Journal of Pharmacology, 222 (1992) 153-156 0 1992ElsevierSciencePublishersB.V. All rights reserved0014-2999/92/$05.00
European
153
EJP52723
Antagonism of vecuronium by one of its metabolites in vitro Karin S. Khuenl-Brady, Clinics
for Anaesthesia
and General
Intensive
Peter Mair and Johann Koller Care Medicine,
University
of Innsbruck,
Innsbruck,
Austria
Received23 March 1992,revisedMS received17 July 1992,accepted4 August 1992
The neuromuscular-blocking agent vecuronium bromide undergoes hydrolysis to three pharmacologically active metabolites (3-desacety1,17-desacetyl and 3,17-desacetyl vecuronium) which might modify the neuromuscular-blocking action of their parent compound. In order to elucidate the possible role of the interaction between vecuronium and its metabolites in the complications reported after long-term use of vecuronium in intensive care unit (ICU) patients, the relative potency of vecuronium, 3-desacetyl and 3,17-desacetyl vecuronium was determined in the rat hemidiaphragm in vitro and the mode of interaction of the above-mentioned compounds investigated. Dose-response relationships were established for each substance alone and for combinations of vecuronium with its metabolites. The relative potency at the EDmax,, levels (o/o maximal effect) were in the order of 1: 1.2: 27 for vecuronium, the 3-desacetyl derivative and the 3,17-desacetyl derivative, respectively. The mode of interaction characterized by isobolographic and algebraic (functional) analysis showed vecuronium and 3-desacetyl vecuronium to interact in an additive fashion while the combined effect of the parent compound and its 3,17-desacetyl derivative was less than additive, indicating antagonism. Neuromuscular relaxants; Vecuronium; 3-Desacetyl vecuronium; 3,17-Desacetyl vecuronium; Metabolites (antagonism)
1. Introduction Vecuronium bromide undergoes hydrolysis to three different pharmacologically active compounds. These metabolites are 3-desacetyl (Org 72681, 17-desacetyl (Org NC58) and 3,17-desacetyl vecuronium (Org 7402) (Savage et al., 1980). Recently, long-term application of vecuronium to intensive care unit (ICU) patients has been reported to cause the development of vecuronium resistance (Coursin et al., 1989) as well as an apparent increased sensitivity to the compound, resulting in prolonged paralysis (Segredo et al., 1990). Since all three metabolites are pharmacologically active (Durant et al., 1979; Savage et al., 19801, they might contribute to the neuromuscular-blocking effects of vecuronium. However, how they affect the neuromuscular blockade induced by their parent compound depends on the mode of their mutual interactions, which could be addition, potentiation, or antagonism. In order to elucidate the possible role of the interaction between vecuronium and its metabolites in the complications reported after long-term use of vecuronium in ICU patients, we determined the relative potency of vecuronium, 3-des-
Correspondenceto: KS. Khuenl-Brady,Clinicsfor Anaesthesiaand General Intensive Care Medicine, Anichstrasse35, A-6020 Innsbruck, Austria. Tel. 43-512-5042400,fax 43-512-5042440.
acetyl and 3,17-desacetyl vecuronium in vitro and investigated the mode of interaction between the parent compound and these two metabolites in the rat hemidiaphragm. We studied only the first and terminal breakdown products, and did not use the 17-desacetyl derivative as it is an instable intermediary product in the formation of the terminal metabolite 3,17-desacetyl vecuronium (Marshall et al., 1983).
2. Materials
and methods
After institutional approval, male Sprague-Dawley rats of 200-300 g bodyweight were anaesthetized with thiopentone and decapitated. The phrenic nerves and hemidiaphragms were dissected and mounted in an organ bath containing Krebs-Ringer solution (pH 7.357.451, aerated with 5% CO, in oxygen and maintained at 37°C. The hemidiaphragms were stimulated indirectly via a Grass SS88 stimulator with supramaximal square-wave impulses of 0.2 ms duration at a frequency of 0.1 Hz. The resulting force of contraction was quantified with a Grass FT-03 transducer and continuously recorded on a Graphtec printer after applying a preload that resulted in maximal contractions (2-5 g). Each preparation was allowed to stabilize for at least 20 min until a stable twitch response was achieved. After each
154
measurement, diaphragms were washed at least three times and were allowed to stabilize again. A train-offour stimulation (2 Hz for 2 s) was given after the last washout to show the unchanged experimental conditions and complete neuromuscular recovery. 2.1. Experimental design
In the first part of the study the dose-response relationship of vecuronium, 3-desacetyl and 3,17-desacetyl vecuronium was determined in seven diaphragms with each substance in cumulative fashion. Increments of 50 nmol of vecuronium or 3-desacetyl vecuronium .or of 100 nmol of 3,17-desacetyl vecuronium were added to the bath, the next dose being given only after maximum blockade had developed. Dose-response curves for each substance were constructed by log-logit transformation, and the EDmaxz, EDmax,, and EDmax,s (% maximal response) were derived by linear regression analysis of the logit of the maximum blockade plotted against the logarithm of the dose. In the second part of the study the interaction between vecuronium and its metabolites was studied by determining the dose-response relationship of the 3desacetyl and 3,17-desacetyl derivatives in the presence of partial blockade induced by 2.2 pmol vecuronium. These experiments were carried out in the same way as those in the first part of the study (seven experiments with each combination). The EDmax=, EDmax,, and EDmax,, values were derived from the log-logit doseresponse curves for the metabolites in the presence of vecuronium. The data were analysed by isobolographic (Loewe, 1955) and algebraic (functional) analysis (Berenbaum, 1977). In the isobolographic analysis, the EDmax,, values of the interacting agents are plotted on the dose co-ordinates of the isobologram and connected with a straight line - the isobol or “additive line”. If the EDmax,, of a combination of two compounds is located on the isobol, simple additive effects can be assumed. A deviation of the combined EDmax,, point to the left indicates more than additive effects (potentiation), while a deviation to the right signifies less than additive effects (antagonism). In the case of a simple additive interaction, the same principle can be
TABLE
1
Dose-response relationship of vecuronium and its metabolites in the rat diaphragm.
Vecuronium 3-De-sacetyl vecuronium 3,17-Desacetyl vecuronium
EDmax, (~mol/l) 2.2 3.1 70.2
EDmax,, (~mol/l) 3 3.7 83.6
EDmaxrs (pmol/l) 4.2 4.4 99.4
expressed using the EDmax,, equation:
values by the following
Vecuronium (combined) 3-desacetyl vecuronium (comb.) + = 1.0 Vecuronium (alone) 3-desacetyl vecuronium (alone)
If the sum of this expression is less than 1.0, the interaction is synergistic, whereas if it is greater than unity the interaction is antagonistic. Slopes of dose-response curves were compared with an analysis of covariance, and P < 0.05 was considered significant. 3. Results The log-logit linear regression analysis of the doseresponse data revealed the following regression equations: y = 3.3 X - 1.64 (r* = 0.89, R.S.S. = 1.05), y = 4.3 X - 2.56 (r* = 0.55, R.S.S. = 2.62) and y = 6.3 x 5.8 (r* = 0.52, R.S.S. = 10.6) for vecuronium, 3-desacetyl and 3,17-desacetyl vecuronium, respectively. The slopes of the curves did not differ significantly from each other. The EDmax,, EDmax,, and EDmax,, values for vecuronium and its metabolites derived from the first part of this study are listed in table 1. The relative potency of vecuronium and its breakdown products at their EDmax,, levels were in the order of 1: 1.2 : 27 for vecuronium, the 3-desacetyl derivative and the 3,17-desacetyl derivative, respectively, which is close to that reported by Marshall et al. (1983) in the cat. The interaction (second part of the study) was characterized by isobolograms (fig. 1) showing vecuroB
A
Combined
EDmax
3.0 2.2
V e C
0 )rmol
pm&l
63.6
41.2 3,17desaceIyl
Vecuronlum
Fig. 1. Isobolograms of the interaction between vecuronium (Vet) and 3-desacetyl vecuronium or 3,17-desacetyl vecuronium. The diagonal solid lines, “additive” lines or isobql, connect the EDmax,, concentration of vecuronium on the ordinate with that of 3-desacetyl vecuronium (A) and 3,17-desacetyl vecuronium (B) on the abscissa. Broken lines perpendicular to the abscissa are drawn from the points of the EDmax,, concentration of 3-desacetyl and 3,17-desacetyl vecuronium observed in the presence of the vecuronium concentrations indicated by the values from which the broken lines perpendicular to the ordinate are drawn. The intersection of the broken lines is located on the “additive” line (A) or right of the “additive” line (B), indicating additive and less than additive (antagonistic) effects of vecuronium and 3-desacetyl or 3,17-desacetyl vecuronium, respectively.
15.5
2.2 pm01 Vet
Twitchheight
50 nmol3,17-des
Vet
200 nmol3,17-des
Vet
1
Fig. 2. Original tracing from an experiment. The first arrow indicates partial blockade induced with 2.2 pmol/l of vecuronium (Vet). The addition of a small dose (50 nmol/l) of 3,17-desacetyl vecuronium (3,17-des Vet, second arrow) partially antagonized the vecuronium-induced neuromuscular blockade, followed by a decrease in blockade after administration of 200 nmol/l of 3,17-desacetyl vecuronium (third arrow).
nium and 3-desacetyl vecuronium to interact in an additive fashion and vecuronium and its 3,17-desacetyl derivative to interact in a less than additive, i.e. antagonistic, fashion. The fractional (algebraic) analysis of the above interaction also demonstrated addition and antagonism by virtue of the sum of fractional doses equaling 1.0 and 1.2 in the combinations of vecuronium with the 3-desacetyl and 3,17-desacetyl compounds, respectively. The antagonism between vecuronium and 3,17-desacetyl vecuronium was further demonstrated in a “typical” experiment (fig. 21, in which partial reversal of the vecuronium-induced blockade occurred after the addition of 50 nmol of the latter compound, followed by a decrease in blockade after addition of 200 nmol of 3,17-desacetyl vecuronium.
4. Discussion The results of the present study show that the neuromuscular-blocking effect of vecuronium in combination with its 3-desacetyl derivative is additive whereas the combination of low concentrations of vecuronium and its 3,17-desacetyl metabolite results in a less than additive effect, namely antagonism. At higher concentrations this metabolite, being a weak neuromuscularblocking agent, facilitates neuromuscular blockade, and probably interacts with vecuronium in an additive fashion. In an earlier study, Ohta et al. (1985) described the “paradoxical” antagonism between vecuronium and its 17- and 3,17-desacetyl derivatives. However, their data and conclusions could be questioned on grounds of the experimental conditions used. Ohta et al. (1985) described that the blockade induced by the above breakdown products “increased gradually and never became stable”. It was therefore not possible for Ohta et al. (1985) to establish a dose-response relationship in
the presence or absence of vecuronium and to subject the data to isobolographic analysis, which is a recommended and commonly used method (Berenbaum, 1989) to characterize this type of drug interaction. For the explanation of the above observations the following possibilities should be considered. The observed partial antagonism of vecuronium induced by its 3,17-desacetyl derivative is caused by the cholinesterase-inhibiting activity of the latter compound, the interaction being similar to that observed for hexamethonium and D-tubocurarine by Rang and Rylett (1984). Other alternatives include presynaptic transmitter release or increased muscle contractility due to the activity of the metabolite. An appreciable inhibition of cholinesterase cannot be excluded, although it is less likely since all known steroidal neuromuscular-blocking agents are reported to be relatively weak inhibitors (I,,-, > 10m5 M) of acetylcholinesterase (Foldes and Deery, 1986). Presynaptic transmitter release or direct effects on muscle contractility usually require intracellular activity, as with aminopyridines, caffeine or quinine. Since neuromuscular-blocking agents, including vecuronium and its metabolites, are strong quaternary bases, it is unlikely that they could interfere directly with intracellular mechanisms. We assume therefore that vecuronium and its 3,17desacetyl derivative could be viewed as competitive antagonists with different affinities for and different dissociation rates from postsynaptic nicotinic cholinoceptors. As Stephenson and Ginsborg (1969) and Ferry and Marshall (1973) pointed out earlier, during the interaction of two antagonists with different affinity and dissociation rates, the addition of a second, rapidly dissociating antagonist to a preparation where a slowly dissociating antagonist is already present may increase
156
the free fraction of receptors available to the agonist (i.e. acetylcholine), and thereby promote apparent reversal of the existing neuromuscular blockade. This concept can be applied to the interaction between vecuronium and its 3,17-desacetyl derivative. In this case, the metabolite is the weaker, low-affinity antagonist whose rapid dissociation causes the partial reversal of the neuromuscular blockade induced by the parent compound. Only a small proportion of a dose of vecuronium underwent biotransformation in anaesthetized surgical patients (Bencini et al., 1986; Wierda et al., 1989). About 510% of the injected dose of vecuronium was recovered from body fluids as the 3-desacetyl derivative, whereas the other two metabolites have not been demonstrated after the surgical use of vecuronium. It is conceivable, however, that after long-term administration of high doses of vecuronium to critically ill patients, the severely altered sensitivity of the patients and profound changes in the biotransformation and elimination patterns caused by disease may result in subparalytic concentrations of 3,17-desacetyl vecuronium, which partially counteract the effects of the parent compound and in this way may contribute to the development of resistance. In conclusion, we have shown that 3-desacetyl vecuronium interacts additively with vecuronium while its 3,17-desacetyl derivative partially antagonizes the neuromuscular effects of the parent compound in vitro. The exact mechanism of this interaction and the extent to which these effects could explain the complications associated,with the long-term use of muscle relaxants, especially in patients in the KU, remains to be elucidated in human studies. References Bencini, A.F., A.H.J. Scaf, Y.J. Sohn, C. Meistelman, A. Lienhart, U.W. Kersten, S. Schwarz and S. Agoston, 1986, Disposition and
urinary excretion of vecuronium bromide in anesthetized patients with normal renal function and renal failure, Anesth. Analg. 65, 245. Berenbaum, M.C., 1977, Synergy, additivism and antagonism in immunosuppression, Clin. Exp. Immunol. 28, 1. Berenbaum, M.C., 1989, What is synergy?, Pharmacol. Rev. 41, 93. Coursin, D.B., G. Klasek and S.L. Goelzer, 1989, Increased requirements for continuously infused vecuronium in critically ill patients, Anesth. Analg. 69, 518. Durant, N.N., LG. Marshall, D.S. Savage, D.J. Nelson, T. Sleigh and I.C. Carlyle, 1979, The neuromuscular and autonomic blocking activities of pancuronium, Org NC 45, and other pancuronium analogues, in the cat, J. Pharm. Pharmacol. 31, 831. Ferry, C.B. and A.R. Marshall, 1973, An anti-curare effect of hexamethonium at the mammalian neuromuscular junction, Br. J. Pharmacol. 47, 353. Foldes, F.F. and A. Deery, 1986, Interaction of neuromuscular blocking agents with human cholinesterases and their binding in plasma, in: Handbook of Experimental Pharmacology, Vol 75: New Neuromuscular Blocking Agents, ed. D.A. Kharkevich (Springer, Berlin, Heidelberg, New York, Tokyo) p, 228. Loewe, S., 1955, Isobols of dose-effect relation in the combination of nikethamide (coramine) and phenobarbital, J. Pharmacol. 115, 6. Marshall, LG., A.J. Gibb and N.N. Durant, 1983, Neuromuscular and vagal blocking actions of pancuronium bromide, its metabolites, and vecuronium bromide (ORG NC45) and its potential metabolites in the anaesthetized cat, Br. J. Anaesth. 55, 703. Ohta, Y., M. Kinjo, K. Ono, K. Morita and F. Kosaka, 1985, Paradoxical antagonism of neuromuscular block by vecuronium metabolites, Anesthesiology 63, A357. Rang, H.P. and R.J. Rylett, 1984, The interaction between hexamethonium and tubocurarine on the rat neuromuscular junction, Br. J. Pharmacol. 81, 519. Savage, D.S., T. Sleigh and I. Carlyle, 1980, The emergence of ORG NC45, 1-((2P,3~,5~,16P,17P)-3,17-bis(acetyloxy)-2-(l-piperidinyl)-androstan-16-yl)-1-methylpiperidinium bromide, from the pancuronium series, Br. J. Anaesth. 52, 3s. Segredo, V., M.A. Matthay, M.L. Sharma, L.D. Gruenke, J.E. Caldwell and R.D. Miller, 1990, Prolonged neuromuscular blockade after long term administration of vecuronium in two critically ill patients, Anesthesiology 72, 566. Stephenson, R.P. and B.L. Ginsborg, 1969, Potentiation by an antagonist, Nature 222, 790. Wierda, J.M.K.H., A.H.J. Scaf, P.J. Hennis, M. Belopavlovic, W.D. Kloppenburg, A.M.L. Hart, T.M. Beaufort and S. Agoston, 1989, The influence of hypothermia on the pharmacokinetics of vecuronium bromide, Br. J. Anaesth. 63, 627P.
European
153
EJP52723
Antagonism of vecuronium by one of its metabolites in vitro Karin S. Khuenl-Brady, Clinics
for Anaesthesia
and General
Intensive
Peter Mair and Johann Koller Care Medicine,
University
of Innsbruck,
Innsbruck,
Austria
Received23 March 1992,revisedMS received17 July 1992,accepted4 August 1992
The neuromuscular-blocking agent vecuronium bromide undergoes hydrolysis to three pharmacologically active metabolites (3-desacety1,17-desacetyl and 3,17-desacetyl vecuronium) which might modify the neuromuscular-blocking action of their parent compound. In order to elucidate the possible role of the interaction between vecuronium and its metabolites in the complications reported after long-term use of vecuronium in intensive care unit (ICU) patients, the relative potency of vecuronium, 3-desacetyl and 3,17-desacetyl vecuronium was determined in the rat hemidiaphragm in vitro and the mode of interaction of the above-mentioned compounds investigated. Dose-response relationships were established for each substance alone and for combinations of vecuronium with its metabolites. The relative potency at the EDmax,, levels (o/o maximal effect) were in the order of 1: 1.2: 27 for vecuronium, the 3-desacetyl derivative and the 3,17-desacetyl derivative, respectively. The mode of interaction characterized by isobolographic and algebraic (functional) analysis showed vecuronium and 3-desacetyl vecuronium to interact in an additive fashion while the combined effect of the parent compound and its 3,17-desacetyl derivative was less than additive, indicating antagonism. Neuromuscular relaxants; Vecuronium; 3-Desacetyl vecuronium; 3,17-Desacetyl vecuronium; Metabolites (antagonism)
1. Introduction Vecuronium bromide undergoes hydrolysis to three different pharmacologically active compounds. These metabolites are 3-desacetyl (Org 72681, 17-desacetyl (Org NC58) and 3,17-desacetyl vecuronium (Org 7402) (Savage et al., 1980). Recently, long-term application of vecuronium to intensive care unit (ICU) patients has been reported to cause the development of vecuronium resistance (Coursin et al., 1989) as well as an apparent increased sensitivity to the compound, resulting in prolonged paralysis (Segredo et al., 1990). Since all three metabolites are pharmacologically active (Durant et al., 1979; Savage et al., 19801, they might contribute to the neuromuscular-blocking effects of vecuronium. However, how they affect the neuromuscular blockade induced by their parent compound depends on the mode of their mutual interactions, which could be addition, potentiation, or antagonism. In order to elucidate the possible role of the interaction between vecuronium and its metabolites in the complications reported after long-term use of vecuronium in ICU patients, we determined the relative potency of vecuronium, 3-des-
Correspondenceto: KS. Khuenl-Brady,Clinicsfor Anaesthesiaand General Intensive Care Medicine, Anichstrasse35, A-6020 Innsbruck, Austria. Tel. 43-512-5042400,fax 43-512-5042440.
acetyl and 3,17-desacetyl vecuronium in vitro and investigated the mode of interaction between the parent compound and these two metabolites in the rat hemidiaphragm. We studied only the first and terminal breakdown products, and did not use the 17-desacetyl derivative as it is an instable intermediary product in the formation of the terminal metabolite 3,17-desacetyl vecuronium (Marshall et al., 1983).
2. Materials
and methods
After institutional approval, male Sprague-Dawley rats of 200-300 g bodyweight were anaesthetized with thiopentone and decapitated. The phrenic nerves and hemidiaphragms were dissected and mounted in an organ bath containing Krebs-Ringer solution (pH 7.357.451, aerated with 5% CO, in oxygen and maintained at 37°C. The hemidiaphragms were stimulated indirectly via a Grass SS88 stimulator with supramaximal square-wave impulses of 0.2 ms duration at a frequency of 0.1 Hz. The resulting force of contraction was quantified with a Grass FT-03 transducer and continuously recorded on a Graphtec printer after applying a preload that resulted in maximal contractions (2-5 g). Each preparation was allowed to stabilize for at least 20 min until a stable twitch response was achieved. After each
154
measurement, diaphragms were washed at least three times and were allowed to stabilize again. A train-offour stimulation (2 Hz for 2 s) was given after the last washout to show the unchanged experimental conditions and complete neuromuscular recovery. 2.1. Experimental design
In the first part of the study the dose-response relationship of vecuronium, 3-desacetyl and 3,17-desacetyl vecuronium was determined in seven diaphragms with each substance in cumulative fashion. Increments of 50 nmol of vecuronium or 3-desacetyl vecuronium .or of 100 nmol of 3,17-desacetyl vecuronium were added to the bath, the next dose being given only after maximum blockade had developed. Dose-response curves for each substance were constructed by log-logit transformation, and the EDmaxz, EDmax,, and EDmax,s (% maximal response) were derived by linear regression analysis of the logit of the maximum blockade plotted against the logarithm of the dose. In the second part of the study the interaction between vecuronium and its metabolites was studied by determining the dose-response relationship of the 3desacetyl and 3,17-desacetyl derivatives in the presence of partial blockade induced by 2.2 pmol vecuronium. These experiments were carried out in the same way as those in the first part of the study (seven experiments with each combination). The EDmax=, EDmax,, and EDmax,, values were derived from the log-logit doseresponse curves for the metabolites in the presence of vecuronium. The data were analysed by isobolographic (Loewe, 1955) and algebraic (functional) analysis (Berenbaum, 1977). In the isobolographic analysis, the EDmax,, values of the interacting agents are plotted on the dose co-ordinates of the isobologram and connected with a straight line - the isobol or “additive line”. If the EDmax,, of a combination of two compounds is located on the isobol, simple additive effects can be assumed. A deviation of the combined EDmax,, point to the left indicates more than additive effects (potentiation), while a deviation to the right signifies less than additive effects (antagonism). In the case of a simple additive interaction, the same principle can be
TABLE
1
Dose-response relationship of vecuronium and its metabolites in the rat diaphragm.
Vecuronium 3-De-sacetyl vecuronium 3,17-Desacetyl vecuronium
EDmax, (~mol/l) 2.2 3.1 70.2
EDmax,, (~mol/l) 3 3.7 83.6
EDmaxrs (pmol/l) 4.2 4.4 99.4
expressed using the EDmax,, equation:
values by the following
Vecuronium (combined) 3-desacetyl vecuronium (comb.) + = 1.0 Vecuronium (alone) 3-desacetyl vecuronium (alone)
If the sum of this expression is less than 1.0, the interaction is synergistic, whereas if it is greater than unity the interaction is antagonistic. Slopes of dose-response curves were compared with an analysis of covariance, and P < 0.05 was considered significant. 3. Results The log-logit linear regression analysis of the doseresponse data revealed the following regression equations: y = 3.3 X - 1.64 (r* = 0.89, R.S.S. = 1.05), y = 4.3 X - 2.56 (r* = 0.55, R.S.S. = 2.62) and y = 6.3 x 5.8 (r* = 0.52, R.S.S. = 10.6) for vecuronium, 3-desacetyl and 3,17-desacetyl vecuronium, respectively. The slopes of the curves did not differ significantly from each other. The EDmax,, EDmax,, and EDmax,, values for vecuronium and its metabolites derived from the first part of this study are listed in table 1. The relative potency of vecuronium and its breakdown products at their EDmax,, levels were in the order of 1: 1.2 : 27 for vecuronium, the 3-desacetyl derivative and the 3,17-desacetyl derivative, respectively, which is close to that reported by Marshall et al. (1983) in the cat. The interaction (second part of the study) was characterized by isobolograms (fig. 1) showing vecuroB
A
Combined
EDmax
3.0 2.2
V e C
0 )rmol
pm&l
63.6
41.2 3,17desaceIyl
Vecuronlum
Fig. 1. Isobolograms of the interaction between vecuronium (Vet) and 3-desacetyl vecuronium or 3,17-desacetyl vecuronium. The diagonal solid lines, “additive” lines or isobql, connect the EDmax,, concentration of vecuronium on the ordinate with that of 3-desacetyl vecuronium (A) and 3,17-desacetyl vecuronium (B) on the abscissa. Broken lines perpendicular to the abscissa are drawn from the points of the EDmax,, concentration of 3-desacetyl and 3,17-desacetyl vecuronium observed in the presence of the vecuronium concentrations indicated by the values from which the broken lines perpendicular to the ordinate are drawn. The intersection of the broken lines is located on the “additive” line (A) or right of the “additive” line (B), indicating additive and less than additive (antagonistic) effects of vecuronium and 3-desacetyl or 3,17-desacetyl vecuronium, respectively.
15.5
2.2 pm01 Vet
Twitchheight
50 nmol3,17-des
Vet
200 nmol3,17-des
Vet
1
Fig. 2. Original tracing from an experiment. The first arrow indicates partial blockade induced with 2.2 pmol/l of vecuronium (Vet). The addition of a small dose (50 nmol/l) of 3,17-desacetyl vecuronium (3,17-des Vet, second arrow) partially antagonized the vecuronium-induced neuromuscular blockade, followed by a decrease in blockade after administration of 200 nmol/l of 3,17-desacetyl vecuronium (third arrow).
nium and 3-desacetyl vecuronium to interact in an additive fashion and vecuronium and its 3,17-desacetyl derivative to interact in a less than additive, i.e. antagonistic, fashion. The fractional (algebraic) analysis of the above interaction also demonstrated addition and antagonism by virtue of the sum of fractional doses equaling 1.0 and 1.2 in the combinations of vecuronium with the 3-desacetyl and 3,17-desacetyl compounds, respectively. The antagonism between vecuronium and 3,17-desacetyl vecuronium was further demonstrated in a “typical” experiment (fig. 21, in which partial reversal of the vecuronium-induced blockade occurred after the addition of 50 nmol of the latter compound, followed by a decrease in blockade after addition of 200 nmol of 3,17-desacetyl vecuronium.
4. Discussion The results of the present study show that the neuromuscular-blocking effect of vecuronium in combination with its 3-desacetyl derivative is additive whereas the combination of low concentrations of vecuronium and its 3,17-desacetyl metabolite results in a less than additive effect, namely antagonism. At higher concentrations this metabolite, being a weak neuromuscularblocking agent, facilitates neuromuscular blockade, and probably interacts with vecuronium in an additive fashion. In an earlier study, Ohta et al. (1985) described the “paradoxical” antagonism between vecuronium and its 17- and 3,17-desacetyl derivatives. However, their data and conclusions could be questioned on grounds of the experimental conditions used. Ohta et al. (1985) described that the blockade induced by the above breakdown products “increased gradually and never became stable”. It was therefore not possible for Ohta et al. (1985) to establish a dose-response relationship in
the presence or absence of vecuronium and to subject the data to isobolographic analysis, which is a recommended and commonly used method (Berenbaum, 1989) to characterize this type of drug interaction. For the explanation of the above observations the following possibilities should be considered. The observed partial antagonism of vecuronium induced by its 3,17-desacetyl derivative is caused by the cholinesterase-inhibiting activity of the latter compound, the interaction being similar to that observed for hexamethonium and D-tubocurarine by Rang and Rylett (1984). Other alternatives include presynaptic transmitter release or increased muscle contractility due to the activity of the metabolite. An appreciable inhibition of cholinesterase cannot be excluded, although it is less likely since all known steroidal neuromuscular-blocking agents are reported to be relatively weak inhibitors (I,,-, > 10m5 M) of acetylcholinesterase (Foldes and Deery, 1986). Presynaptic transmitter release or direct effects on muscle contractility usually require intracellular activity, as with aminopyridines, caffeine or quinine. Since neuromuscular-blocking agents, including vecuronium and its metabolites, are strong quaternary bases, it is unlikely that they could interfere directly with intracellular mechanisms. We assume therefore that vecuronium and its 3,17desacetyl derivative could be viewed as competitive antagonists with different affinities for and different dissociation rates from postsynaptic nicotinic cholinoceptors. As Stephenson and Ginsborg (1969) and Ferry and Marshall (1973) pointed out earlier, during the interaction of two antagonists with different affinity and dissociation rates, the addition of a second, rapidly dissociating antagonist to a preparation where a slowly dissociating antagonist is already present may increase
156
the free fraction of receptors available to the agonist (i.e. acetylcholine), and thereby promote apparent reversal of the existing neuromuscular blockade. This concept can be applied to the interaction between vecuronium and its 3,17-desacetyl derivative. In this case, the metabolite is the weaker, low-affinity antagonist whose rapid dissociation causes the partial reversal of the neuromuscular blockade induced by the parent compound. Only a small proportion of a dose of vecuronium underwent biotransformation in anaesthetized surgical patients (Bencini et al., 1986; Wierda et al., 1989). About 510% of the injected dose of vecuronium was recovered from body fluids as the 3-desacetyl derivative, whereas the other two metabolites have not been demonstrated after the surgical use of vecuronium. It is conceivable, however, that after long-term administration of high doses of vecuronium to critically ill patients, the severely altered sensitivity of the patients and profound changes in the biotransformation and elimination patterns caused by disease may result in subparalytic concentrations of 3,17-desacetyl vecuronium, which partially counteract the effects of the parent compound and in this way may contribute to the development of resistance. In conclusion, we have shown that 3-desacetyl vecuronium interacts additively with vecuronium while its 3,17-desacetyl derivative partially antagonizes the neuromuscular effects of the parent compound in vitro. The exact mechanism of this interaction and the extent to which these effects could explain the complications associated,with the long-term use of muscle relaxants, especially in patients in the KU, remains to be elucidated in human studies. References Bencini, A.F., A.H.J. Scaf, Y.J. Sohn, C. Meistelman, A. Lienhart, U.W. Kersten, S. Schwarz and S. Agoston, 1986, Disposition and
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