1 Faculté de Pharmacie, Université de Montréal C.P. 6128, Succ. Centre-Ville, Montréal (QC), Canada H3C 3J7. 2 Département dAnesthésie, Faculté de Médecine, Université de Montréal C.P. 6128, Succ. Centre-Ville, Montréal (QC), Canada H3C 3J7 france.varin@umontreal.ca
This research was supported by the Canadian Institutes of Health Research (MA-10274). A CIHR-Rx&D studentship was awarded to Julie Laurin.
Accepted for publication: July 10, 2002
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Abstract |
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Methods. The in vitro rate of degradation of each isomer of mivacurium and the in vitro rate of formation of their primary (monoesters and alcohols) and secondary (alcohols) metabolites were examined using human plasma from six healthy volunteers. The in vitro rate of degradation of the monoester metabolites was also assessed. All these determinations were made using a stereospecific high-performance liquid chromatography assay.
Results. The in vitro rate of disappearance of the two active isomers of mivacurium was very rapid, with mean values for the trans trans and cis trans isomers of 0.803 and 0.921 min1 respectively. These values are twofold faster than published in vivo data. The in vitro rate of disappearance was much slower for the cis cis isomer, with a mean value of 0.0106 min1. The cis trans isomer was converted exclusively to cis monoester and trans alcohol, while only metabolites in the trans and cis configuration were found for the trans trans and cis cis isomers respectively. Mean in vitro rates of disappearance for the trans and cis monoester were 0.00750 and 0.000633 min1 respectively.
Conclusions. The in vitro rates of hydrolysis of the active isomers of mivacurium confirm that plasma cholinesterases play a major role in their in vivo degradation, but that in vivo elimination is slowed by extravascular distribution. Mivacurium hydrolysis is stereoselective, the ester group in the trans configuration being more accessible to enzymatic attack. This stereoselective pattern, along with the relatively slow breakdown of the cis cis isomer, sheds light on the in vivo disposition of the cis alcohol metabolite.
Br J Anaesth 2002; 89: 8328
Keywords: blood, plasma; metabolism, mivacurium; metabolism, stereoselective; neuromuscular block, mivacurium
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Introduction |
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The present study was designed to elucidate the individual degradation pathways of each isomer of mivacurium and its monoester metabolites in human plasma, and represents the first attempt to propose an overall pattern for the in vivo disposition of mivacurium.
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Methods |
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Plasma collection
Blood was obtained from six healthy fasting volunteers after they had given written informed consent. The study protocol was approved by the Hotel-Dieu Hospital Ethics Committee (Montreal, Canada). Among the volunteers, there were three men and three women and their age varied between 23 and 40 yr. Approximately 100 ml of blood was obtained in a heparinized syringe. After collection, the blood was centrifuged at 1650 g for 15 min and the plasma was separated and kept on an ice-water bath until incubation.
Incubation
The three isomers of mivacurium and the mixture of monoester metabolites were incubated separately at 37°C in fresh plasma obtained from every volunteer. The in vitro study was performed within 1 h of blood collection to ensure integrity of the medium, especially with respect to enzyme activity. For each analyte, a starting solution (10 ml) was prepared by adding an appropriate amount of corresponding analyte to each preincubated individual plasma. Final concentrations of 4500 ng ml1 (4.37 µmol litre1) for trans trans mivacurium, 2500 ng ml1 (2.43 µmol litre1) for cis trans mivacurium, 500 ng ml1 (0.49 µmol litre1) for cis cis mivacurium and 3000 ng ml1 (5.15 µmol litre1) for monoester metabolites were prepared. These final concentrations were chosen to mimic the maximum plasma concentrations observed in patients after an intubating dose of the commercial mixture of the three isomers.3 According to the relative proportions of the trans and cis monoester in the mixture, the monoester metabolite starting solution corresponded to 1989 ng ml1 (3.41 µmol litre1) and 789 ng ml1 (1.35 µmol litre1) of trans and cis monoester respectively. Each starting solution was separated in nine aliquots of 1 ml, to which 20 µl of echothiophate iodide 0.04 M was added at predetermined times to stop the reaction and to prevent further degradation of the compound. The time points for stopping the reaction after addition of the analyte were 0, 1, 2, 3, 4, 5, 6, 7 and 8 min for the trans trans and cis trans mivacurium metabolites; 0, 15, 30, 45, 60, 75, 90, 105 and 120 min for the cis cis mivacurium metabolites; and 0, 60, 120, 180, 240, 300, 360 and 420 min for the monoester metabolites. After adding the inhibitor, the aliquots were frozen using dry ice and then stored at 70°C until HPLC analysis.
Sample preparation and chromatographic apparatus and conditions
Mivacurium isomers and their metabolites were determined by HPLC using a method similar to that published for the determination of cisatracurium and its metabolites in human urine.7 Bond Elut phenyl solid-phase extraction cartridges (Varian, Harbor City, CA, USA) were conditioned with acetonitrile 1 ml and 5 mM sulphuric acid 1 ml. The plasma sample (0.75 ml) and internal standard (75 µl of laudanosine 1 µg ml1) were combined in the reservoir and then aspirated through the sorbent. A vacuum of 5080 kPa was applied to the manifold of the Vac-Elut chamber (Analytichem International, Harbor City, CA, USA) throughout the extraction procedure. The cartridges were washed sequentially with 5 mM sulphuric acid 0.75 ml and a mixture of methanol:water (50:50, 0.75 ml). Analytes were eluted with 80 mM sodium sulphate 2x300 µl in 5 mM sulphuric acid:acetonitrile (40:60). The eluents were then reduced to half of their volume by evaporation using a Speed-Vac concentrator (Model SC210A; Savant Instruments, Farmingdale, NY, USA). Aliquots were injected directly into the HPLC system according to the following procedure.
The chromatographic system consisted of a Constametric 4100 pump (LDC Analytical, Riviera Beach, FL, USA) which was programmed to deliver 14 mM sodium sulphate in 0.5 mM sulphuric acid:acetonitrile (40:60) for 5 min followed by 70 mM sodium sulphate in 0.5 mM sulphuric acid:acetonitrile (40:60) for 6 min, returning to the initial condition to re-equilibrate for 3 min. The flow rate was 2.0 ml min1 and the mobile phase was not allowed to recirculate. Samples were injected using an auto-injector (SIL-9A; Shimadzu, Kyoto, Japan). Separation of mivacurium isomers and their metabolites was performed on a 5 µm Spherisorb strong cation exchanger column (150x4.6 mm internal diameter; Phenomenex, Torrance, CA, USA). Peaks were detected with a Hewlett Packard 1046 A fluorescence detector (Hewlett Packard, Waldbroom, Germany) at excitation and emission wavelengths of 202 and 320 nm respectively. A Shimadzu C-R3A integrator was used.
The method proved to be sensitive, with a lower limit of quantitation of 4.88 ng ml1 (0.0047 µmol litre1) for each isomer of mivacurium and for the quaternary alcohol metabolites, and 10.36 ng ml1 (0.0232 µmol litre1) and 4.11 ng ml1 (0.0071 µmol litre1) for the trans and cis monoesters respectively. The assay proved to be linear up to 5000 ng ml1 (4.86 µmol litre1) for the isomers of mivacurium, 5000 ng ml1 (11.20 µmol litre1) for the quaternary alcohol metabolites, 2652 ng ml1 (4.55 µmol litre1) for the trans monoester and 1052 ng ml1(1.81 µmol litre1) for the cis monoester. The coefficient of determination (r2) was always higher than 0.9930 for each analyte. The method proved to be reproducible for each analyte, with a coefficient of variation always less than 20% over the linear range.
Data analysis
The rate of in vitro disappearance (kin vitro) of each analyte from human plasma was determined by fitting the plasma data to a non-compartmental model with bolus input using a non-linear least-squares computer program (WinNonlin® software; Pharsight Scientific Consulting, Palo Alto, CA, USA) that applied linear regression to all data points. The in vitro elimination half-life (T in vitro) was calculated using 0.693 kin vitro1. The mass balance (number of moles disappeared vs number of moles formed) was examined in all subjects.
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Results |
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After incubation in human plasma, the cis cis isomer of mivacurium disappeared at a much slower rate than that of the other two isomers. After 120 min of incubation, the hydrolytic reaction was incomplete, with 30% of the initial cis cis mivacurium plasma concentration remaining. Only metabolites in the cis configuration resulted from hydrolysis of this isomer. Hence, this isomer was equally (on a molar basis) converted to cis monoester and cis alcohol metabolites (Fig. 2C). The resulting mean kin vitro value was 0.0106 min1 (Table 1).
After a 7-h incubation, the trans monoester metabolite was completely converted to trans alcohol metabolite, whereas only 20% of the cis monoester metabolite was converted to cis alcohol metabolite (Fig. 2D). The resulting mean kin vitro value was 0.00750 min1 for the trans monoester metabolite and 0.000633 min1 for the cis monoester metabolite (Table 1). The mean terminal elimination half-life (T in vivo) obtained in eight healthy patients undergoing elective surgery2 is also presented in Table 1 for each isomer of mivacurium and for the monoester metabolites.
As demonstrated in Figure 2, the mass balance (number of moles disappeared vs number of moles formed) was good, the values varying from 86 to 101%. The slight variation was probably the result of the error inherent in the analytical method (±15%).
In view of these results, a pattern is proposed for the stereoselective degradation of mivacurium (Fig. 3).
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Discussion |
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There is no clear explanation for the flattening of the curve for cis trans degradation in plasma. However, we are sure that this finding is not an artefact. There are two good reasons why analytical sensitivity can be ruled out. First, the levels are well above the lower limit of quantitation (LLOQ; at least four times the LLOQ). Secondly, the flattening of the cis trans concentration curve was consistent in all patients but never observed for the trans trans isomer. After 4 min, the measured plasma concentration of the cis trans isomer averaged 50 ng ml1 (0.0486 µmol litre1), which represents approximately 2% of the initial concentration. One possible explanation for the flattening of the curve would be that the rate of ester hydrolysis is different for the cis and trans positions. Hydrolysis at the trans position would be complete at 4 min and the flattening of the curve could be attributed to slower hydrolysis at the cis position.
After the hydrolysis of mivacurium isomers into monoester and alcohol metabolites, the monoester metabolites are in turn degraded into alcohol metabolites. However, as the kin vitro values for the monoesters are at least 10-fold slower than those for the isomers, the relative importance of this secondary metabolic pathway to the formation of the alcohol metabolite is thought to be negligible.
Our results also show that the cis monoester metabolite does not undergo any significant in vitro hydrolysis in comparison with the trans monoester metabolite. This finding supports the results obtained for the isomers, i.e. that the ester group in the trans configuration is more susceptible to attack by the enzyme. The presence of greater steric hindrance when the ester group is in the cis configuration may explain the greater resistance to enzymatic degradation.
For drugs undergoing extensive hydrolysis in human plasma (non-organ-based elimination), the in vitro rate of degradation (kin vitro) is often used as a substitute in pharmacokinetic models assuming peripheral elimina tion.810 In addition, direct comparison of in vitro and in vivo elimination half-lives provides information about the relative contribution of organ-based elimination (e.g. by the kidneys and liver) to the overall body elimination. Our results indicate that, for the two most active isomers (trans trans and cis trans), the T in vitro is 2-fold faster than the T
in vivo, confirming that hydrolysis by plasma cholinesterases plays a major role in the degradation of these two isomers. The possibility that this apparent discrepancy resulted from the fact that two different populations of subjects were studied cannot be ruled out. However, Wiesner and colleagues6 recently reported in vitro half-lives similar to those obtained in this study. Another explanation for the slower in vivo elimination could be that extravascular distribution of these two isomers delays their overall elimination from the body. The presence of plasma cholinesterases in the cerebrospinal fluid has been demonstrated in animals,11 although their activity may differ markedly between tissues and plasma. Despite the fact that the two active isomers of mivacurium (trans trans and cis trans) are eliminated rapidly by plasma cholinesterases, a contribution of the peripheral compartment to their elimination cannot be excluded. Recent data have confirmed the presence of an arterialvenous gradient of approximately 30% across muscle tissue in anaesthetized patients.12
In contrast, the T in vivo for the cis cis isomer is approximately 2-fold faster than the T
in vitro. This finding is an indication that the overall elimination of this isomer is not solely the result of its hydrolysis by plasma cholinesterases. Indeed, the contribution of renal function to the overall elimination of this isomer has been demonstrated by Head-Rapson and colleagues.13 Moreover, Lien and colleagues5 have shown that the clearance of this isomer is not related to plasma cholinesterase activity.
For the monoester metabolites, the in vivo and in vitro values were similar for the trans monoester, indicating a minor contribution of organ-based elimination to total body disposition. Conversely, the in vitro half-life of the cis monoester was considerably slower than that observed in vivo. However, this value should be interpreted cautiously as the duration of incubation did not allow accurate determination of the half-life. Nonetheless, our findings suggest that the contribution of enzymatic hydrolysis to the overall in vivo elimination of the cis monoester metabolite is negligible.
When examining the in vivo formation and elimination of the alcohol and monoester metabolites after an i.v. bolus of the commercial mixture of mivacurium in human patients, Lacroix and colleagues3 noted that the cis alcohol metabolite was detected minimally and transiently. An initial mixing peak was observed at approximately 30 s for both the cis and the trans alcohol metabolite. This was attributed to the alcohols (known degradation or synthesis by-products of the isomers) that are already contained in the injectable solution. Of importance, the levels of the cis alcohol were not quantifiable (below the LLOQ) after only 8 min, while levels almost 10-fold higher allowed characterization of the kinetics of the trans alcohol for up to 250 min. As the later portion of the plasma concentrationtime curve was more compatible with in vivo formation of the metabolites, no explanation can be provided for this finding. Our in vitro results now shed some light on the in vivo disposition of the cis alcohol metabolite. First, less than 2% of the cis alcohol is formed after in vitro degradation of the cis trans isomer. Secondly, the in vitro rate of formation of cis alcohol from the cis monoester is questionable. Finally, as the cis cis isomer represents only 6% of the injectable mixture of mivacurium, its contribution to the in vivo formation of the cis alcohol is certainly minimal.
In conclusion, the in vitro rates of hydrolysis of the active isomers confirm that plasma cholinesterases play a major role in their in vivo degradation, but also suggest that their in vivo elimination is slowed by extravascular distribution. Mivacurium hydrolysis is stereoselective, the trans configuration being more amenable to ester hydrolysis than the cis configuration. This stereoselective pattern, along with the relatively slow breakdown of the cis cis isomer, explains the in vivo disposition of the cis alcohol metabolite. Hydrolysis is facilitated by the presence of another esterified moiety at the opposite end of the molecule, the hydrolysis of the cis cis and trans trans isomers being considerably faster than that of their respective monoesters. From the results of this in vitro study we propose an overall stereoselective pattern for the degradation of mivacurium that allows the contribution of organ-based elimination to be dissociated from that of non-organ-based elimination.
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Acknowledgements |
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References |
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