Does ester hydrolysis change the in vitro degradation rate of cisatracurium and atracurium?

M. Weindlmayr-Goettel*,1, H. Gilly1,2 and H. G. Kress1,2

1Department (B) of Anaesthesiology and General Intensive Care, University of Vienna, Waehringer-Guertel 18–20, A-1090 Vienna and 2L. Boltzmann-Institute of Experimental Anaesthesiology and Research in Intensive Care Medicine, Vienna, Austria*Corresponding author

Accepted for publication: December 13, 2001


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Background. We assessed the role of ester hydrolysis as an additional degradation mechanism to Hofmann elimination in the breakdown of cisatracurium and atracurium.

Methods. Cisatracurium and atracurium were incubated in phosphate buffer (pH 7.4, 37°C) with and without the addition of carboxylesterase. Control measurements with an added esterase inhibitor were performed separately. Cisatracurium/atracurium and their degradation products, laudanosine and monoquaternary acid, were analysed using high-pressure liquid chromatography.

Results. Degradation of cisatracurium and atracurium proceeded exponentially, and after addition of carboxylesterase, no significant differences in the degradation rates were found. Neither an increase in carboxylesterase activity nor the addition of esterase inhibitor showed any effect. However, areas under the peaks of the chromatogram representing monoquaternary acid increased during incubation with esterase.

Conclusion. The rate-limiting step in the degradation of cisatracurium/atracurium is Hofmann elimination. Ester hydrolysis is involved in the second degradation step that forms monoquaternary acid, but its contribution to the total elimination rate is negligible.

Br J Anaesth 2002; 88: 555–62

Keywords: neuromuscular block, cisatracurium; neuromuscular block, atracurium; metabolism, Hofmann elimination; metabolism, ester hydrolysis


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Atracurium besylate and its 1R-cis, 1R'-cis isomer, cisatracurium, are non-depolarizing neuromuscular blocking drugs of intermediate duration of action. Both drugs undergo Hofmann elimination, a process dependent on pH and temperature. This unique pharmacological property provides an organ-independent degradation pathway. Atracurium and, in particular, cisatracurium with its 3- to 5-fold higher neuromuscular blocking potency but lower histamine releasing potential, are widely used clinically.1

The degradation of cisatracurium and atracurium via Hofmann elimination has been thoroughly investigated. Two comprehensive reviews with special emphasis on the pharmacokinetics and the main degradation pathways of cisatracurium and atracurium were published in 1999.1 2 In vitro and in vivo studies suggested that Hofmann elimination produces 30–70% of the total drug elimination in man and mammals.1

It has been shown3 that the rate of Hofmann elimination depends on the composition and ion activity/concentration of the incubation medium, which at least in part, may explain the rather large differences in elimination rates between plasma and various buffer solutions, even at identical pH and temperature.4 5

The role of ester hydrolysis as a second degradation pathway is controversial. In humans, some authors consider ester hydrolysis to be a major degradation pathway.4 6 Other authors assume the contribution of ester hydrolysis to the metabolism of atracurium to be small but nevertheless relevant, but they cannot find any significant role for this pathway in the metabolism of cisatracurium.710

The exact nature and type of esterases involved in ester hydrolysis of cisatracurium and atracurium is unknown, but most likely, the non-specific aliesterase (carboxylesterase) facilitates the hydrolysis.4 No data are yet available on the potential effect of carboxylesterase on the degradation rates of these two neuromuscular blocking drugs. The aim of the present study was, therefore, to determine whether and how the in vitro degradation rates of cisatracurium and atracurium are influenced by ester hydrolysis.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Compounds and reagents
Cisatracurium besylate and atracurium besylate were supplied by Glaxo-Wellcome Pharma, Vienna, Austria. Acetonitrile (Promochem, Wesel, Germany) was high-pressure liquid chromatography (HPLC) grade. Carboxylesterase (CE) from porcine liver (EC 3.1.1.1) and bis-(p-nitrophenyl) phosphate (BPNP) were obtained from Sigma, St Louis, MO, USA. All other reagents were obtained from E. Merck (Darmstadt, Germany) and were of analytical grade. Buffers for calibration of the pH-meter were obtained from Radiometer (Copenhagen, Denmark).

Incubations
The commercially available solution of cisatracurium (2.15 mmol litre–1) was diluted (1:2) with 5 mmol litre–1 H2SO4. A volume of 20 µl of the diluted solution was then added to 10 ml of the incubation buffer, mixed well, and incubated at 37°C. Similarly, the commercially available solution of atracurium (8 mmol litre–1) was diluted (1:10) with H2SO4 5 mmol litre–1, and 25 µl of the diluted solution were added to 10 ml of the incubation buffer.

The buffer was a 50 mmol litre–1 phosphate buffer (Na2HPO4+NaH2PO4) with NaCl 136.8 mmol litre–1 and KCl 2.7 mmol litre–1.8 The pH was adjusted to 7.4 at 37°C with either the acidic or the basic component of the buffer as necessary. At the end of the incubation period, the pH was rechecked and the incubation experiment rejected if the measured pH value differed from 7.40 by more than ±0.05.

Incubations of cisatracurium were performed in phosphate buffer (experimental series C1, cisatr), and in phosphate buffer after addition of 20 (C2, cisatr+20CE) and 100 (C3, cisatr+100CE) unit litre–1 carboxylesterase (esterase EC 3.1.1.1). In order to check whether protein per se influences the degradation of cisatracurium, separate measurements with the same concentration of enzyme were performed in the presence of the esterase inhibitor, BPNP. The series with the combined addition of carboxylesterase and esterase inhibitor BPNP (CE 20 unit litre–1 and BPNP 10 µmol litre–1, and CE 100 unit litre–1 and BPNP 50 µmol litre–1, respectively) are denoted as C4 (cisatr+20CE+10BPNP) and C5 (cisatr+100CE+50BPNP). Incubations with buffers containing only cisatracurium and esterase inhibitor BPNP (10 µmol litre–1) are denoted C6 (cisatr+10BPNP).

Corresponding incubations of atracurium in phosphate buffer are denoted as A1 (atr) and the incubation series in phosphate buffer after addition of CE 20 and 100 unit litre–1 are denoted as A2 (atr+20CE) and A3 (atr+100CE), respectively. The series with the combined addition of carboxylesterase and esterase inhibitor BPNP (CE 20 unit litre–1 and BPNP 10 µmol litre–1, and CE 100 unit litre–1 and BPNP 50 µmol litre–1, respectively) are denoted as A4 (atr+20CE+10BPNP) and A5 (atr+100CE+50BPNP). Incubations with buffers containing only atracurium and esterase inhibitor BPNP (BPNP 10 µmol litre–1) are denoted A6 (atr+10BPNP).

Before the addition of cisatracurium or atracurium, the incubation solutions were pre-incubated with the supplements for 30 min at 37°C. After defined time intervals (0, 10, 20, 30, 60, 90, 120, 180, and 240 min), 0.5 ml aliquots were withdrawn, acidified with 10 µl of H2SO4 1 mol litre–1 and frozen (–20°C) until measurement. The concentrations of cisatracurium, atracurium, and laudanosine were measured by HPLC using an assay developed. Five independent experiments were performed for each incubating condition.

HPLC analysis
HPLC was performed using the model 126 HPLC pump (Beckman Instruments, San Ramon, CA, USA) with an autosampler model AS-950 (Jasco, Tokyo, Japan).

Separation of atracurium/cisatracurium and laudanosine was carried out on a 5 µ Hypersil C18 column 125x4 mm. The mobile phase concentration was: 30% acetonitrile, 60% Na2SO4 24 mmol litre–1 in H2SO4 5 mmol litre–1, and 10% methanol. The flow rate was 0.6 ml min–1. The fluorometric detector FP-920 (Jasco) was set to 280 nm for excitation and to 320 nm for emission. The lower limit of detection was atracurium or cisatracurium 5 ng ml–1 (4 nmol litre–1) and laudanosine 2 ng ml–1 (5.6 nmol litre–1). The atracurium isomers (cis–cis, cis–trans, and trans–trans) were identified and separately determined.

Monoquaternary acid, the atracurium metabolite of ester hydrolysis, was not available for us to prepare standard stock solutions for calibration purposes. It could not, therefore, be quantitatively determined. To estimate monoquaternary acid formation, the peak area (as obtained from the HPLC chromatogram) was presented as an arbitrary unit. For the identification of these peaks in the chromatogram, the HPLC chromatogram described by van den Brom12 was used. As the monoquaternary acid concentration should progressively increase during hydrolysis at low pH (acid hydrolysis), cisatracurium was incubated in HCl solution (pH 3). Increasing peak areas found by subsequent HPLC analysis at 2–8 h intervals during an incubation period of 24 h, confirmed that we were semi-quantitatively measuring monoquaternary acid concentration.

Data analysis
The kinetic model used for data analysis has been described previously.3 The degradation pathway of the parent compounds (atracurium or cisatracurium) via Hofmann elimination is depicted in Figure 1A. The model includes sequential formation of two laudanosine molecules from one atracurium molecule, with only one molecule of laudanosine being formed in the first step of the degradation of atracurium.





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Fig 1 Proposed pathway of cisatracurium degradation. (A) Hofmann elimination as the only degradation mechanism. (B) Ester hydrolysis as the proposed degradation pathway for the parent drug. (C) Hofmann elimination in the first elimination step, ester hydrolysis for break down of quaternary monoacrylate in the second step. The degradation of atracurium follows the same pathway.

 
A single rate constant (k1) defined both the degradation rate of cisatracurium or atracurium and the rate of formation of the first molecule of laudanosine. The second molecule of laudanosine is formed from quaternary monoacrylate, which undergoes further elimination. The corresponding rate constant was designated k2.

The model was fitted to the time-dependent, measured concentrations of atracurium or cisatracurium and to the sum of both laudanosine molecules in each experiment; the respective half-lives were calculated as T1/2=ln(2)/k. As a parameter for the goodness of fit, the coefficient of determination was used. For the calculations, Scientist software (MicroMath, Salt Lake City, UT, USA) was used.

In our mathematical model, ester hydrolysis, as shown in Figure 1B, was not taken into account by assigning a particular elimination rate constant, as it is not possible to differentiate the individual contribution of Hofmann elimination and ester hydrolysis to the degradation process based on the measurement of the concentration of cisatracurium or atracurium and laudanosine. However, in the presence of carboxylesterase, the degradation rate must increase if ester hydrolysis occurs, and detection of the monoquaternary acid metabolite can be considered to be caused by enzymatic ester hydrolysis only.

Results are reported as mean (SD). Statistical evaluation was performed using the t-test (df=(10–6)=4). Level of significance was assumed to be 0.05.


    Results
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The initial concentration of cisatracurium in the incubation solution was 1.80 (0.05) µmol litre–1, that of atracurium was 1.47 (0.07) µmol litre–1, and that of laudanosine was very low (0.056 (0.064) µmol litre–1 in cisatracurium solutions and 0.069 (0.033) µmol litre–1 in atracurium solutions). The cis–cis isomeric group constituted 66.6%, cis–trans 28.6%, and trans–trans isomers 4.8% of the atracurium mixture.

Results obtained by incubating cisatracurium and atracurium with and without additives at pH 7.4 and 37°C are given in Table 1. The degradation of cisatracurium and atracurium proceeded exponentially in all incubation solutions. The degradation rate was always faster for cisatracurium than for atracurium. The corresponding rate constants (k1), and the calculated half-lives in the plain solutions and after addition of carboxylesterase, carboxylesterase and BPNP, or BPNP alone are also given in Table 1. The proposed model for the degradation of cisatracurium or atracurium and the formation of laudanosine fitted the data well under all experimental conditions.


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Table 1 Rate constants (mean (SD)) n=5, characterizing the degradation of cisatracurium (cisatr) or atracurium (atr) and the formation of the first molecule of laudanosine (k1) in plain phosphate buffer and after addition of carboxylesterase (CE), carboxylesterase and esterase inhibitor BPNP, or BPNP. k2, rate constant for the formation of the second molecule of laudanosine. T1/2, half-lives calculated for the rate constants k1. C1 and A1, cisatr/atr in plain phosphate buffer. C2/C3 and A2/A3, cisatr/atr after addition of carboxylesterase 20 or 100 unit litre–1. C4/C5 and A4/A5, combined addition of carboxylesterase 20 unit litre–1 and BPNP 10 µmol litre–1 or carboxylesterase 100 unit litre–1 and BPNP 50 µmol litre–1. C6 and A6, cisatr/atr after addition of BPNP 10 µmol litre–1. P values: k1 was compared in group C1 or A1 (cisatr/atr in phosphate buffer) with groups C2–C6 and A2–A6, respectively. P values for k1: C2 vs C3, 0.135; C4 vs C5, 0.635; A2 vs A3, 0.213; A4 vs A5, 0.180
 
The half-lives (T1/2) of cisatracurium/atracurium in plain phosphate buffer were 33.3 and 39.4 min cisatracurium and atracurium, respectively. After addition of carboxylesterase 20 unit litre–1, no differences in the degradation rate could be found (T1/2: cisatr+20CE, 32.7 min; atr+20CE, 43.1 min). An increase in carboxylesterase activity from 20 to 100 unit litre–1 did also not affect the degradation rates (T1/2: cisatr+100CE, 37.5 min; atr+100CE, 37.9 min), or the concentration of monoquaternary acid. The addition of the esterase inhibitor BPNP did not change the half-lives (T1/2: cisatr+20CE+10BPNP, 32.4 min; cisatr+100CE+50BPNP, 35.7 min; atr+20CE+10BPNP, 38.3 min; atr+100CE+ 50BPNP, 45.6 min) nor the areas under the monoquaternary acid peaks. Control measurements with BPNP showed only marginal differences in degradation (T1/2: cisatr+10BPNP, 32.4 min; atr+10BPNP, 36.7 min).

As a representative example for the time course of degradation, the concentrations of cisatracurium and laudanosine in phosphate buffer without and with the addition of carboxylesterase 20 unit litre–1 are illustrated in Figure 2A. No differences in the respective degradation rates were apparent.



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Fig 2 Time course of cisatracurium degradation and formation of laudanosine (A), and formation of monoquaternary acid (B). (A) Continuous and dashed lines show the decay of cisatracurium and formation of laudanosine as calculated from the model. (B) The dashed and dotted lines show time course of monoquaternary acid concentration and were fitted using a spline function. Note that concentration of monoquaternary acid is given in arbitrary units. Data shown represent the means of the corresponding concentration of each group.

 
Those peak areas of the chromatogram that were assumed to represent the monoquaternary acid, increased rapidly during incubation in the presence of esterase. The time course, indicative of the change in concentration of this metabolite, is shown in Figure 2B. After an initial increase during the first 60 min of incubation, the concentration of the metabolite slowly decreased. In the absence of carboxylesterase, no change in monoquaternary acid concentration could be detected.


    Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
In vitro investigations in various buffers and plasma4 6 8 9 suggest that cisatracurium and atracurium undergo temperature and pH-dependent Hofmann elimination forming laudanosine and a quaternary monoacrylate (Fig. 1A). In the second degradation step, another molecule of laudanosine and a molecule of diacrylic acid ester are produced from quaternary monoacrylate. The degradation rates determined in vitro and in vivo were reported to differ significantly. Tsui and co-workers8 found a half-life for a mixture of the cis–cis isomers in humans of 20.5 min; but using atracurium, Stiller and co-workers4 found a half-life of 58 min for atracurium in Sörensen buffer, compared with 21 min in plasma.

The role of ester hydrolysis and its relevance to the in vivo elimination process have not been extensively studied. Welch and co-workers9 reported the degradation rate of cisatracurium in Sörensen buffer to be similar in the presence or absence of a non-specific plasma esterase. Nevertheless, the different elimination rates found in in vivo and in vitro experiments have been interpreted by several authors to be the result of ester hydrolysis.4 7 11 Esterase can promote the ester hydrolysis of the parent molecule to produce monoquaternary alcohol and monoquaternary acid (Fig. 1B). Monoquaternary alcohol then undergoes further Hofmann degradation to form laudanosine. But, if the parent molecule (cisatracurium/atracurium) is degraded primarily by Hofmann elimination, then the esterase would break down the resulting quaternary monoacrylate (Fig. 1C), forming monoquaternary alcohol, which is an unstable molecule and would be further degraded by ester hydrolysis to the more stable monoquaternary acid.9

In our mathematical model for the degradation of cisatracurium/atracurium and the generation of laudanosine, the first rate constant k1 was calculated from the measured concentrations of the parent drug. Accordingly, the rate constant k1 is a reliable estimate for the generation of the first molecule of laudanosine. Analysing the role of ester hydrolysis in the context of the suggested degradation pathways (Fig. 1B and C), by the addition of carboxylesterase, the rate constant k1 should increase if ester hydrolysis splits cisatracurium or atracurium to a significant degree (Fig. 1B). If ester hydrolysis occurs only in the second step of degradation (splitting of quaternary monoacrylate, Fig. 1C), k1 would not change, whereas the formation rate of the second laudanosine molecule, k2, should.

As only the total concentrations of laudanosine can be measured, the calculated rate k2 of the formation of the second laudanosine molecule must be considered an approximation. We did not therefore use k2 as a quantitative measure in characterizing the role of ester hydrolysis. However, this degradation pathway does evidently exist because the monoquaternary acid concentration increased in the presence of carboxylesterase (Fig. 2B).

Our experiments did not yield a significant change in the rate constant for the elimination of cisatracurium/atracurium (Table 1) or in the concentrations of monoquaternary acid, with either carboxylesterase 20 or 100 unit litre–1 added to the incubation solution. This result suggests a negligible contribution of ester hydrolysis to the degradation of cisatracurium/atracurium and evidently confirms the degradation pathway schematically shown in Figure 1C.

The peaks representing monoquaternary alcohol did not change during incubation of cisatracurium/atracurium (data not shown). These results correspond with the assumption of Welch and co-workers9 that monoquaternary alcohol converted rapidly to the more stable monoquaternary acid and laudanosine, and also that a certain amount of ester hydrolysis must be involved in the second stage of atracurium degradation in human plasma.9

The degradation rate of atracurium proved to be somewhat slower than that of cisatracurium (Table 1). Atracurium is a mixture of 10 stereoisomers differing in their respective degradation rates. Tsui and co-workers8 showed that in vitro degradation of the cis–cis isomers (representing about 67% of the atracurium isomers) occurred somewhat faster (T1/2=57 min) than that of the cis–trans (T1/2=60 min) and trans–trans isomers (T1/2=66 min). Therefore, atracurium (containing about 14% cisatracurium) is to be expected to degrade more slowly than cisatracurium in vitro.

As reported previously,3 the in vitro degradation rate of atracurium via Hofmann elimination strongly depends on the ion composition and ion concentrations of the incubation buffers used, and this fact alone may well explain the observed differences between the degradation rates of atracurium in vivo and in vitro. Our present results strongly support this suggestion, as they clearly show that ester hydrolysis does not play a major role in the degradation of atracurium.

Species-specific differences in the relative roles and activities of esterases are known to exist.11 In the rat, Welch and co-workers9 reported that ester hydrolysis played a major role in the decomposition pathway of atracurium (Fig. 1B). However, in respect of the enzymatic breakdown of cisatracurium and atracurium, it should be emphasized that the results obtained from animal experiments must not be extrapolated to humans.

In conclusion, our data suggest a measurable, but minimal role of ester hydrolysis in the degradation process of cisatracurium and atracurium. Its clinical relevance has to be discussed with caution, as the potential involvement and yet unknown contribution of nucleophilic substances like acetylcysteine13 in the degradation of neuromuscular blockers have yet to be investigated.


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
1 Kisor DF, Schmith VD. Clinical pharmacokinetics of cisatracurium besilate. Clin Pharmacokinet 1999; 36: 27–40[ISI][Medline]

2 Atherton DPL, Hunter JM. Clinical pharmacokinetics of the newer neuromuscular blocking drugs. Clin Pharmacokinet 1999; 36: 169–89[ISI][Medline]

3 Weindlmayr-Goettel M, Kress HG, Hammerschmidt F, Nigrovic V. In-vitro degradation of atracurium and cisatracurium depends on the composition of the incubating solutions. Br J Anaesth 1998; 81: 409–14[ISI][Medline]

4 Stiller RL, Cook DR, Chakravorti S. In-vitro degradation of atracurium in human plasma. Br J Anaesth 1985; 57: 1085–8[Abstract]

5 Fisher DM, Canfell PC, Fahey MR, et al. Elimination of atracurium in humans: contribution of Hofmann elimination and ester hydrolysis versus organ-based elimination. Anesthesiology 1986; 65: 6–12[ISI][Medline]

6 Merrett RA, Thompson CW, Webb FW. In-vitro degradation of atracurium in human plasma. Br J Anaesth 1983; 55: 61–6[ISI][Medline]

7 Neill EAM, Chapple DJ. Metabolic studies in the cat with atracurium: a neuromuscular blocking agent designed for non-enzymic inactivation at physiological pH. Xenobiotica 1982; 12: 203–10[ISI][Medline]

8 Tsui D, Graham GG, Torda TA. The pharmacokinetics of atracurium isomers in-vitro and in humans. Anesthesiology 1987; 67: 722–8[ISI][Medline]

9 Welch RM, Brown A, Ravitch J, Dahl R. The in-vitro degradation of cisatracurium, the R, cis-R’-isomer of atracurium, in human and rat plasma. Clin Pharmacol Ther 1995; 58: 132–42[ISI][Medline]

10 Kisor DF, Schmith VD, Wargin WA, Lien CA, Ornstein E, Cook DR. Importance of the organ-independent elimination of cisatracurium. Anesth Analg 1996; 83: 1065–71[Abstract]

11 Nigrovic V, Auen M, Wajskol A. Enzymatic hydrolysis of atracurium in-vivo. Anesthesiology 1985; 62; 606–9[ISI][Medline]

12 van den Brom RHG. Verslag van een studie naar de klinische farmacokinetiek van atracurium besylaat en enkele metabolieten in de mens. Thesis, Groningen, 1985

13 Amann A, Rieder J, Fleischer M et al. The influence of atracurium, cisatracurium, and mivacurium on the proliferation of two human cell lines in vitro. Anesth Analg 2001; 93: 690–6[Abstract/Free Full Text]





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