variation in pressures and HR during 1 h of monitoring are larger than the change because of injection at time=0. (SAP, systolic arterial pressure; MAP, mean arterial pressure; DAP, diastolic arterial pressure.)

 

View this table:
[in this window]
[in a new window]
  		Table 3 Relative changes in circulatory variables (HR and MAP) as percentage of basal values (value at the time of injection of the reversal agent). Data are only presented for the cyclodextrin derivatives showing shorter recovery time than neostigmine/atropine (Org 26142 and Org 25969). For both reversal agents two dosages are reported (N=4 for each dose). Traces of one particular experiment are depicted in Figure 3.

 
In the experiments in which the neostigmine/atropine combination was given, two animals showed signs of abdominal discomfort, but none of the cyclodextrin derivatives produced this effect.

In the experiments in which the neostigmine/atropine combination was used, MAP and HR showed changes that were significantly higher than 10% of the baseline values.

After the use of the cyclodextrin derivatives and the neostigmine/atropine combination, no signs of residual block or recurarization were observed in any of the experiments.


   Discussion
Top
 Footnotes
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Previous work has shown that cyclodextrins are highly water soluble, biologically well tolerated, and are not biologically active.6 7 They are a group of cylindrical oligosaccharides with a hydrophobic internal cavity and a hydrophilic exterior containing 6 ({alpha}), 7 (ß), or 8 ({gamma}) dextrose molecules. Their molecular weights are 973, 1135, and 1297, respectively; but this increases when side chains are attached (Table 1). Cyclodextrins have a well-defined lipophilic internal cavity and are able to encapsulate lipophilic guest molecules such as steroids, forming a host–guest inclusion complex also known as chemical encapsulation.7 The binding of the guest molecule in the host cyclodextrin occurs because of van der Waals forces and hydrophobic and electrostatic interactions.7 The present study shows that the {gamma}-cyclodextrin derivatives, in particular Org 26142 at a dose of 0.59 and 1.18 mg kg–1 and Org 25969 at a dose of 0.5 and 1.0 mg kg–1, cause rapid and effective reversal of neuromuscular block induced by rocuronium in the anaesthetized Rhesus monkey vs spontaneous recovery. Reversal of rocuronium-induced block by Org 26142 and Org 25969 is also faster, more efficient, and without cardiovascular side effects compared with reversal of the currently used combination of neostigmine and atropine. The other synthetic cyclodextrin derivatives investigated were less effective than Org 26142 and Org 25969 in reversing rocuronium-induced neuromuscular block. Some of these compounds might be able to reverse neuromuscular block at very high doses. Only 1 or 2 experiments were performed with some of the cyclodextrin derivatives used in this study. For Org 25288, only two experiments were carried out with different doses, but even at high dose it showed relatively long recovery times. For several other derivatives only two experiments were performed at one dosage level as they were also clearly inferior to Org 26142 and Org 25969. The recovery with Org 25166 and Org 25288 at a dose of 30 mg kg–1 , as depicted in Figure 2, appears to be slower than spontaneous recovery. The inter-individual variation of spontaneous recovery among the monkeys is the likely reason that these test drugs appear slower than the average spontaneous recovery. Some appear faster in Figure 2 for the same reason, but prove to lack effect if evaluated in the paired fashion as depicted in Figure 1B.

The level of block of ~90%, at which reversal was induced, was chosen because, in clinical practice, reversal of neuromuscular block is considered to be achievable when a single twitch or the first twitch of the train-of-four stimulation has recovered spontaneously to 10% of the control twitch height.9

There were no observable side effects or significant effects on blood pressure or HR caused by any of the nine synthetic cyclodextrin derivatives (Table 3). In contrast, in the experiments with neostigmine/atropine combination, two of the animals showed abdominal discomfort (retching). None of the nine cyclodextrins produced signs of residual block or recurarization. As the main target of this study was to evaluate which compounds showed the strongest reversal effect, no additional effort was performed to detect residual block with other stimulation modes (more powerful for the detection of residual block than single twitch stimulation). In the experiments in which neostigmine/atropine combination was used, MAP and HR increased significantly (more than 10% of the baseline values) in two of the four experiments.

In vitro studies in the isolated mouse hemidiaphragm showed that the potency of the natural {alpha}-, ß-, and {gamma}-cyclodextrins to reverse neuromuscular block induced by the steroidal NMBA rocuronium correlates with their cavity sizes, resulting in a clear measurable effect for {gamma}-cyclodextrins.7 The best fit as far as cavity size is concerned can be improved by adding side chains to the molecule. The electric charge distribution in these side chains interacting with the charges in the guest molecule, results in even greater mutual affinity. This encapsulation of rocuronium by the cyclodextrin derivative Org 25969 was confirmed in the X-ray crystal structure of the complex.7

It is assumed that the reversal effect is because of reduction of the concentration of rocuronium in the solution around the hemidiaphragm preparation and subsequently in the biophase at the motor endplate.6 7 Synthetic derivatives of, in particular, {gamma}-cyclodextrin, could theoretically improve the complex formation of the cyclodextrin derivative and rocuronium. It is expected that in vivo this complex formation will promote liberation of acetylcholine receptors, and activity will reappear. As cyclodextrins have no direct or indirect actions on cholinergic transmission, it is unlikely that during reversal muscarinic side effects will occur.6 7 In support of this suggestion, no signs of muscarinic side effects were observed after cyclodextrin treatment in this study. Cyclodextrin–rocuronium complexes are highly hydrophilic and it has been demonstrated that Org 25969 is excreted fast and dose-dependently in the urine of anaesthetized guinea pigs.10

We conclude that chemical encapsulation of rocuronium represents a new concept for reversal of the effects of NMBAs.11 As a result of our findings, Org 25969 is the synthetic cyclodextrin derivative that has been chosen for further investigation of this concept.


   Footnotes
Top
 Footnotes
Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
{dagger} Declaration of interest. This study was supported by a grant from Organon. A. Bom is employed by Organon and L.H.D.J. Booij is a member of their scientific advisory board. Back


   References
Top
 Footnotes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
1 Hunter JM. New neuromuscular blocking drugs. N Engl J Med 1995; 332: 1691–9[Free Full Text]

2 Pedersen T, Viby-Mogensen J, Ringsted C. Anaesthetic practice and postoperative pulmonary complication. Acta Anaesthesiol Scand 1992; 36: 812–18[Web of Science][Medline]

3 Shorten GD. Postoperative residual curarisation: incidence, aetiology and associated morbidity. Anaesth Int Care 1993; 21: 782–9[Medline]

4 Mencke T, Echternach M, Kleinschmidt S, et al. Laryngeal morbidity and quality of tracheal intubation: a randomized controlled trial. Anesthesiology 2003; 95: 1049–56

5 Viby-Mogensen J, Jørgensen BC, Ørding H. Residual curarization in the recovery room. Anesthesiology 1979; 50: 539–41[CrossRef][Web of Science][Medline]

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

7 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: 266–270[Medline]

8 Szejtli J. Medicinal applications of cyclodextrins. Med Res Rev 1994; 14: 353–86[Medline]

9 Berg H, Roed J, Viby-Mogensen J, et al. Residual neuromuscular block is a risk factor for postoperative pulmonary complications: a prospective, randomised, and blinded study of postoperative pulmonary complications after atracurium, vecuronium and pancuronium. Acta Anaesthesiol Scand 1997; 41: 1095–103[Web of Science][Medline]

10 Epemolu O, Mayer I, Hope F, Scullion P, Desmond P. Liquid chromatography/mass spectrometric bioanalysis of a modified {gamma} cyclodextrin (Org 25969) and 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]

11 Van Egmond J, van de Pol, Booij L, et al. Neuromuscular blockade induced by steroidal NMBs can be rapidly reversed by Org 25969 in the anaesthetised monkey. Eur J Anaesthesiol 2001; 18: A100[CrossRef]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Br J AnaesthHome page
A. Srivastava and J. M. Hunter
Reversal of neuromuscular block
Br. J. Anaesth., July 1, 2009; 103(1): 115 - 129.
[Abstract] [Full Text] [PDF]

Home page
 Br J AnaesthHome page
A. L. Molina, H. D. de Boer, M. Klimek, M. Heeringa, and J. Klein
Reversal of rocuronium-induced (1.2 mg kg-1) profound neuromuscular block by accidental high dose of sugammadex (40 mg kg-1)
Br. J. Anaesth., May 1, 2007; 98(5): 624 - 627.
[Abstract] [Full Text] [PDF]

Home page
 Anesth. Analg.Home page
S. B. Groudine, R. Soto, C. Lien, D. Drover, and K. Roberts
A Randomized, Dose-Finding, Phase II Study of the Selective Relaxant Binding Drug, Sugammadex, Capable of Safely Reversing Profound Rocuronium-Induced Neuromuscular Block
Anesth. Analg., March 1, 2007; 104(3): 555 - 562.
[Abstract] [Full Text] [PDF]

Home page
 Anesth. Analg.Home page
M. Naguib
Sugammadex: Another Milestone in Clinical Neuromuscular Pharmacology
Anesth. Analg., March 1, 2007; 104(3): 575 - 581.
[Abstract] [Full Text] [PDF]

Home page
 Br J AnaesthHome page
H. D. de Boer, J. van Egmond, F. van de Pol, A. Bom, J. J. Driessen, and L. H. D. J. Booij
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
Br. J. Anaesth., November 1, 2006; 97(5): 681 - 686.
[Abstract] [Full Text] [PDF]

Home page
 Br J AnaesthHome page
J. M. Hunter and E. A. Flockton
The doughnut and the hole: a new pharmacological concept for anaesthetists.
Br. J. Anaesth., August 1, 2006; 97(2): 123 - 126.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
96/2/201    most recent
aei306v1
Right arrow E-Letters: Submit a response to the article
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (17)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by de Boer, H. D.
Right arrow Articles by Booij, L. H. D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by de Boer, H. D.
Right arrow Articles by Booij, L. H. D. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?