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PANCURONIUM AND ITS METABOLITES: PHARMACOLOGY, PHARMACODYNAMICS, PHARMACOKINETICS AND ANTAGONISM PROMOTORES PROF DR J F CRUL PROF R D MILLER PANCURONIUM AND ITS METABOLITES: PHARMACOLOGY, PHARMACODYNAMICS, PHARMACOKINETICS AND ANTAGONISM

PROEFSCHRIFT TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE GENEESKUNDE AAN DE KATHOLIEKE UNIVERSITEIT TE NIJMEGEN, OP GEZAG VAN DE RECTOR MAGNIFICUS PROF. DR. P. G. A. B. WIJDEVELD VOLGENS BESLUIT VAN HET COLLEGE VAN DECANEN IN HET OPENBAAR TE VERDEDIGEN OP DONDERDAG 2 APRIL 1981 DES NAMIDDAG TE 4.00 UUR

DOOR

LEONARDUS HENRICUS DOMITIANUS JOSEPH BOOIJ

GEBOREN TE DORDRECHT

1981 DRUKKERIJ-UITGEVERIJ BRAKKENSTEIN, NIJMEGEN Copyright 1981 L. H D. J. Booij

No part of ibis thesis may be in any way reproduced without written permission of the author. In memory of my father For Ria, Josquin and Celesta "From early enthusiasm through temporary damnation, relaxants have progressed to a status of great respect and importance to all of medicine."

- William K. Hamilton (1979) Professor and Chairman of Anesthesia, University of California, San Francisco, U.S.A.

6. FOREWORD

During general anesthesia, approximately 60 to 70 percent of all patients receive a nondepolarizing muscle relaxant. With the development of safer and more ideal relaxants, as well as the increased understanding of their mechanism of action, the admin­ istration of the relaxants by experienced anesthetists is without great risk. Nevertheless, Stanley A. Feldman made the following statement in 1980:

"Although the neuromuscular blocking drugs are seldom the cause of overt mortality or morbidity, there is little doubt that residual paralysis in the recovery phase is a frequent cuase of uncomfortable and danger­ ous side effects."

These uncomfortable and dangerous side effects have been observed in many cases where pancuronium has been administered. It has been suggested that the metabolism of pancuronium was the causative factor. This thesis is a collection of studies performed with the objective of demonstrating the safety of pan­ curonium, in spite of its metabolism.

The kind co-operation and stimulation received from the mem­ bers of the Department of Anesthesiology of the Catholic Universi­ ty of Nijmegen were of great value in the preparation of this thesis. The help of the many people who contributed during the performance of the experiments, the search for literature, the drawing of the figures, and the discussions during the work meet­ ings is gratefully appreciated. Special acknowledgement is extend ed to the technical assistance received from Francien van der Pol and Wim Kleinhands during the studies. Carla Vermeulen generously edited and typed the manuscript. Dr. David S. Savage (Organon Ltd., Newhouse, Scotland) kindly provided me with the pancuronium derivatives. The members of the 'international muscle relaxant group' frequently showed their interest in our work, especially Dr. Ronald D. Miller, who was my main assessor. Last but not least, I thank Ria, Josqum, and Celesta for their patience and for their consideration during the work on this thesis and its many studies.

7. CONTENTS

FOREWORD ι

Chapter 1 INTRODUCTION H

Chapter 2 METABOLISM, PHARMACODYNAMICS AND PHARMACOKINETICS OF PANCURONIUM; A REVIEW OF THE LITERATURE 15 Chapter 3 POTENCY OF PANCURONIUM AND ITS METABOLITES IN THE ISOLATED ARM TECHNIQUE IN HUMAN VOLUNTEERS 19 3.1. The isolated arm technique 19 3.2. Comparison of pancuronium and its metabolites in the isolated arm technique 20

Chapter 4 PHARMACODYNAMICS AND PHARMACOKINETICS OF PANCURONIUM AND ITS METABOLITES IN ANESTHETIZED MAN 25 4.1. The cumulative dose response curve technique 25 4.2. The comparative potency and pharmacokinetics of pancuronium and its metabolites m anesthetized man 26

Chapter 5 INTERACTION BETWEEN PANCURONIUM AND EACH OF ITS DEACYLATED METABOLITES IN IN VIVO AND IN VITRO RAT PREPARATIONS 31

Chapter 6 REVERSIBILITY OF PANCURONIUM-INDUCED NEUROMUSCULAR BLOCKADE 37 6.1. The effect of Cholinesterase inhibiting drugs; A literature review 37 6.2. Aminopyndines and neuromuscular transmission; A literature review 41 6.3. Antagonism of pancuronium and its metabolites by neostigmine in cats 48 6.4. Analysis of drug combinations 52 6.5. Potentiation of neostigmine and pyridostigmine by 4-aminopyridine in the rat 60 6.6. 4-атіпоругіаіпе potentiates neostigmine and pyridostigmine in man 64

8. 6.7. Comparative reversal of pancuronium by neostigmine, edrophonium, 4-aminopyridine and their combinations in cats 69. 6.8. Do neostigmine and 4-aminopyridine inhibit the antibacterial activity of antibiotics? 76.

Chapter 7 CONCLUSIONS AND CLINICAL IMPLICATIONS 81.

Chapter 8

SUMMARY 85.

SAMENVATTING 87.

CURRICULUM VITAE 91.

9. "Much of the information on the action of drugs affecting neuro- musaular transmission is derived from studies in animals. But it has been clearly established that these drugs aot different in the cat, dog, rat, chicken and man."

- Ronald L. Katz (1967) Professor and Chairman of Anesthesia, University of California, San Francisco, U.S.A.

10. CHAPTER 1 INTRODUCTION Muscle relaxation during general anesthesia is mainly achieved by neuromuscular transmission blocking agents. These are charac­ teristically either depolarizing or nondepolarizing. The depolar­ izing neuromuscular blocking agents show intrinsic activity on the acetylcholine receptors of the motor end-plate. The nondepolarizing blockers only compete with acetylcholine for the receptors but have no intrinsic activity. Pancuronium is presently the most frequently used nondepolarizing relaxant. Contrary to d-tubocurarine, meto- curine and gallamine, pancuronium undergoes metabolism primarily in the liver (Agoston et al. 1973а'Ь; Buzello 1975). Some of the unexpected side effects and complications after the administration of pancuronium may therefore be related to the products of this metabolism. Other side effects and complications can be explained by interaction with other drugs such as antibiotics (Pittinger et al. 1970, 1972; Booi] et al. 1978; Rutten et al. 1981), local anes­ thetics (Telivuo et al. 1970), quinidine (Miller et al. 1967), ketamine (Johnston et al. 1974), furosemide (Miller et al. 1978a; Azar et al. 1980), inhalation anesthetics (Miller et al. 1971,1972; Ngai 1975), and Imipramine (Edwards et al. 1979). Other side effects are explainable by pre-existing disturbances in acid-base balance (Crul-Slui]ter et al. 1974; Miller et al. 1975), changes in temperature (Zaimis et al. 1958; Ham 1977; Miller et al. 1978b), and electrolyte shifts (Giesecke et al. 1968; Ghoneim et al. 1970), while still other side effects are the result of an action on other organ systems (Saxena et al. 1971; Nana et al. 1973; Domeneck et al. 1976; Kumar et al. 1978). Prolonged paralysis and problems in antagonizing the blockade by neostigmine and pyridostigmine are seen occasionally and are then poorly understood. This happens most usually after prolonged administration of pancuronium in patients with renal and/or hepatic diseases (Miller et al. 1976). Especially in these cases, impaired metabolism and cumulation of pancuronium and its metabolites are suggested. It is a great lack that the amount and time course of metabolism is not exactly known because of lack of a specific quan­ titative method of separation and analysis of pancuronium and its metabolites. With aspecific semiquantitative methods (Kersten et al. 1973), total metabolism is estimated to be 30-45%. 25-35% of the pancuronium is metabolized into 3-OH-pancuronium and 5-10% into 17-OH and 3,17-diOH-pancuronium together (fig. 1). The main excretion is through the kidneys, while during renal failure the liver can partially compensate for this by increased uptake (Agoston et al. 1977). On a theoretical basis, problems may occur if the pancuronium metabolites also possess neuromuscular blocking properties. Accumu­ lation of metabolites may then contribute to the blockade. The pharmacodynamics and pharmacokinetics of the metabolites are there­ fore important. Problems can also occur if active metabolites are less reversible by acetylcholinesterase inhibitors, or if they interact with each other and pancuronium in a synergistic way, i.e. potentiate each other. A study of the pharmacology, pharmacodyna­ mics, pharmacokinetics and antagonism of pancuronium and its meta-

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Figure 1. Strurtural formulas for pancur­ onium and its metabolites. bolites xs therefore of great clinical importance.

To overcome the problem of the lack of a specific assay, we studied pancuronium and its metabolites when administered separately both in man and in animals.

Although we recognize that with all these compounds (except in the case of 3,17-diOH-pancuronium) metabolism will take place, and that conclusions drawn are not absolute, it was presumed that the compounds when administered individually were not broken down to a major degree during the time course of the experiment. The concen­ tration of compound measured by a fluorimetrie method (Kersten et al. 1973) was thereby considered to be that of the compound injected. We realize the limitation of this presumption, but believe this to be the only method presently available for investigation of the influence of the metabolism of pancuronium. Only when the 3,17- diOH-denvative is used will no further metabolism take place, thus, conclusions drawn from this drug are not valid. Since the behavior of the other two metabolites is analogous to that of 3,17-diOH-pan- curomum, the presumption made earlier is acceptable.

In the following chapters, the role of the pancuronium metabo­ lites and their action and reversal are discussed. In these chapters, many different species were studied using various tech­ niques. This was necessary since for ethical reasons some of the studies could not be performed in man. Either in vitro or in vivo studies were performed, the choice being based upon technical appropriateness.

References

Agoston S., Kersten U.W., and Heyer D.K.F. (1973a): The fate of pancuronium bromide m the cat. Acta Anaesthesiol. Scand. 17, 129-135. Agoston S., Vermeer G.Α., Kersten U.W , and Meyer D.K.F. (1973ь). The fate of pancuronium bromide in man. Acta Anaesthesiol. Scand. 17, 267-275. Agoston S., Crul E.J., Kersten U.S., Houwertjes M.C., and Scaf, A.H.J. (1977):

12. The relationship between disposition and duration of action of congeneric series of steroidal neuromuscular blocking agents. Acta Anaesthesiol. Scand. 21, 24-30. Azar I.. Cotrell J., Gupta В., and Tumdorf H. (1980): Furosemide facilitates recovery of evoked twitch response after pancuronium. Anesth. Analg. 59,55-57. Booij L.H.D.J., Miller R.D., and Crul J.F. (1978): Neostigmine and 4-aminopyri- dine antagonism of lincomycin-pancuronium neuromuscular blockade in man. Anesth. Analg. 57, 316-321. Buzello W. (1975): Stoffwechsel von pancuronium beim menschen. Der Anaesthesist 24, 13-16. Crul-Sluijter E.J., and Crul J.F. (1974): Acidosis and neuromuscular blockade. Acta Anaesthesiol. Scand. 18, 224-236. Domeneck J.S., Garcia R.C., Sasiain J.M.R., Loyola A.Q., and Oroz J.S. (1976): Pancuronium bromide, an indirect sympathicomimetic agent. Br. J. Anaesth. 48, 1143-1148. Edwards R.P., Miller R.D., Roizen M.F., Ham J., Way W.L., Lake C.R., and Roderick L.L. (1976): Cardiac response to Imipramine and pancuronium during anesthesia with halothane and enflurane. Anesthesiology 50, 421-425. Ghoneim H.M., and Long J.P. (1970): The interaction between magnesium and other neuromuscular blocking agents. Anesthesiology 32, 23-27. Giesecke A.H., Morris R.E., Dalton M.D., and Stephen C.R. (1968): Of magnesium, muscle relaxants, toxemic parturients and cats. Anesth. Analg. 47, 689-695. Ham J., Miller R.D., Benêt L., Matteo R.S., and Roderick L.L. (1978): Pharmaco­ kinetics and pharmacodynamics of d-tubocurarine during hypothermia in the cat. Anesthesiology 49, 324-329. Johnston R.R., Miller R.D., and Way W.L. (1974): The interaction of ketamine with neuromuscular blocking drugs. Anesth. Analg. 53, 496-501. Kersten U.K., Meyer D.K.F., and Agoston S. (1973): Fluorimetrie and chromato­ graphic determination of pancuronium bromide and its metabolites in biological materials. Clin. Chim. Acta 44, 59-66. Kumar S.M., Kothany S.P., and Zsigmond E.K. (1978): Effects of pancuronium on plasma-free norepinephrine and epinephrine in adult cardiac surgical patients. Acta Anaesthesiol. Scand. 22, 423-429. Miller R.D., Way W.L., and Katzung B.G. (1967): The potentiation of neuromuscu­ lar blocking agents by quinidine. Anesthesiology 28, 1036-1041. Miller R.D., Way W.L., and Dolan W.M. (1971): Compative neuromuscular effects of pancuronium, gallamine and succinylcholine during forane and halothane anesthesia in man. Anesthesiology 35, 509-514. Miller R.D., Way W.L., Dolan W.M., Stevens W.C. and Eger E.I. II (1972): The dependence of pancuronium and d-tubocurarine induced neuromuscular blockades on alveolar concentration of halothane and forane. Anesthesiology 37, 573-581. Miller R.D. (1975): Factors affecting the action of muscle relaxants. In: Katz, R.L. (Ed.) Muscle relaxants, pp. 163-191. Excerpta Medica, North Holland. Miller R.D. and Cullen D.J. (1976): Renal failure and postoperative respiratory failure: recurarization? Br. J. Anaesth. 48, 253-256. Miller R.D., and Roderick L.L. (1978a): Diuretic induced hypokalemia, pancuronium neuromuscular blockade and its antagonism by neostigmine, Br. J. Anaesth. 50, 541-544.

13. Miller R.D., Agoston S., van der Pol F., Booij L.H.D.J., Crul J.F., and Ham J. ОЭТвЬ): Hypothermia and the pharmacokinetics and pharnuicodynamics of pancur­ onium in the cat. J. Pharmacol. Exp. Ther. 207, 532-538. Nana Α., Cardan E., and Domokos M. (J973): Blood catecholamines changes after pancuronium. Acta Anaesthesiol. Scand. 17, 83-87. Ngai S.H. (1975): Action of general anesthetics in producing muscle relaxation- interaction of anesthetics with relaxants. In: Katz R.L. (Ed.) Muscle relax­ ants. pp. 279-298. Excerpta Medica, North Holland. Pittinger C.B., Eryasa Y., and Adamson R. (1970): Antibiotics induced paralysis. Anesth. Analg. 49, 487-501. Pittinger C.B., and Adamson R. (1972): Antibiotic blockade of neuromuscular function. Ann. Pharmacol. Rev. 24, 169—184. Rutten J.M.J., Booij L.H.D.J., Rutten C.E.J., and Crul J.F. (1981): The compar­ ative neuromuscular blocking effects of some aminoglycoside antibiotics. Acta Anaesthesiol. Belg. Accepted for publication. Saxena R.R. and Bontà I.L. (1971): Specific blockade of cardiac muscarinic receptors by pancuronium bromide. Arch. Int. Pharmacodyn. 189, 410-412. Telivuo L., and Katz R.L. (1970): The effects of modem intravenous local analgesics on respiration during partial neuromuscular blockade in man. Anaesthesia 25, 30-35. Zaimis E., Cannard Т.Н., and Price H.L. (1958): Effect of lowered muscle temperature upon neuromuscular blockade in man. Science 128, 34-35.

14. CHAPTER 2 METABOLISM, PHARMACODYNAMICS AND PHARMACOKINETICS OF PANCURONIUM, A REVIEW OF THE LITERATURE Since many drugs bearing acetyl groups are metabolized by esterase activity to hydroxy1 groups, it was assumed that pancuron­ ium is also converted to corresponding hydroxyl analogues. There were three metabolites possible: 3-OH-pancuronium, 17-OH-pancuron- lum, and 3,17-diOH-pancuronium. By combined thin layer chromato­ graphy and fluonmetry (Kersten et al. 1973), all three metabolites are indeed demonstrated in animals and in man (Agoston et al. 1973а'ь; Buzello 1975). Estimations for the amount of metabolism are made from the analysis of plasma, urine, and bile. 35-45% of the injected pancur­ onium is thought to be metabolized: 30-35% into 3-OH-pancuronium, and 5-10% into 17-OH-pancuronium and 3,17-diOH-pancuronium together (Agoston et al. 1973а<Ь; Buzello 1975). Of the possible metabolites, 17-OH-pancuronium possesses 1/50 the potency of pancuronium in blocking the neuromuscular transmission (Feldman et al. 1970; Norman et al. 1971). No data could be found in the literature for the other compounds. On intravenous injection, pancuronium exerts its maximal effect within 2-8 minutes (Dobkin et al. 1971, Katz 1971). 95% depression of twitch response is caused by pancuronium 0.0625 mg/kg body weight (Somogyi et al. 1978; Krieg et al. 1980). In most cases, complete blockade is observed with 0.08 mg/kg, which in patients without renal or hepatic diseases is recovered to 10% of the control in about 65 minutes (Katz 1971) . Five minutes after injection, 60% of the injected amount has disappeared from the plasma, 80% after 30 minutes, and 90% after 1 hour, resulting in half-life tunes in the distribution phase (t^a) of 4-10 minutes, and in the elimination phase (t^$) of 108-147 minutes (Agoston et al. 1973b). After 30 hours 43% is excreted in the urine and 11% m the bile. It is assumed that liver uptake plays an important role in termination of the pancuronium activity (Agoston et al. 1977a). Pharmacokinetic models have been developed for pancuronium both as 2- and 3-compartjnent open models (MacLeod et al. 1976; Somogyi et al. 1976; Hull et al. 1978; Buzello et al. 1978).

In patients with renal failure, the duration of the blockade is prolonged by 50% (Miller et al. 1973), but the intensity of the blockade is not different. This is explained by decreased plasma clearance, and longer t'ja and t%ß (Somogyi et al. 1977a). Conse­ quently, there is a decrease in pancuronium elimination (MacLeod et al. 1976), and thus a higher plasma level during the elimination phase. After the first injection, the depots are not yet filled up, and since the duration of blockade is largely dependent on re­ distribution of the relaxant, the duration is not significantly prolonged. With each subsequent repeating dose, the duration of blockade is prolonged because less redistribution can then occur. With total biliary obstruction, t^B is prolonged and plasma clearance decreased, leading to a longer duration of action (Somogyi et al. 197І^1 ). In the case of cirrhosis of the liver, t^a and t^g are significantly prolonged (Duvaldestin et al. 1978a). With cirrhosis of the liver, hyperhydration may be present as a

15. result of a low plasma protein concentration. This in turn may on one hand cause a decreased central volume of distribution, and,on the other hand, result in less protein binding. Since 80% of the in­ jected pancuronium is normally bound to plasma proteins (Thompson 1976), the plasma concentration of the unbound pancuronium may then be elevated.

In hypothermia, a reduction in pancuronium requirement with prolonged duration has been shown in cats (Miller et al. 1977) . The reasons have been proven to be the decrease in urinary and biliary excretion, decrease in pancuronium metabolism, and increase in sensitivity of the neuromuscular junction (Miller et al. 1978) .

There is a good correlation between percentage depression of the twitch and the plasma level of pancuronium, both after bolus injection and continuous infusion (Somogyi et al. 1976; Agoston et al. 1977b,· Shanks et al. 1978; Shanks et al. 1979). After complete blockade, the twitch response starts to recover (99% block) at a plasma level of about 0.21 pg/ml(Somogyi et al. 1976; Agoston et al. 1977b). Pancuronium crosses the placenta (Booth et al. 1977). An estimated 2-3% of the injected pancuronium reaches the fetus m this way (Duvaldestin et al. 1978°), causing a distribution ratio of cord vein to maternal blood of 0.22. Fetal uptake of pancuron­ ium is suggested by a cord arterial to cord venous ratio of 0.66. In pregnant women, the elimination of pancuronium is slower than in non-pregnant women (Duvaldestin et al. 1978'-').

The pharmacokinetics of pancuronium and its pharmacodynamics are also influenced by the type of anesthetic used (Miller et al. 1979). These differences are due partly to circulatory changes and partly to alteration of the sensitivity of the neuromuscular junc­ tion (Miller et al. 1979). With age there is a decrease in elimin­ ation of pancuronium from the plasma with a resulting increased duration of action (MacLeod et al. 1979).

In our studies, we determined the pharmacodynamics and pharma­ cokinetics of pancuronium and its metabolites in anesthetized man (chapters 3 and 4).

Refevenoes

Agoston S., Kersten U.W., and Meyer D.K.F. (1973a): The fate of pancuronium bromide in the cat. Acta Anaesthesiol. Scand. 17, 129-135. Agoston S., Vermeer G.Α., Kersten U.W., and Meyer D.K.F. (1973b): The fate of pancuronium m man. Acta Anaesthesiol. Scand. 17, 267-275. Agoston S., Crul E.J., Kersten U.W., Houwercjes M.C., and Scaf A.H.J. (1977a): The relationship between disposition and duration of action of a congeneric series of steroidal neuromuscular blocking agents. Acta Anaesthesiol. Scand. 21, 24-30. Agoston S., Crul J.F., Kersten U.W., and Scaf A.H.J. (1977ь): Relationship of the serum concentration of pancuronium to its neuromuscular activity in man. Anesthesiology 47, 509-512. Booth P.N., Watson M.S., and McLeod K. (1977): Pancuronium and the placental barrier. Anaesthesia 32, 320-323.

16. Buzello W. (1975): Stoffwechsel von Pancuronium beLm Menschen. Der Anaesthesist 24, 13-16. Buzello W., and Ruthven-Murray J. (1976): Der Konzentrationsverlauf von Pancur­ onium im Serum anurischer Patienten. Der Anaesthesist 25, 440—443. Dobkin A.B., Evers W., Ghanooni S., Levy A.A., and Thomas E.T. (1971): Pancur­ onium bromide (Pavulon) evaluation of its clinical pharmacology. Can. Anaesth. Soc. J. 18, 512-535. Duvaldestin P., Agoston S., Henzel D., Kersten U.W., and Desmonts J.M. (1978a): Pancuronium pharmacokinetics in patients with liver cirrhosis. Br. J. Anaesth. 50, 1131-1136. Duvaldestin P., Demetriou M., Henzel D., and Desmonts J.M. (1978^): The placen­ tal transfer of pancuronium and its pharmacokinetics during Caesarian section. Acta Anaesthesiol. Scand. 22, 327-333. Feldman S.A., and Tyrell M.F. (1970): A new steroid muscle relaxant dacuronium- NBftg (Organon). Anaesthesia 25, 349-355. Hull C.J., van Beem H.B.H., McLeod K., Sibbald Α., and Watson M.J. (1978): A pharmacodynamic model for pancuronium. Br. J. Anaesth. 50, 1113-1123. Katz R.L. (1971): Clinical neuromuscular pharmacology of pancuronium. Anesthesiology 34, 550-556. Kersten U.W., Meyer D.K.F., and Agoston S. (1973): Fluorometric and chromato­ graphic determination of pancuronium bromide and its metabolites in biological materials. Clin. Chir. Acta 44, 59-66.

Krieg N.. Crul J.F. and Booij L.H.D.J. (1980): Relative potency of Org NC45, pancuronium, alcuronium and d-tubocurarine in anaesthetized man. Br. J. Anaesth. 52, 783-788. McLeod K., Watson M.J., and Rawlins M.D. (1976): Pharmacokinetics of pancuronium in patients with normal and impaired renal function. Br. J. Anaesth. 48, 341- 345. McLeod K., Hull C.J., and Watson M.J. (1979): Effects of ageing on the pharmaco­ kinetics of pancuronium. Br. J. Anaesth. 51, 435-438. Miller R.D., Stevens W.C., and Way W.C. (1973): The effect of renal failure and hyperkalemia on pancuronium neuromuscular blockade. Anesth. Analg. 52,661-666. Miller R.D., and Roderick L.L. (1977): Pancuronium induced neuromuscular block­ ade and its antagonism by neostigmine at 29, 37 and 410C. Anesthesiology 46, 333-335. Miller R.D., Agoston S., van der Pol F., Booij L.H.D.J., Crul J.F., and Ham J. (1978): Hypothermia and the pharmacokinetics and pharmacodynamics of pancur­ onium in the cat. J. Pharmacol. Exp. Ther. 207, 532-538. Miller R.D., Agoston S., van der Pol F., Booij L.H.D.J., and Crul J.F. (1979): Effect of different anesthetics on the pharmacokinetics and pharmacodynamics of pancuronium in the cat. Acta Anaesthesiol. Scand. 23, 285-290. Norman J., and Katz R.L. (1971): Some effects of the steroid muscle relaxant dacuronium bromide in anaesthetized patients. Br. J. Anaesth. 43, 313-319. Shanks C.A., Somogyi A.A., and Triggs E.J. (1978): Plasma concentrations of pancuronium during predetermined intensities of neuromuscular blockade. Br. J. Anaesth. 50, 235-239. Shanks C.A., Somogyi A.A., Triggs E.J. (1979): Dose-response and plasma concen­ tration-response relationship of pancuronium in man. Anesthesiology 51, 111Ы118.

17. Somogyi A.A., Shanks CA., and Triggs E.J. (1976): Clinical phanoacokinetics of pancuronium bromide. Eur. J. Clin. Pharra. 10, 367—372. Soraogyi A.A., Shanks CA., and Triggs E.J. (1977a): The effect of renal failure on the disposition and neuromuscular blocking action of pdncuronium bromide. Eur. J. Clin. Pharmacol. 12, 23-29. Somogyi A.A., Shanks CA., and Triggs E.J. (1977°): Disposition kinetics of pan­ curonium bromide in patients with total biliary obstruction. Br. J. Anaesth. 49, 1103-1108. Somogyi A.A., Shanks CA., and Triggs E.J. (1978): Combined Г bolus and infu­ sion of pancuronium bromide. Br. J. Anaesth. 50, 575-582. Thompson J.M. (1976): Pancuronium binding by serum proteins. Anaesthesia 31, 219-227.

18. CHAPTER 3

POTENCY OF PANCUROMUM AND ITS METABOLITES IN THE ISOLATED ARM TECHNIQUE IN HUMAN VOLUNTEERS

3.1. The isolated arm technique

From anunal studies it is difficult to predict the potency and duration of action of neuromuscular blocking agents in man. Es­ pecially for the duration of action, a species difference clearly exists. This difference is not only dependent on the species used, but also on the relaxant studied. Therefore, there is the need for an in vivo human preparation to determine the potency and duration of action of relaxants before they are used clinically in patients. With the normally-used techniques, total body paralysis will occur and the volunteers must thus be artificially ventilated. Aside from technical problems and risk to the volunteers, this induces psychological strain and possible hypoventilation with their re­ spective influence on the blockade. To solve this problem, intra­ venous administration of a small amount of relaxant (diluted in a physiological saline solution) into the veins of an occluded arm has been described (Torda et al. 1966). This technique has been further developed for the diagnosis of myasthenia gravis (Foldes 1968; Foldes et al. 1968). Use of the method for the study of non­ depolarizing muscle relaxants was initiated (Feldman et al. 1970), and is at present accepted for the determination of relative poten­ cy and relative duration of action of such drugs (Bencini et al. 1980) .

An indwelling catheter is inserted in a vein on the dorsal side of the hand. Then an inflatable tourniquet is placed around the upper arm and inflated at a pressure above systolic blood pressure, after elevation of the arm for a few minutes to drain the blood. A small airount of the relaxant to be studied, diluted in 30 to 40 ml physiological saline, is administered through this cathe­ ter over a period of 2 minutes. The decrease in the evoked twitch response of the adductor pollicis muscle on supramaximal stimula­ tion of the ulnar nerve is recorded. Five minutes after the injec­ tion, the tourniquet is deflated. It has been demonstrated that ischemia of 5 minutes' duration does not influence the results and that 5 minutes is also sufficient to guarantee diffusion of the re­ laxants from the vessels to the receptors (Agoston, personal comm­ unication) . Since the amount of relaxant administered is small, there is no generalized paralysis after deflation of the cuff.

The effect is measured by quantitation of the adduction force of the thumb by a force-displacement transducer. This adduction is evoked by supramaximal stimulation of the ulnar nerve near the wrist at a rate of 0.1 Hz and 0.2 msec duration. If the volunteer so wishes, an ulnar nerve block near the elbow can be applied to avoid the experience of pain from the stimulation.

By studying a great number of volunteers with different doses, it is possible to construct a dose response curve with this tech­ nique. When a comparison is made with an already known relaxant, an estimate of the potency can be made. If the dose response curves

19. of the drugs to be compared are parallel, the study of one point from the dose response curve is sufficient to determine the relative potencies. Although the duration of action depends on the way the drug is administered, i.e. in the isolated arm, as an intravenous bolus, or by continuous infusion (Agoston et al. 1979), by comparison an esti­ mate of the duration of action can also be made with this technique. In the following section we used this technique to study the separ­ ate effects of pancuronium and its metabolites m man.

Peferenees

Agoston S., Feldman S.A., Miller R.D. (1979): Plasma concentrations of pancuron- ішп and neuromuscjlar blockade after injection into the isolated arm, bolus in­ jection, and continuous infusion. Anesthesiology 5), 119-122. Bencini Α., Agoston S , and Ket J. (1980)· Use of the human "isolated arm" pre­ paration to indicate qualitative aspects of a new neuromuscular blocking agent,

Org NCA5. Br. J. Anaesth. 52, 43S-48S. Feldman S.A., and Tyrrell M.F. (1970): A new theory of the termination of action of the muscle relaxants. Proc. Roy. Soc. Med. 63, 692-695. Foldes F.F. (1968). Regional intravenous neuromuscular block: a new diagnostic and experimental tool. Proc. 4th World Congr. Anaesthesiol. London, pp.425- Foldes F.F., Klonymus D.H., Maisel W., and Osserman K.E (1968): A new curare test for the diagnosis of myasthenia gravis. J.A.M.A 203, 649-653. Torda T.A.G., and Klonymus D.H. (1966): Regional neuromuscular block. Acta Anaesthesiol. Scand. Suppl. 24, 177-192.

3.2. Comparison of pancuronium and its metabolites in the isolated arm technique Leo H.D.J. B0013, M.D., Ronald D. Miller, M.D., Jan F. Crul, M.D., Ph.D.,3. Agoston, M.D.,Ph.D., and Stanley A. Feldman, M.D Submitted to the British Journal of Anaesthesia.

Summarij Of the pancuronium metabolites, only the 3,17-diOH derivative has so far been used clinically (Feldman et al. 1970; Norman et al. 1971). Therefore, the potency of the other derivatives remained to be determined in man. From in vitro studies in the rat hemidiaplragm preparation and in vivo studies in the rat tibialis muscle prepara­ tion, it is known that pancuronium is about twice as potent as 3-0H- pancuronium and 40 to 50 times as potent as 17-OH- and 3,17-diOH- pancuronium (Booij, unpublished observations). In order to have some indication of the potency and duration of action, pancuronium and its metabolites were compared in the isolated arm preparation in awake non-anesthetized man. The dosages used were based on observa­ tions in the rat and on the known potency of pancuronium.

20. Methods The isolated arra technique of Feldman and Tyrell (1970) was used in four of the authors (LB, RM, JC and SA) to determine the relative magnitude and duration of neuromuscular blockade induced by pancuronium or its metabolites. The ulnar nerve was stimulated near the wrist through thin-wall needle electrodes by a Grass S48 stimulator. Single supramaximal stimuli were administered at 0.1 Hz and a duration of 0.2 msec. Force of thumb adduction was measured with a Statham force displacement transducer and recorded on a poly­ graph. By means of two surface electrodes on the hypothenar region, and an indifferent surface electrode on the palmar side of the fore­ arm, the evoked compound electromyography (EMG) was also recorded on the polygraph (Lee et al., 1977).

All studies were performed with the subject in the supine posi­ tion. An indwelling needle was inserted into a vein on the forearm, and a pneumatic tourniquet placed around the upper arm. A dose of relaxant which produced between 80 and 99% depression of twitch ten­ sion was selected and diluted in physiological saline solution to 40 ml. When twitch tension and EMG were constant, the tourniquet was inflated to 200 mmHg, and the drugs were injected into the fore­ arm over two minutes. The tourniquet was kept inflated for five minutes to allow retrograde spread of the drug into the capillary bed, and was then released. Percent depression to twitch tension, time from the end of relaxant administration until 50% recovery of the depressed twitch tension (duration), and time from 25 to 75% recovery of twitch tension (slope) were calculated from both the mechanical and the EMG responses. All volunteers received each compound at an interval of one week. Comparison between the com­ pounds was done by analysis of variance. Results

Since there was quite a difference in height and weight of the subjects, it was decided that the doses administered be expressed in mg/m2.

The body surface areas and the doses of relaxants administered are listed in table 1.

Table 1. Dose of drugs used in mg/m2 B.S.A.

Pane. 3-OH 17-OH 3,17-OH Subject *B.S.A. (mg/m2) (mg/m2) (mg/m2) (mg/m2)

LB 1.99 0.35 0.35 10.05 7.54 RM 1.83 0.16 0.51 3.28 10.93 JC 2.05 0.15 0.73 9.76 5.85 SA 1.87 0.11 0.27 3.21** 4.28

Mean 1.94 0.19 0.47 7.71 8.11 * B.S.A. = body surface area in m2. **Not included in calculation because of a block of leas than 50% of control twitch tension.

21. The mean depressions of mechanical twitch from pancuronium, 3-0H-, 17-0H-, and 3,17-diOH-pancuronium were 80 + 9 (S.E.), 96 + 2, 90+1, and Θ7 + 8%, respectively. EMG was depressed 83+2, 86+4, 84+4, and 76 + 13%, res­ pectively. These neuromuscular blockades were statistically not significantly different from each other (p>Q.05). From the mean doses listed in table 1, we calculated pancuronium to be 2.4 times (0.47/0.19) more potent than 3-OH-pancuronium, 40 times (7.71/0.19) more potent than 17-OH-pancuronium, and 42 times (8.11/0.19) more potent than 3,17-diOH-pancuronium. Although the duration of neuromuscular blockade tended to be shorter with 17-OH-pancuronium, no statistically significant diff­ erence existed between the four drugs (p>0.05) (fig. 1).

Ί ,: ¡i; ;il ι ¡r I I i ι i ! M li M, ¡'I lil LLL ρ 3 OH 17 ОМ 3 17 ОН

Figure 1. Duration of neuromuscular block­ ade by pancuronium and its 3-OH-, 17-0H-, and 3,1 7-diOH-nietabolites. The slopes of recovery were also not statistically different (p>0.05) among the four drugs (fig. 2).

0 JA m,

3 OH 17 OH 3 17 OH

Figure 2. Slopes of recovery of pancuronium and its 3-OH-, 17-OH, and 3,17-diOH-metabolites.

22. Discussion Since 17-0Н-рапсигопішп and 3,17-diOH-pancuronium only have about 1/40 the potency of pancuronium, they are not of clinical sig­ nificance. However, 3-OH-pancuronium is approximately half as po­ tent as pancuronium, and could therefore contribute to the neuro­ muscular blockade of pancuronium. Using semiquantitative techniques (thin-layer chromatography), Agoston et al. (1973а'Ь) and Buzello (1975) have estimated that 20 to 30% of pancuronium is metabolized to 3-OH-pancuronium. When enzyme induction occurs, the extent to which pancuronium is metabolized could be even greater. An analyti­ cal technique which can accurately measure concentrations of pancur­ onium and its metabolites in body fluids is required to provide better information as to how much 3-OH-pancuronium contributes to the neuromuscular blockade of pancuronium.

As observed in previous muscle relaxant studies (Katz, 1971), and also in the present study, much variability in the dose of re­ laxant required for 80-99% depression of twitch tension occurred from sub3ect to subject (table 1). However, the manner in which the variability occurred could not clearly be explained. A subject who was particularly sensitive to pancuronium may be comparatively insensitive to one of the metabolites, and vice versa. For example, subject LB required twice as much 3-OH-pancuronium as subject JC for an 80-99% depression of twitch tension. Yet subject JC required twice as much 3-OH-pancuronium as subject LB (table 1). We are pre­ sently unable to explain this type of variability.

The potency ratios between pancuronium and its metabolites stated above only represent one point on a dose response curve. We believe that establishing potency ratios based on one dose was appropriate because the magnitudes of block were similar (no statis­ tically significant difference) among the four drugs studied. If the dose response curves deviated from parallelism, then the potency ratios between pancuronium and its metabolites may be different at different points on the dose response curve. We were unable to det­ ermine complete dose response curves with this method. The durations of neuromuscular blockade were all similar. So even if a clinically significant amount of 3-OH-pancuroniun were formed in a patient receiving pancuronium, the duration of blockade should not be significantly prolonged. If they potentiate each other, the extent of metabolism may account for part of the varia­ bility in magnitude and duration of neuromuscular blockade from pan­ curonium. Of the metabolites, only 17-OH-pancuronium has previously been studied in man. Using the isolated arm technique, Feldman and Tyrell (1970) found a slope of recovery of 8.4 + 0.8 (S.E.) minutes for 17-OH-pancuronium, which is not much different from the 9.5 + 1.0 minutes observed in our study. In summary, we conclude that the duratiors of neuromuscular blockade by pancuronium and its metabolites in the isolated arm preparation are very similar, and that only the 3-OH-pancuronium derivative is potent enough to be of clinical significance.

23. References

Agoston S., Kersten U.U., and Meijer D.K.F. (1973a): The fate of pancuronium bromide in the cat. Acta Anaesthesiol. Scand. 17, 129—135. Agoston S., Vermeer G.Α., Kersten U.K., and Meijer D.K.F. (1973b): The fate of pancuronium bromide in man. Acta Anaesthesiol. Scand. 17, 267—275. Buzello K. (1975): Stoffwechsel von Pancuronium beim Menschen. Der Anacsthesist 24, 13-16. Feldman S.A., and Tyrrell M.F. (1970): Anew steroid muscle relaxant: Dacuronium - N.B.68. Anaesthesia 25, 349-355. Feldman S.A., and Tyrrell M.F. (1970): A new theory of the termination of action of the muscle relaxants, Proc. Roy. Soc. Med. 63, 692-695. Katz R.L. (1971): Clinical neuromuscular pharmacology of pancuronium. Anesthes­ iology 34, 550-556. Lee C, Katz R.L., Lee A.S.J., and Glaser В. (1977): A new instrument for con­ tinuous recording of the evoked compound electromyogram in the clinical setting. Anesth. Analg. 56, 260-270. Norman J., and Katz R.L. (1971): Some effects of the steroidal muscle relaxant dacuronium bromide, in anaesthetized patients. Br. J. Anaesth. 43, 313-319.

24. CHAPTER 4

PHARMACODYNAMICS AND PHARMACOKINETICS OF PANCURONIUM AND ITS METABOLITES IN ANESTHETIZED MAN

4.1. The cumulative dose response curve technique

Dose response curves are usually constructed by single dose administrations followed by linear regression analysis of the total number of doses and responses. This requires that a large number of patients be studied. When the response can be clearly recorded and is also dose-dependent, the cumulative dose response technique can be used. Dose response curves are then constructed by incre­ mental administration of cumulating small amounts of the drug. In the case of the nondepolarizing neuromuscular blocking agents, each incremental dose is administered when 3 subsequent twitches have the same height (Donlon et al. 1974). With short-acting compounds, the twitch depression may already be recovering before the next dose is effective. When longer-lasting drugs are studied, this problem does not occur. Since pancuronium and its metabolites are long-act­ ing drugs, we used this technique in our studies. The validity of the method has previously been demonstrated (Bavarese et al. 1974), and has since been reported more extensively (Donlon et al. 1980). We confirmed this validity on several occasions during studies we per­ formed (Booi], Krieg et al., unpublished observations).

The compound studied must always be injected at a constant speed in a fast-running intravenous drip. Stable conditions for depth of anesthesia, temperature, and acid-base balance should exist during the whole procedure. With this method, no information can be obtained on the duration of action and the recovery speed of the drug. Such data remain to be obtained from single bolus administra­ tions . References

Donlon J.W., All H.H., and Savarese, J.J. (1974). A new approach to the study of four nondepolarizing relaxants in man Anesth. Analg. 2-4, 13-16. Donlon J.W. Jr., Savarese J.J., All H.H., and Teplik R.S. (1980): Human dose- response curves for neuromuscular blocking drugs: A comparison of two methods of construction and analysis. Anesthesiology 53, 161-166. Savarese J.J., Donlon J.W., and Ali H H. (1974): Human dose response curves for neuromuscular blocking agents: A comparison of two methods of construction and analysis. In: Abstracts of Scientific papers, 1974 Annual meeting of the American Society of Anesthesiologists, Washington D.C. pp. 121-122.

25. The Comparative Potency and Pharmacokinetics of Pancuronium and Its Metabolites in Anesthetized Man1 2

RONALD D MILLER, SANDOR AGOSTON, L H D J BOOIJ, URSULA W KERSTEN, J F CRUL and JAY HAM Department oí Anesthesiology Catholic University, Ni/megen the Netherlanös (RDM SA L H D J В J F С ) Departments of Experimental Anesthesia and Clinical Pharmacology State University of Groningen Groningen the Netherlands (RDM SA U W К J and Departments of Anesthesia and Pharmacology University of California San Francisco California (RDM JH> Accepted for publication June 12, 197Θ

ABSTRACT Miller, Ronald O., Sandor Agoston, L. H. D. J. either pancuronium or one of its metabolites Bool), Ursula W. Kersten, J. F. Crul and Jay was given as an ι ν bolus Onset time and Ham: The comparative potency and pharmaco­ duration of neuromuscular blockade from 3-OH- kinetics of pancuronium and its metabolites in and 3,17-OH-pancuronium did not differ signifi­ anesthetized man J Pharmacol Exp Ther cantly from that of pancuronium, 1 7-OH-pan- 207: 539-543, 1978 curomum had a shorter duration of action than did pancuronium Although pancuronium To determine the potency of pancuronium and tended to have a slightly longer elimination half- its metabolites, 3-OH-, 1 7-OH- and 3,17-OH- life, the pharmacokinetics of the four drugs did- pancuromum, cumulative dose-response curves not differ significantly The elimination half-lifes were determined in five anesthetized patients were 110, 68, 73 and 71 mm for pancuronium with each drug Pancuronium (ED50 = 0 041 and its 3-OH, 17-OH and 3,17-OH derivatives, mg/kg) was 2 times more potent lhan 3-OH- respectively We conclude that although pan­ pancuromum (ED50 = 0 082 mg/kg), 50 times curonium is more potent than its 3-OH, 1 7-OH more potent than 17-OH-pancuronium (ED50 and 3,1 7-OH metabolites, the pharmacokinetics = 20 mg/kg) and 54 times more potent than of these three metabolites do not differ from 3,17-OH-pancuronium (ED50 = 2 15 mg/kg) each other and from that of pancuronium In 21 other patients, one equipotent dose of

In cats and man, pancuronium is metabolued of an injected dose of pancuronium appears as by deacetylation to 3-hydroxy (З-ОН-рапсигоп- 3-OH-pancuronium in the urine and bile of man lum), 17-hydroxy (17-OH-pancuronium) and (Agoston et al 1973b, Buzello, 1975, Somogyi et 3,17-hydroxy (3,17-OH-pancuronium) bisquater- al, 1977), however, less than Ъ% of an injected nary derivatives (Agoston et al, 1973a,b, Buz- dose of pancuronium appears in the urine of ello, 1975, Somogyi et al, 1977) As much as 25% man as 17-OH- or 3,17-OH-pancuromum (Buz­ ello, 1975; Somogyi et al., 1977) Although 17-OH-pancuronium has been evaluated clini­ Received for publuation Detemher 1Я 1977 cally (Feldman and Tyrrell, 1970, Norman and 1 This work was presented in part at the Annual Meeting of the Association of University AneslhetiM-s Apnl 14 \Ч~Н in Kalz, 1971), the potency and pharmacokinetics Тш son and the "ith Kuropean ('onpress of Anaesthesiologv of all three metaboliteb relative to pancuronium September 4-9 1478 in Рагіч Frante have not been determined We analyzed the Work was performed while R [) Miller was Visiting Professor at the above institutions in the Nelherlands pharmacokinetics and compared the magnitude

26. and duration of neuromuscular blockade from was administered as an ι ν bolus in the remaining 21 these metabolites with pancuronium in anesthe­ patients These doses are approximately equipotent as tized man. determined from the dose-response curves described above From mechanical and electromyographic Methods twitch recordings, we calculated onset time defined as that time from relaxant administration untd peak de­ Forty-one patients, 48 ± 5 (S D ) years of age and pression of the twitch, magnitude and duration of 66 ± 3 kg in weight with no hepatic or renal abnor­ neuromuscular blockade Duration was defined as that malities were studied They had no cardiovascular time from relaxant administration until recovery of abnormalities with the exception of six patients who 50% of the depressed twitch For example, if an 80% had systolic hypertension, however, none had a sys­ depression of twitch tension occurred, duration would tolic blood pressure greater than 150 mm Hg No other be that time from relaxant administration until recov­ drugs known to interact with neuromuscular blocking ery to 40% depression of twitch tension (40%/80% - drugs were being taken by these patients Approxi­ 50%) Slope of recovery was determined as time from mately 1 hr before anesthesia, they received atropine, 25 to 75% recovery from the depressed twitch tension 0 25 mg, dropen dol, 5 mg, and pmtramide, 11 25 mg Venous blood samples were drawn from an indwell­ ι m , after which anesthesia was induced with thiopen­ ing cannula 2, 5, 7, 10, 15, 20, 30, 45, 60, 90, 120, 150 tal, 1 5 to 2 0 mg/kg, γ-hydroxybutync acid, 60 mg/kg, and 180 mm after the bolus intravenous relaxant ad­ and fentanyl, I 5 μg/kg ι ν By controlled ventilation ministration Pancuronium, 3-OH-pancuronium, PaCOs was maintained between 31 and 40 mm Hg A 17-OH-pancuronium, or 3,17-OH-pancuronium con­ Grass S-44 stimulator was used to administer supra­ centrations in plasma were determined fluonmetn- maximal square-wave bipolar pulses of 0 2 msec du­ cally using the method described by Kersten et al ration to the ulnar nerve at the wnst through thin wall (1973) of which the lower limit of sensitivity is 0 02 needle electrodes at 0 1 Hz The resultant force of yg/ml This method does not distinguish between un­ thumb adduction was quantità ted with a force-dis­ changed pancuronium and its deacetylated metabo­ placement transducer and recorded on a polygraph lites resulting in reported pancuronium concentrations (Miller et al, 1977) In addition, by means of two being actually a combination of the concentration of surface electrodes on the hypothenar region and an pancuronium and its metabolites The extent to which indifferent surface electrode on the palmar side of the the reported pancuronium concentration is metabolite forearm, the electromyograph (EMG) was recorded on and not pancuronium would depend on the rate of a polygraph using instrumentation described by Lee metabolism which is not known The values obtained etat (1977) were analyzed by computer using weighted nonlinear The first section of the study was designed to de­ least squares regression analysis according to a two- termine comparative potency between pancuronium compartment open model (Van Rossum, 1971) The and its metabolites In the first 20 patients, cumulative data for each subject were fitted to a function of the dose-response curves as described by Uonlon et al equation С = Ae "' +• Be ^ where С representa the (1974) were determined for pancuronium and its bis- plasma relaxant concentration, A and В are h\brid quatemary den vati ves No difference was found in intercept terms having units of concentration, and the dose-response curves determined by the cumulative exponents a and β are hybrid rate constants having method vs the single bolus method (Savárese et al units of reciprocal time From these functions appar­ 1974) Briefly, the patients received a bolus injection ent elimination half-life (íi/¿^), volume of central com­ of pancuronium, 0 025 mg/kg, 3-OH, 0 04 mg/kg, partment ( V\), total volume of distribution at steady 17 OH, 0 θ mg/kg or J,17-OH, 0 8 mg/kg ι ν followed state ( Vdv.) and total plasma clearance were calculated by incremental doses Before the next incremental (Van Rossum, 1971, Greenblatt and Koch Weser, dose was given, the twitch response to the previous 1975) These results and data obtained from the re­ dose was allowed lo stabilize as judged by a recording cordings of mechanical and electromyographic twitch of three consecutive twitches of equal tension Five were analyzed further by analysis of variance and patients were studied with each drug while only one unpaired Student's t test (Goldstein, 1964) relaxant was studied in each patient The total amount of drug given for each response was calculated and Results plotted on a log dose response graph from which linear regression was analyzed From these curves, the ED50 In the first phase of the study, the cumulative (dose of relaxant which causes a 50% decrease in twitch dose method was used to obtain dose-response tension) doses were determined, doses causing either curves The ED50 values for pancuronium, no response or a 100% depression of twitch tension 3-OH-, 17-OH- and ЗД7-ОН-рапсигопіит were were not included in the calculation 0 041, 0 082, 2 0 and 2 15 mg/kg, respectively To determine the onset time and duration of neu­ romuscular blockade and pharmacokinetics of pancu­ (fig 1) Thus, pancuronium is two times more ronium and its metabolites, either pancuronium, 0 04 potent than 3-OH-pancuronium (0.082/0 041 = mg/kg, 3-OH pancuronium, 0 1 mg/kg, 17-OH pancu­ 2), 50 times more potent than 17-OH-pancuron- ronium, 2 mg/kg, or 3,17-OH-pancuronium, 2 mg/kg, lum (2 0/0 041 = 50) and 53 7 times more potent

27. than 3,17-OH-pancuronium (2.15/0.041 = 53.7). ± 5.5%, respectively. With the EMG a 61 ± 11, With the EMG as the measure, the ED50 values 77 ± 4, 52 ± 10 and 49 ± 6% depression occurred were the same as with the mechanical twitch respectively. No significant difference existed except for 17-OH-pancuronium which was 1.57 between the magnitude of twitch depression mg/kg. from these doses of pancuronium and its three In the second phase of the study, onset time metabolites. Also, depressions of the mechanical and duration of neuromuscular blockade and twitch were not significantly different from those pharmacokinetics of pancuronium and its deace- depressions of the EMG. tylated metabolites were determined from the Although pancuronium tended to have longer i.v. bolus administration of equipotent doses of onset time than any of its metabolites, these these drugs. The magnitude of maximal depres­ differences were not significant (fig. 2). Although sion of the mechanical twitch from pancuron­ 17-OH-pancuronium, had a significantly shorter ium, 0.04 mg/kg, 3-OH-pancuronium, 0.1 mg/kg, duration of neuromuscular blockade (P < .05), 17-OH-pancuronium, 2.0 mg/kg and 3,17-OH- the slopes of recovery of the four drugs were not pancuronium, 2.0 mg/kg, all given as an i.v. significantly different (fig. 2). There were no bolus, were 70 ± 11 (S.E.), 79 ± 7, 56 ± 5 and 4S significant differences between the results ob-

о о PANCURONIUM 3-ЭН PANCURONIUM Û Δ 17-ОН PANCURONIUM ^ Ζ 100 ρ о <Р' / • · L· Д 3,'7.0М PANCURONIUM / / О (Л À А/ 80 - с J . fr* IE Af- У 60 - 0 / А / /· /Ь / А 40 - 53 7' /· /Δ / * и О / У-, <•> п ь/ »/* К 6 / О /, / / /* Д / 2 0 1 1 I 1 1 1 І A I I 002 0 04 0 07 0 1 Ü2 0 5 10 2 0 4 0 DOSE (mg/kg) Fig. 1. Correlation between dose ot muscle relaxant and depression ot mechanical twitch tension. The lines represent analysis of linear regression. The correlation coefficients were 0.87, 0.92. 0.93 and 0.96 for pancuronium and its ЗОН, 17-OH and 3,17-OH derivatives, respectively

ВЯ ONSET I I SLOPE ¡53 DURATION

0 ^ 3-0H 17-OH 3,17-OH Fig. 2. Onset limes, duration of neuromuscular blockade and slope of recovery (mean ± SE.) for pancuronium and its Э-ОН, 17-OH and 3,1 7-OH derivatives using the mechanical Iwilch as the measure Onset is defined as that time from relaxant administration to peak depression of twitch tension. Duration is that time from relaxant administration until recovery of 50% of the depressed twitch tension. Slope of recovery is defined as lhat time from 25 to 75% recovery ol the depressed twitch tension. ' Significantly different from pancuronium.

28. tamed from the force displacement transducer niques (thin-layer chromatography), Agoston et and the EMO. al. (1973a,b) and Buzello (1975) identified as We were unable to demonstrate any signifi­ much as 25% of an injected dose of pancuronium cant difference in the pharmacokinetics of pan­ as the 3-OH derivative in the urine and bile. The curonium and its three metabolites (fig. 3). Al­ 17-OH and 3,17-OH derivatives are approxi­ though the elimination half-life tended to be mately 50 times less potent than pancuronium longer and clearance less with pancuronium, the (fig. 1) and appear in the urine and bile in an differences were not significant (table 1) amount less than 5% of an injected dose of pancuronium (Agoston et al., 1973b, Buzello, Discussion 1975; Somogyi et al, 1977) Consequently, only the 3-OH derivative appears in sufficient amounts and is potent enough to contribute The 3-OH derivative is half as potent as pan­ significantly to the neuromuscular blockade of curonium (fig. 1). Using semiquantitative tech- pancuronium. The results of this study are similar to those of studies performed previously which examined the pharmacokinetics of pancuronium. We found a mean apparent elimination half-life of 110 min from a dose of 0.04 mg/kg (table 1), while McLeod et al. (1976) found a half-life of 96 mm from a dose of about 0.057 mg/kg of pancuronium and Agoston et al. (1973b) found the elimination half-life to range from 108 to 147 mm from a dose of 0.06 mg/kg Somogyi et al. (1976) found the elimination half-life to range from 89 to 161 mm (mean 132 min) from a dose of 6 mg. Somogyi et al. (1976) also found the Vdv, to be 261 mg/kg while we found a value of 241 mg/kg. McLeod et al. (1976) also found the V'I and clearance of pancuronium to be 79 ml/kg, and approximately 1.1 ml/kg/min, respectively, while we found these values to be 72 and 1.76 ml/kg/min, respectively (table 1). Although our data seem to fit the two-com­ partment open model, we cannot exclude the possiblity that in other circumstances, other models may be more appropriate. For example, if sampling had been continued for longer than 180 mm, perhaps an even slower elimination phase might have been observed as with rf-tub- 25 50 75 100 125 150 175 ocurarme (Gibaldi et al., 1972, Miller et al., time (mm) 1977). However, .Somogyi et al. (1976, 1977) Fig. 3. Correlation between lime and plasma concen­ found that the use of a tn-exponential equation tration of pancuronium and its 3-OH, 17-OH or 3.17- did not improve significantly on the fit obtained OH derivatives The symbols and bracket represent mean ± S E The lines were computed from pharma­ by a bi-exponential equation when sampling was cokinetic data described m the text continued for 400 min.

TABLE 1 Pharmacokinetics of pancuronium and its metabolites (mean + S.E.) Total Plasma Drug V, Vd,, Dose Clearance mg/kg ml/kg ml/kg mllkg/mm Pancuronium 004 110 ± 20 72 ± 10 241 ± 27 1 76 ± 0.3 3-OH 0 10 68 ± 7 66 ± 4 205 ± 37 2.58 ± 0.7 17-OH 20 73 ±6 57 ± 15 203 ± 8 2.27 ±02 3.17-OH 20 71 ±8 87 r 12 251 ± 35 2 87 + 0 6

29. The precise extent to which these metabolites Acknowledgementb contribute to the neuromuscular blockade of The authors gratpfullv acknowltdßt Iht advice of Dodcrs J M Vanltosbum Issile 7 Hencl and I^ewis H Sheinerwith pancuronium depends on the amount and rate the pharmatokmeLit analvsis I he authors ilso thank Waller of metabolism although the abo\ e figures prob I Wa\ MI) Klmond I Ьвег II MI) Donald Stanskl MI) Dorothy Urban and I rud> CrarrelLson for their editorial ably underestimate the extent to whit h pancu advue romum is metabolized Although up to 25^ of an HtferenreN injected dose of pancuronium has been identified AÍONSTON S Km-STfN V Vc AND MAIII-R D к (ι I he as the 3 OH derivative in bile and urine only 40 fate of pancuronium hromidt in Iht cat Acta Anaesth to 60S of the total dose of pancuronium was Sc-ind 17 124 115 1471a recovered (Agoston et al 1971b Вшеііо 1975 A( O'TTON S VfRMtíR ( A KFRSTPN V \V AM) Mt I lb R D К У I he tale ol pancuronium bromide in man Acta Somogyi et al, 1977) Τ he remaining 40 to 60% Anaesth Stand 17 ·!(>" 2 τ IS lb of pancuronium which was not recovered may Biznto W Der St f/fwechse! von pancuronium beim have been significantly metabolized and the rate men-st hen ^naesthesisl 24 1 1 К IS > DONICIN I \\ All HH AND SAVORI-SI· I Ι λ ntw at which pancuronium is metabolized is not approach le the sludv of four noncltp ilarmng relaxants in known The above figures refer to urine and bile man Anesth Anale (( leve ) 51 414 ) 14 IT-I analyzed after 12 (Buzello, 1975) 24 (Somogyi tl-LiiMAN S A AND IYRHMI M l· Anew steroid musc le relaxant dacuronium — NIÌW* [Organon) Anaesthtsia 25 et al 1977) and 30 hr (Agoston et al 197 )b) 144 i')') ITO In order to pro\ ide more precise information GIRAI ш M I LVY (, AND HAYTON W Kinetics of the elimination and neuromuscular blocking effect of d tubo about the amount and rate of pancuronium me curarme in man Aneslhesiolo(,v 36 21J 21Й \4™¿ tabolism, a better method of analysis is required (IOLDSTUN A Biostalislics pp "0-^2 Hr) 1S4 Iht Mac The fluonmetnc method measures the concen millnn ( ompany New Vork I%4 GR^NHIATT I) I AND Kuc Η Wi-stR J ( Imitai pharrna tration of the total bisquaterndry ammonium cokinetics N Ьngl 1 Med 293 702 "(Г) 1СГ> compound and does not distinguish between un KfRSThN V W MnitR DKP AM) Ac OSTON S Fluori changed pancuronium and its deacetylated me metric and chromatographic determination of pancuronium bromide and iLs meltholites in biological materials ( lin tabohtes which may lead to inaccurate conclu ( him Acta 44 ЭД Ы 19"! sions in some studies For example McLeod LFF С KATZ H I AND Iti As I A new instrument foi (1976) found that renal failure markedly pro continuous Γοοο^ιημ of the evoked compound eleclromv ograph in the clinical setting Anesth Analg К leve I 56 longed the elimination half life of pancuronium 2MI >"(> 14-7 and assumed that the delayed elimination from MtlMH) К WATSON M I AND HAWLINS M I) Phairm cokinetics of pancuronium in patients with normal ant plasma represents accumulation of pancuron impaired renal function Brit I Amesth 4H I'll 14Γ> ΙΤΙ> mm However it may in fact represent accu MiiifR Η I) МАТТЮ К S ШМІ I / AND SOHN Y I mulation of relatively inactive metabolites In 1 he pharmacokinetics of d tuboturarine in man with anc without renal failure J Phamiacol i-xp Fher 202 1 7 our study it is possible that the pancuronium 14/- and 1 OH pancuronium plasma decay curves NORMAN J AND ΚΑΤ/ It I Some effects tf the steroida represent some contribution from 17 OH and muscle relaxant dacuronium hre midi in aneslhelmd pa ticnLs Hnt J /Vnaeslh 43 )П 114 ГП 3,17 OH pancuronium If the metabolic conver SAVARI-SF I I DONI os J V AND Αι ι H H Hum in close Sion of pancuronium to its metabolites is sequen rtspon.se curves for neuromuscular bloc king agents A com tial, an underestimation of the rate of decay of panson cif two methods of construction and analysis lì Abstrae Ls of Scientitic Papen* IS"·! Annual Meeting of th pancuronium from plasma may occur ( ι e the Ameritan Society of AnesthesmlogisLs Washington D С elimination half life may be shorter) This may pp 121 122 1474 account for the slightly longer (although statis SoMoe γι A A SHANKS ( \ AND I Rie с s I- J Disposino kinetics of pancuronium bromide in patienLs with lota tically insignificant) elimination half life of pan biharv obstruction Brit J Anaesth 49 1101 110" 197 curomum Because the semiquantitative studies SOMCM γι A A SHANKS С A AND I Rie с s F J С Imita pharmacokinetics of pancuronium bromide Ь ur J Clin of Agoston et al (1973b) Buzello (1975) and I htmiacol 10 )()7 172 197b Somogyi et al (1977) suggest limited metabo VAN Rosst M ) M Signifie ante of pharmacokinetics for drti| design and the planning of dosage regimen-s In Drug De lism of pancuronium and because of the possible г shorter half lives of the metabolites we think sign pp 49 ) 50J Aeadtmic Press Ine New York 1971 this is unlikely However, definitive proof awaits Send reprint requests to Ronald D Miller M D Depart a more precise method for measuring pancuron ment of Anesthesia 5Л4Ь University of California San Fran mm concentrations cisto Calif 94143

30. CHAPTER 5 INTERACTION BETWEEN PANCURONIUM AND EACH OF ITS DEACYLATED METABOLITES IN IN VIVO AND IN VITRO RAT PREPARATIONS Leo H.D.J. Booij, M.D., Ronald D. Miller, M.D., Laura Roderick, Francien van der Pol, Jan F. Crul, M.D., Ph.D. Submitted to Anesthesiology

Abs tract We studied the interaction of pancuronium and its metabolites in vivo and in vitro rat muscle preparations. In vivo, the effect of the metabolites was additive to the effect of pancuronium. In vitro, however, a slight antagonism was observed. A cardiovascular explanation was proposed. It is concluded that the interaction of pancuronium and its metabolites is not important to the activity of pancuronium. Prolonged pancuronium blockade and problems with re­ versibility can thus not be explained from such an interaction.

Introduotion Prolonged neuromuscular blockade from pancuronium, and problems in reversing the blockade may be the result of several factors. Different pharmacokinetics, as compared to normal, in patients with renal failure or hepatic diseases have been demonstrated (McLeod et al. 1976; Somogyi et al. 1977а'Ь). Hypothermia produces a aifrer- ent pharmacokinetic behavior (Miller et al. (1У78а), and acid-base balance disturbances mainly influence the pharmacokinetics as well (Crul-Sluijter et al. 1974; Miller et al. 1976). Pancuronium is par­ tially metabolized by deacetylation (Agoston et al. 1973a'^; Buzello 1975). The resulting hydroxy derivatives possess neuromuscular blocking activity (Miller et al. 1978':') . Nevertheless, theoretical­ ly only the 3-OH metabolite may contribute to the pancuronium block­ ade because of the amount produced and its potency. The metabolites do not differ from pancuronium pharmacokinetically (Miller et al. 19781:,) . For reversal of equal degrees of blockade, the 17-OH and 3,17-OH derivatives need significantly more neostigmine, but all can be reversed completely (Booij et al. 1979). Therefore, prolonged duration of action and problems in reversing pancuronium blockades are not likely to be the result of the potency, pharmacokinetics, or reversibility of the individual metabolites. A remaining possibili­ ty may be an interaction between pancuronium and its metabolites. In this study we determined whether such an interaction exists in both in vitro and in vivo rat preparations.

Methods A. In vitro rat hemidiaphragm preparation Rat hemidiaphragms suspended in a Krebs solution at a constant temperature of 370C, bubbled with a carbon dioxide 5% and oxygen 95% mixture, were supramaximally stimulated. The st- mli were delivered by a Grass S44 nerve stimulator at a rate of 0.' > Hz and 0.2 msec duration. When twitch height remained constar a dose of pancuron-

31. iura or one of its metabolites was added to tbe bathing fluid, resulting in a depression of twitch height. Incremental doses of the same com­ pound were added and the resulting twitch depression recorded. Linear regression analysis of the data resulted in cumulative dose response curves for pancuronium and each of its metabolites. In the next group of hemidiaphragms, pancuronium or one of the metabolites was added to the bathing fluid in a 10% blocking dose. Incremental a- mounts of pancuronium were again administered, and linear regres­ sion analysis of the data performed. The dose response curves pancuronium were compared by analysis of covariance. ECso's (con­ centration resulting in 50% depression of twitch tension) were calculated from the regression line.

B. In vivo rat muscle preparation Rats were anesthetized with pentobarbital 40 mg/kg intrapen- toneally. Via a tracheostomy, ventilation was controlled by a Braun air pump. The left jugular vein was cannulated for drug administra­ tion. Arterial blood pressure was continuously monitored through a cannula in the left carotid artery. The tibialis muscle tendon was freed and connected to a force displacement transducer with the rest­ ing tension adjusted to 20 gm. A Grass S44 stimulator delivered supramaximal stimuli to the sciatic nerve at a rate of 0.1 Hz and 0.2 msec duration. The resulting twitch tension was recorded on a polygraph, together with the arterial blood pressure and heart rate. Rectal and muscle temperature were maintained at between 37 and 380C. When twitch tension was constant for 10 minutes, a constant 10 to 15% twitch depression was obtained by continuous infusion of pancur­ onium or one of its metabolites. Then incremental doses of pancur­ onium were administered to construct a cumulative dose response curve. Each consecutive dose was administered when 3 succeeding twitches were of the same height. Linear regression analysis of the data resulted in dose response curves. Analysis of covariance was performed to compare the regression lines with each other. EDSQ'S (dose resulting in 50% depression of twitch height) were calculated from the regression lines.

Results A. In vitro rat hemidiaphragm Cumulative dose response curves for pancuronium and each of its deacylated metabolites (fig. 1) did not deviate from parallelism (p>0.50). Except for 17-0H- and 3,17-diOH-pancuronium (p>0.05), they were significantly different from each other (p<0.01). Pancur­ onium was 2.36 times more potent than 3-OH-pancuronium, 50.52 times more potent than 17-0H-pancuronium, and 45.24 times more potent than 3,17-diOH-pancuronium. Their ЕВзд'з were 1.878 yg/ml, 4.435 pg/ml, 94.876 pg/ml, and 84.954 pg/ml, respectively. In 8 hemidiaphragms, pancuronium 1.27 pg/ml-1 (the blocking dose) was added to the bathing fluid, resulting in 6.5 ± 3.9% depression of the twitch height, and a cumulative dose response curve for pancuronium was again determined (fig. 2). In another 8 diaphragms, 3-OH-pancuronium 2.75 vig/ml-1 was added to the bathing fluid, resulting in a 3.3 ± 3.3% depression of twitch height, and a cumulative dose response curve for pancuronium was

32. determined (fig. 2). The same was done in 12 hemidiaphramgs with 66.1 vg/ml 3,IT-diOH-pancuronium, resulting in 4.2 ± 4.4% depression (fig. 2). Although they were shifted to the left, the obtained dose response curves did not deviate from parallelism (p>0.50), and were not different from each other (p>0.50) (fig. 2). All these loading doses were equipotent to 1.27 pg/ml-! pancuronium. When a correction was made for the additional dose, the shift of the dose response curves disappeared completely and the curves were not different from the original pancuronium dose response curve (p>0.50). ЕОзо'з were calculated from the dose response curves and corrected for the load­ ing dose (table 1). There was no difference between these EDßo's; if anything, there was antagonism of pancuronium by its metabolites in vitro.

Table 1. EDso's for pancuronium with and without a loading dose of pancuronium, 3-OH-pancuronium, 17-OH-pancuronium, or 3,17-diOH- pancuronium equipotent to 1.27 pg/ml pancuronium. ED50 ED50 corrected

pancuronium 1.88 1.88 pancuronium + pancuronium 0.83 2.10 pancuronium + 3-OH-pancuronium 0.79 2.06 pancuronium + 17-OH-pancuronium 1.00 2.27 pancuronium + 3,17-diOH-pancuronium 1.01 2.28

B. In vivo rat tibialis muscle preparation Constant infusions of pancuronium causing a constant 15.2 + 5.2% twitch depression, 3-OH-pancuroniuin resulting in 14.5 ± 3.7% twitch depression, 17-OH-pancuronium giving a 14.5 ± 10.3% twitch depression, and 3,17-diOH-pancuronium leading to 12.5 ± 5.9% depression were res­ pectively administered in the successive groups of rats. The depres­ sion of twitch height by these continuous infusions were not differ­ ent from each other (p>0.50). Then a cumulative dose response curve for pancuronium was made on top of this (fig. 3). The resulting curves did not deviate from parallelism (p>0.50). Although there was a tendency towards significant difference when 3-OH-pancuronium was infused (p<0.10), there was only a difference for 17-OH and 3,17-diOH from pancuronium (p<0.01). This means that in vivo there may be an antagonistic effect by the metabolites on the activity of pancuronium.

Ог s eus s г en Pancuronium is metabolized in the human body. It is most likely that the metabolites are the 3-(JH, 17-OH, and 3,17-diOH derivatives. In plasma, urine, and bile, only 3-OH-pancuronium has so far been detected in relevant amounts. With improvement of tne detection methods, rate and exact amount of metabolism may be determined. That all proposed metabolites possess neuromuscular blocking activity

(Miller et al. 1978b) Was confirmed in this study (fig. 1). When

33. юо

S 75

_ 50

25

15 25 6 8 10 20 30 50 70 100 dose (nig/ml) Figure I. Cumulative concentration response curves for pancuronium and its metabolites in the rat hemidiaphragm preparation (0—0: pancuronium; X—X: 3-OH-pancuronium; ·-·: J 7-OH-pancuronium; '-СУ- 3, 17-diOH-pancuroniuni).

100

Ä 75

о

50

с о и 03 ω 25 α. (U •α

-τ 1 1— 05 1.0 1.5 20 Pancuronium (rtjg/ml)

Figure 2. In vitro cumulative concentration response curves for pancuronium when a small amount of a metabolite or pancuronium is added to the bathing fluid (o—o: pancuronium; X—X: 3-OH-pancuronium;·-·: 1 7-0H-pancuronium; ..-LJ: 3,17-di0H-pancuronium).

34. 100 с о I 75 .с и 1 " 50 о с о и tu ^0 α> •σ

7 5 15 30 45 75 Pancuronium (/ug/kg) Figure 3. Cumulative dose response curves for pancuronium m vivo on top of a constant 10% blockade by pancuronium or one of its metabolites in rats (O—0: pancuronium; X—X. 3-0H-pancur- onium; ·—·: 17-OH-pancuronium; — : 3,17-di0H-pancuronium). adnunistered simultaneously in vitro, each of the metabolites and pancuronium had an additive effect. In vivo there was an antagon­ izing effect of the metabolites upon pancuronium. This was especially so with 17-OH and 3,17-diOH-pancuronium. These two com­ pounds possess more cardiovascular effects than pancuronium and 3-0H- pancuronium (Booij, unpublished observations), and may thereby in­ fluence the availability of pancuronium at the receptor. In patients receiving 17-OH or 3,17-diOH-pancuronium, mydriasis and bradycardia were observed in most cases (Booij, unpublished observations). Con­ trary to mixtures of other nondepolarizing relaxants (Wong et al. 1971; Park et al. 1974), the effects of pancuronium and its metabol­ ites in vitro are only additive. In vivo there is a tendency to­ wards antagonism. This means that the metabolism of pancuronium is, considering its interaction, not an important factor in explaining prolonged pancuronium blockade or problems with reversal.

References

Agoston S., Kersten U.W., and Meyer D.K.F. (1973a). The fate of pancuronium bromide in the cat. Acta Anaesth. Scand. 17, 129-135. Agoston S., Vermeer G.Α., Kersten U.W., and Meyer D.K.F. (1973b): The fate of pancuronium bromide m man. Acta Anaesth. Scand. 17, 267-275. Booij L.H.D.J., Miller R.D., Jones M.J., and Stanski D.R. (1979): Antagonism of pancuronium and its metabolites by neostigmine in cats. Anesth. Analg. 58, 483-486. Buzello W. (1975): Stoffwechsel von Pancuronium beim Menschen. Der Anaesthesist 24, 13-16.

35. Crul-Sluijter E.J., and Crul J.F. (1974): Acidosis and neuromuscular blockade. Acta Anaesth. Scand. 18, 224-236. McLeod K., Watson M.J., and Rawlins M.D. (1976): Pharmacokinetics of pancuronium in patients with normal and impaired renal failure. Br. J. Anaesth. 48, 341-345. Miller R.D. (1976): Recent developments with muscle relaxants and their antagon­ ists. Can. Anaesth. Soc. J. 26, 83-93. Miller R.D., Agoston S., van der Pol F., Booij L.H.D.J., Crul J.F., and Наш J. (1978a): Hypothermia and the pharmacokinetics and pharmacodynamics of pancur­ onium in the cat. J. Pharmacol. Exp. Ther. 207, 532-538. Miller R.D., Agoston S., Booij L.H.D.J., Kersten U.K., Crul J.F., and Ham J. (1978''): The comparative potency and pharmacokinetics of pancuronium and its metabolites in anesthetized man. J. Pharmacol. Exp. Ther. 207, 539-543. Park W.Y., Balingit P.E., and MacNamara Т.Е. (1974): Interactions of gallamine and pancuronium with tubocurarine under morphine-nitrous oxide-oxygen anesthesia in man. Anesth. Analg. 53, 723-729. Somogyi A.A., Shanks C.A., and Triggs E.J. ((1977a): The effects of renal failure on the disposition and neuromuscular blocking action of pancuronium bromide. Eur. J. Clin. Pharmacol. 12, 23-29. Somogyi A.Ac, Shanks CA., and Triggs E.J. (1977°); Disposition kinetics of pan­ curonium bromide in patients with total biliary obstruction. Br. J. Anaesth. 49, 1103-1108. Wong K.C., and Jones J.R, (1971): Some synergistic effects of d-tubocurarine and gallamine. Anesth. Analg. 50, 285-290.

36. CHAPTER 6 REVERSIBILITY OF PANCURONI UM-INDUCED NEUROMUSCULAR BLOCKADE 6.1. The effect of Cholinesterase inhibiting drugs; A literature review Nondepolarizing neuromuscular blockades can be reversed by cholinesterase-inhibiting drugs (Miller 1976a). In the literature many case reports appeared on recurarisation and irreversibility of blockades (Baraka 1967; Miller et al. 1976ь). Most cases can be explained by inadequate antagonism due to the administration of in­ sufficient amounts of Cholinesterase inhibitors (Lee et al.1977). Prob­ lems with reversibility of pancuronium blockade in other cases are due to factors influencing the effect of the anticholinesterasic drugs. The mechanisms by which the anticholinesterases exert their effect are (Katz 1967): 1. inhibition of true Cholinesterase, 2. increase in acetylcholine release, 3. acetylcholine-like stimulation of motor end-plate receptors, 4. displacement of curarizing drug from the postjunctional receptors, and 5. repetitive firing in the motor nerve. It has not as yet been established which mechanism is the most im­ portant for the anticurarc effect. This may depend partly on the anticholinesterase drug administered (Smith et al. 1952; Blaber et al. 1959; Blaber 1963a; Blaber et al. 1963ь; Barrow et al. 1966; Blaber 1972; Lee et al. 1978; Tiedt et al. 1978; Main 1979). In clinical practice, neostigmine and pyridostigmine are most usually used. Edrophonium is less popular since some authors feel its dura­ tion of action is too short, and its effect unreliable (Katz 1967; Nastuk et al. 1954). This has recently been denied (Lee et al. 1977; Sevan 1979; Kopman 1979).

Neostigmine is about 4 to 5 times as potent as pyridostigmine in antagonizing pancuronium blockade (Fogdall et al. 1973; Gyermek 1975) , while its anticholinesterase activity is 20 times as strong (Randall et al. 1955) . Edrophonium is ï as potent as neostigmine in antagonizing neuromuscular blockade (Randall 1951) with an anti­ cholinesterase activity that is 1/100 to 1/200 that of neostigmine (Bevan 1979; Randall et al. 1955). Pyridostigmine and edrophonium are said to result in comparatively less severe muscarinic side effects. Pyridostigmine should therefore lead to less severe cardio­ vascular side effects (Gyermek 1975). Also, compared to the other compounds, it causes less increase in intestinal motility, and is therefore likely to result in less disrupture of intestinal anasto­ moses (Brown et al. 1973) . Other groups found no difference in cardiovascular side effects between neostigmine and pyridostigmine (Fogdall et al. 1973). Onset time and duration of action of pyrido­ stigmine are longer (5-7 minutes and 40%, respectively) than with neostigmine (Miller et al. 1974). The amount of Cholinesterase inhibitor necessary for antagonism depends on the depth of block and not on the amount of relaxant ad­ ministered (Miller et al. 1972). The activity of neostigmine, and probably also of pyridostigmine, is affected by disturbances in the

37. acid-base balance. In the case of respiratory acidosis, neostigmine is unable to completely reverse a pancuronium neuromuscular blockade (Miller et al. 1978a), while pancuronium in these circumstances is more potent (Crul-Sluijter et al. 1974) . When metabolic alkalosis exists, the effectiveness of neostigmine is decreased and its duration of action shortened (Miller et al. 1978a). A decrease in temperature does not influence the efficiency of neostigmine, but its onset and duration of action are prolonged (Miller et al. 1977a). In the case of hypothermia, however, pancuronium is potentiated by changes in pharmacokinetics and increased receptor sensitivity (Miller et al.

1978b)< Thus, more neostigmine (and probably more pyridostigmine) is necessary to reverse a blockade in hypothermia. For this reason, recuransation upon warming up a patient is unlikely, as has previous­ ly been found (McKlveen et al. 1973).

When renal failure exists, the duration of pancuronium blockade is prolonged, as well as the effects of neostigmine and pyridostig­ mine (Miller et al. ІЭ??13). This has recently been proven pharmaco- kinetically (Cronnelly et al. 1979); the elimination half-life time is prolonged and the total serum clearance diminished. Hypokalaemia increases the amount of neostigmine required to antagonize a pancur­ onium blockade (Miller et al. 1978е).

Edrophonium possesses less severe muscarinic side effects and its onset of action is much faster. Combination with pyridostigmine may be advantageous (Gyermek 1977).

The muscarinic side effects of the Cholinesterase inhibitors are due to accumulation of acetylcholine at the muscarinic receptor (Owens et al. 1978) . Atropine can block this receptor and therefore decrease these side effects. However, atropine has a shorter dura­ tion of action, possibly resulting in a subsequent period of brady­ cardia when its effect has worn off. Muscarinic receptors are pre­ sent in the presynaptic part of the cardiac sympathetic nerve and influence catecholamine release (Sharma et al. 1978). Acetylcholine accumulation inhibits noradrenaline release and causes bradycardia through this mechanism. Since this noradrenaline is released at the ß-adrenergic junction (Lokhandwala 1979), ß-blockers in combination with Cholinesterase inhibitors may cause adverse reactions (Heinonen et al. 1977). Also, interaction between neostigmine and amitrypti- line has been described (Glisson et al. 1978). All these adverse reactions of the anticholinesterases can be diminished by decreasing the dosage. It recently became obvious that this is possible by combining neostigmine or pyridostigmine with 4-aminopyridine (Chapter 6.5 and 6.6). Due to all the afore-mentioned reasons, only one poss­ ibility for failing antagonism of pancuronium remains. Since pancur­ onium is metabolized (Chapter 2), there may for some reason be an ac­ cumulation of the metabolites. These metabolites may not be reversi­ ble by Cholinesterase inhibitors. We have investigated this (Chapter 6.3.) .

Peferences

Baraka A. (1967): Irreversible tubocuranne neuromuscular blockade in the human. Br. J. Anaesth. 39, 891-894. Barrow Ч.Е.Н. and Johson J.K. (1966): A study of the anticholinesterase and anti- curare effects of some Cholinesterase inhibitors. Br. J. Anaesth. 38, 420-431.

38. Bevan D.R. (1979): Reversal of pancuronium with edrophonium. Anaesthesia 34, 614-619. Blaber L.C., and Bowman W.C. (1959): A comparison between the effects of edro­ phonium and choline in the skeletal muscle of the cat. Br. J. Pharmacol. 14, 456-466. Blaber L.C. (1963a): Facilitation of neuromuscular transmission by anticholin­ esterase drugs. Br. J. Pharmacol. 20, 63-73. Blaber L.C, and Bowman W.C. (1963^): Studies on the repetitive discharges evoked in motor nerve and skeletal muscle after injection of anticholinesterase drugs. Br. J. Pharmacol. 20, 326-344. Blaber L.C. (1972): The mechanism of the facilitatory action of edrophonium in cat skeletal muscle. Br. J. Pharmacol. 46, 498-507. Brown E.N., Daughety M.J., and Petty W.C. (1973): Integrity of intestinal anasto­ moses following muscle relaxant reversal with neostigmine. Anesth. Analg. 52, 117-120. Cronnelly R., Stanski D.R., Miller R.D., Sheiner L.B., and Sohn Y.J. (1979): Renal function and the pharmacokinetics of neostigmine in anaesthetized man. Anesthesiology 51, 222-226. Crul-Sluijter E.J., and Crul J.F. (1974): Acidosis and neuromuscular blockade. Acta Anaesthesiol. Scand. 18, 224-236. Fogdall RoP., and Miller R.D. (1973): Antagonism of d-tubocurarine and pancuronium induced neuromuscular blockades by pyridostigmine in man. Anesthesiology 39, 504-509. Glisson S.N., Fajardo L., and El-Etr A.A. (1978): Amitryptiline therapy increases electrocardiographic changes during reversal of neuromuscular blockade. Anesth. Analg. 57, 77-83. Gyermek L. (1975): Clinical studies on the reversal of the neuromuscular blockade produced by pancuronium bromide. I. The effects of glycopyrolate and pyridostig­ mine. Curr. Ther. Res. 18, 377-386. Gyermek L. (1977): Clinical pharmacology of the reversal of neuromuscular blockade. Int. J. Clin. Pharmacol. Biopharm. 15, 356-362. Heinonen J., and Takkunen 0. (1977): Bradycardia during antagonism of pancuronium- induced neuromuscular block. Br. J. Anaesth. 49, 1109-1115. Katz R.L. (1967): Neuromuscular effects of d-tubocurarine, edrophonium and neo­ stigmine in man. Anesthesiology 28, 327-336. Kopman A.F (1979): Edrophonium antagonism of pancuroniunt-induced neuromuscular blockade in man: A reappraisal. Anesthesiology 51, 139-142. Lee С, Mok S., Barnes Α., and Katz R.L. (1977): Absence of "recurarization" in patients with demonstrated prolonged neuromuscular block. Br. J. Anaesth. 49, 485-489. Lee C, Young Ε., and Katz R.L. (1978): Interaction of neuromuscular effects of edrophonium, alpha-bungarotoxin and beta-bungarotoxin. Anesthesiology 48, 311-314. Lokhandwala M.F. (1979): Presynaptic receptor systems on cardiac sympathetic nerves. Life Sci. 24, 1823-1832. Main A.R. (1979): Mode of action of anticholinesterase. Pharmac. Ther. 6, 579- 628.

39. McKlveen J.R., Sokoll M.D., Gergis S.D., and Dretchen K.L. (1973): Absence of recurarization upon rewarming. Anesthesiology 38, 153-156. Miller R.D., Larson C.P. Jr., and Way W.L. (1972): Comparative antagonism of d-tubocurarine-, gallamine- and pancuronium-induced neuromuscular blockade by neostigmine. Anesthesiology 37, 503-509. Miller R.D,, van Nijhuis L., Eger E.I. II, Vitez T.S., and Way W.L. (1974): Comparative times to peak effect and duration of action of neostigmine and pyridostigmine. Anesthesiology 41, 27-33. Miller R.D. (1976a): Antagonism of neuromuscular blockade. Anesthesiology 44, 318-328. Miller R.D., and Cullen D.J. (1976b): Renal failure and postoperative respiratory failure: Recurarization? Br. J. Anaesth. 48, 253-256. Miller R.D., and Roderick L.L. (1977a): Pancuronium-induced neuromuscular block­ ade and its antagonism by neostigmine at 29, 37 and 410C. Anesthesiology 46, 333-335. Miller R.D., and Roderick L. (1977'î): Ligated renal pedicles and duration of action of neostigmine and pyridostigmine. Br. J. Pharmacol. 60, 555-558. Miller R.D. and Roderick L.L. (1978a): Acid-base balance and neostigmine antagon­ ism of pancuronium neuromuscular blockade. Br. J. Anaesth. 50, 317-324. Miller R.D., Agoston S., van der Pol F., Booij L.H.D.J., Crul J.F., and Ham J. (1978°): Hypothermia and the pharmacokinetics and pharmacodynamics of pancuron­ ium in the cat, J. Pharmacol. Exp. Ther. 207, 532-538, Miller R.D., and Roderick L.L, (1978c): Diuretic-induced hypokalaemia, pancuronium neuromuscular blockade and its antagonism by neostigmine. Br. J. Anaesth. 50, 541-544. Nastuk W.L., and Alexander J.T. (1954): The action of 3-hydroxyphenyldimethyl- ethylammonium (Tensilon) on neuromuscular transmission in the frog. J. Pharmacol. Exp. Ther. Ill, 302-328. Owens W.D., Waldbaum L.S., and Stephen CR. (1978): Cardiac dysrhythmia followine reversal of neuromuscular blocking agents in geriatric patients. Anesth. Analg. 57, 186-190. Randall L.O. (1951): Synthetic curare-like agents and their antagonists. Ann. N.Ï. Acad. Sci. 54, 460-479. Randall L.O., Conroy CE., Ferruggia T.M., Kappell B.H., and Knoeppel CR. (1955): Pharmacology of the anticholinesterase drugs mestinon, prostigmin, tensilon and TEPP. Am. J. Med. 19, 673-678. Sharma V.K., and Banerjee S.P. (1978): Presynaptic muscarinic cholinergic recep­ tors. Nature 272, 276-278. Smith СМ., Cohen H.L., Pelikan Ε.К., and Unna R.R. (1952): Mode of action of antagonists to curare. J. Pharmacol. Exp. Ther. 105, 391-399. Tiedt T.N., Albuquerque E.X., Hudson CS., and Rash J.E. (1978): Neostigmine- induced alteration at the mammalian neuromuscular junction. I. Muscle contrac­ tion and electrophysiology. J. Pharmacol. Exp. Ther. 205, 316-339.

40. 6.2 Aminopyridines and neuromuscular transmission; A literature review*

Although 4-aminopyridine (4-AP) was already tested pharmacolo­ gically in the mid-twenties (Dohrn 1925; Dingemanse et al. 1928), it took until the late sixties before great interest was payed to it (Lemeignan et al. 1967, Sobek et al. 1968). Development of more sophisticated electrophysiological techniques and increased atten­ tion to membrane processes were responsible for this. Increased intestinal motility and hypertension upon injection of 4-AP were demonstrated early (Dohrn 1925) as were the local anesthetic and convulsive effects (Dingemanse et al. 1928). These results were confirmed m later experiments (rastier 1948; Tastier et al. 1948;

Abernethy et al. 1958; Fastier et al. 1958а»Ь; von Haxthausen 1955). It was concluded that the aminopyridines produced sympathicomimetic effects (von Haxthousen 1955) both peripherally and centrally. Later on it was demonstrated that this sympathicomimetic effect was not caused by an adrenergic mechanism but by a cholinergic mechanism (Lemeignan 1971) .

In 196 7 it was found that 4-AP antagonized the neuromuscular blocking effects of d-tubocuranne and gallamine. The exact mech­ anism of action remained unknown, although the conclusion from the experiments was that it was neither an anticholinesterase effect nor a direct effect on postsynaptic acetylcholine receptors or muscles (Lemeignan et al. 1967). Sobek demonstrated that the effect was mainly caused by a presynaptic effect (Sobek et al. 1968). In­ creased acetylcholine release was thus postulated. As explanation for this increased release, blockade of potassium channels result­ ing in prolongation of the action potential was found (Hue et al. 1973). Such a blockade has been demonstrated for both inward and outward potassium currents (Pelhate et al. 1974; Yeh et al. 1976a).

Although it was originally stated that 4-AP blocked the potassium channels intra-cellularly (Gillespie et al. 1975; Ulbricht et al. 1976), both intra- and extra-cellular sites of action have now been found (Meves et al. 1975; Yeh et al. 1976ь). Extra-cellularly, 4-AP is able to block open potassium channels, whereas intra-cellularly, 4-AP can block closed channels at depolarization of the membranes (Gillespie 1977) . There is no effect on sodium conductance (Schauf et al. 1976), but an increased calcium movement is observed (Nichol­ son et al. 1976). This is probably due to the prolonged action po­ tential (Leander et al. 1977). The increased intra-cellular calcium then leads to increased quanta! acetylcholine release (Lundh et al. 1977a). The increased acetylcholine release by 4-AP mediated by calcium has been clearly demonstrated both pharmacologically (Vizi et al. 1977; Ules et al. 1978; Lundh 1978; Kim et al. 1980) and morphologically (Tokunaga et al. 1979a'1:,) . 4-AP cannot replace cai- cium ions, which is another proof for calcium mediation (Al-Haboubi et al. 1978). It has recently been proposed that 4-AP acts directly on calcium channels (Molgo et al. 1979). Such a direct calcium effect is also likely to exist in both skeletal and heart muscle where 4-AP can increase contractility. It is likely that in these tissues 4-AP either causes a release of calcium from binding or storage sites, or slows down the binding of calcium intra-cellularly (Yanagisawa et al. 1979). Increased calcium in the muscle then •Part of a paper submitted to Anesthesiology.

41. leads to increased contractility. That calcium is involved can also be concluded from the fact that 4-AP antagonizes dantrolene- induced muscle relaxation by blocking calcium release from the sarcoplasmic reticulum. It can be concluded that 4-AP increases the amount of acetylcholine release from the presynaptic membrane at the motor nerve terminal.

Increased transmitter release by 4-AP is not only seen at the neuromuscular synapse, but also at other sites. Increased contrac­ tility of the guinea pig ileum due to increased acetylcholine release has been shown (Montoki et al. 1978). This cholinergic mechanism is also responsible for the initial sinus bradycardia in isolated sino atrial node preparations of the dog (Yanagisawa et al. 1978). The tachycardia seen with higher amounts of 4-AP is thought to be mediated by adrenergic mechanisms. 4-AP is not only effective in facilitating the transmission in skeletal muscle, but also in smooth muscles with adrenergic and cholinergic terminals (Al-Haboubi et al. 1978). Increase in phrenic nerve activity lead­ ing to ventilatory stimulation may be due to central cholinergic effects (Folgering et al. 1979; See et al. 1978). Besides this, 4-AP increases the release of noradrenaline in various organs such as the rabbit vas deferens (Johns et al. 1976) and ear artery (Glover et al. 1978), the cat spleen (Kirpekar et al. 1977), the rat portal vein (Leander et al. 1977) , and the guinea pig pulmonary artery (Hara et al. 1980). 4-AP even reverses the respiratory de­ pression caused by fentanyl (Sia et al. 1979). Anesthesia induced by ketamine is also reversed by 4-AP (Martmez-Aguirre et al. 1979; Agoston et al. 1980). Another effect that has been demonstrated is the prolongation of action potentials in rat pituitary cell culture and the facilitation of prolactine release (Sand et al. 1980). It must therefore be concluded that 4-AP, by blocking potassium channels, causes a calcium-mediated increase of transmitter release in several synaptic systems both of cholinergic and adrenergic ori­ gin. Thus, 4-AP has many effects, only a few of which will be dis­ cussed here.

Effects on neuromuscular transmission That 4-AP influences neuromuscular transmission was seen in 1967 as an antagonistic effect on d-tubocuranne and gallamine- rnduced neuromuscular blockades (Lemeignan et al. 1967). The mech­ anism was neither due to direct muscle effect, postsynaptic acetyl­ choline receptor effect, nor to an anticholmesterasic effect. However, many groups demonstrated an increase in quantal acetylcho­ line content (Bowman et al. 1977ь, Harvey et al. 1977a; Vizi et al. 1977; Moritoki et al. 1978; liles et al. 1978). Such a quantal content is only increased when the release is evoked (Lundh 1978); there is no increase in spontaneous release. However, increased frequency of stimulation, i.e. above 8 Hz, shows fading of the response. This means that 4-AP only increases release of acetyl­ choline and not its mobilization from storage sites (Molgo et al. 1979; Khan et al. 1979).

4-AP via calcium also exerts a direct muscle effect. The increase in intra-cellular calcium increases contraction of the muscle fibers (Bowman et al. 1977ь; Bowman et al. 1979; Harvey et al. 1977a). Via

42. this mechanism, 4-AP is able to antagonize dantrolene-induced muscle relaxation (Bowman et al. 1977a). Since increased calcium release is thought to be the trigger in the development of malignant hyper­ thermia, it was expected that 4-AP could induce this syndrome in susceptible animals. However, experiments in pigs proven to have malignant hyperthermia could not show such an action (Hall et al. 1980) .

4-AP not only exerts its action when nondepolarizing neuromuscu­ lar blocking agents have impaired neuromuscular transmission, but also when impaired transmission is due to other factors. It has been shown that Eaton-Lambert syndrome can be efficiently treated with 4-AP (Lundh et al. 1977ь; Agoston et al. 1978; Kim et al. 1980ь). After experimental demonstration of the reversibility of botulinum toxin paralysis (Lundh et al. 1977е), some patients have been treated successfully (Ball et al. 1979).

4-AP increases muscle strength in patients with myasthenia gravis (Lundh et al. 1979). Aminoglycosides, polymyxin B, tetracyclines, lincomycine and clindamycme are known to cause neuromuscular blockade and to enhance the effect of nondepolarizing relaxants. Their anta- gonisability with Cholinesterase inhibitors is questionable. 4-AP has proven to be the only reliable and efficient antagonist for these compounds (Booi] et al. 1978; Singh et al. 1978; Burkett et al. 1979; Lee et al. 1979, 1978; Bruckner et al. 1980; Rutten et al. 1981). We studied whether 4-AP antagonized the antibacterial activity of the antibiotics as well (Chapter 6.8).

Although 4-AP has been advocated for clinical use in the antagon­ ism of nondepolarizing neuromuscular blockades (Stoyanov et al. 1976), it is only adequate when administered in amounts that cause central nervous system effects. Thus, it should not be used for this purpose. Since 4-AP enhances acetylcholine release, and Cholinesterase inhibi­ tors decrease the breakdown of acetylcholine, interactions between these compounds may exist. We were able to demonstrate these inter­ actions (Chapter 6.5, 6.6, 6.7). Also, 3,4-di-AP acts by blockade of potassium channels leading to prolongation of the falling phase of the action potential, enhancing acetylcholine release (Kirsch et al. 1978). It is an even better neuromuscular transmission facilitating drug than 4-AP (Durant et al. 1978, 1980; Harvey et al. 1977ь; Molgo et al. 1980).

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47. Antagonism of Pancuronium and Its Metabolites by Neostigmine in Cats Leo H.D.J. Booij, MD/ Ronald D. Miller, MD/f Marjorie J. W. Jones,}: and Donald R. Stanski, MD§

Boou L H D J MILLER R D JONES M J W AND STANSKÎ D R Antagonism of pancuro­ nium and its metabolites by neostigmine m cats Anesth Analg 5Θ 4Θ3-4Θ6 1979

Antagonism by neostigmine of r euromjscular blockade produced by pancuronium or its metabolites was studied in the cat anterior tibialis muscle-peroneal nerve preparation using constant infusions of muscle relaxants The EDso of neostigmine (dose which caused a 50% antagonism) was 16 11 29 and 26 ^g ''kg for pancuronium 3-hydroxypancuronium 17 hydroxypancuromum and 3 17 hydroxypancuromum respectively Times of onset of neostig­ mine action were shorter when antagonizing 17-hydroxypancuronium neuromuscular block­ ade Duration of neostigmine action when antagonizing 1 7- or 3 17-hydroxypancuronium blockade was shorter than with pancuronium or 3-hydroxypancuronium We conclude that more neostigmine is required to antagonize 17 or 3 17-hydroxypancuronium neuromuscular blockade than is required to antagonize pancuronium Conversely less neostigmine was required to antagonize 3-hydroxypancuronium blockade

Key Words NEUROMUSCULAR RELAXANTS pancuronium BIOTRANSFORMATION (DRUG) pancuronium

EVERSAL of a pancuronium neuromuscular mum As much as 20 to 30% of an injected dose of R blockade is occasionally difficult particularly pancuronium may be metabolized into one of its after prolonged administration or in patients with hydroxylated metabolites * In man pancuronium was renal or hepatic dysfunction l 2 One explanation is found to be 2 50 and 52 times as potent as its that delayed plasma clearance of pancuronium may metabolites 3-hydroxypancuronium 17 hydroxy result in a neuromuscular blockade too intense to be pancuronium and 3 17 hydroxypancuromum 6 In this antagonized * Another possibility is that with im­ study we determined the ability of neostigmine to paired routes of excretion, metabolites of pancuro­ antagonize the neuromuscular blockade produced by nium may accumulate and cause a neuromuscular the hydroxylated metabolites of pancuronium blockade in addition to that produced by pancuro- Methods * Senior Clinical and Research Associate of Anesthesia Catholic University Nijmegen The Netherlands This work was performed Twenty seven cats were anesthetized with chlora- while Dr Booi| was a visiting professor in San Francisco supported by a Netherlands Organization for the Advancement of Pure lose 250 mg/kg and urethane 60 mg/kg tntrapen- Research (ZWO) NATO grant toneally Ventilation was controlled thereafter via a t Professor of Anesthesia and Pharmacology University of Cal tracheostomy Both external jugular veins were can- ifornia San Francisco nulated for administration of drugs Arterial blood í Staff Research Associate University of California San Fran CISCO pressure was recorded from a cannula in the carotid tj Clinical Pharmacology Fellow University of California San artery The tibialis anterior muscle tendon was freed Francisco sectioned near its point of attachment and attached to Supported in part by United Slates Public Health Service Grants CM 15571 12 and I ROI CM 26403 01 a Grass FT10 force displacement transducer A stim Received from the Departments of Anesthesia and Pharmacol ulating shielded platinum bipolar electrode was ogy University of California San Francisco San Francisco Cali placed on the sciatic nerve after it had been sectioned forma 94 143 Accepted for publication |uly 5 1979 Reprint requests to Dr Miller Department of Anesthesia (4365) near its point of attachment Supramaximal stimuli

48. were delivered at a rate of 0 15 Hz and 0 2 msec duration The resultant twitch contraction was re­ corded on a polygraph Temperature was maintained between 36 5 and 37 5 С After an intravenous bolus of pancuronium or one of its metabolites, a continu­ ous infusion with the same compound was started at a rate adequate to maintain 90% depression of twitch tension When depression of twitch tension and rate of infusion were constant for at least 20 minutes, a bolus of neostigmine, 5, 10, 20, 30, 50, or 70 /ig/kg was administered intravenously, while the infusion of relaxant continued The TO-jig/kg dose wa$ studied only when reversing a 17-hydroxypancuronium neu­ romuscular blockade Fürth e ι details of this constant- infusion technique have been desenbed previously ' The resultant maximal antagonism of twitch depres­ sion was calculated as a percentage of the preexisting 5 10 20 30 50 Θ0 100 90% twitch depression We also measured the time from the administration of neostigmine to peak effect NEOSTIGMINE

Linear regression analysis and unpaired f-tests were earned out for part of the statistical analyses Dose- 3117-hydroxypancuronium ( ρ > 0 10) (4) Neostig­ response curves of neostigmine while antagonizing mine dose-response curves did not deviate from par­ pancuronium and its metabolites were compared by allelism ( ρ > 0 10) analysis of covanance and the Sheffe's multiple con­ The onset times of neostigmine were shortest when trast procedure β antagonizing a 17-hydroxypancuronium blockade ( ρ < 0 05) (Fig 2) Duration of action of neostigmine Results when antagonizing a 17- or 3,17-hydroxypancuro- mum neuromuscular blockade was less than when The doses of neostigmine required for antagonism pancuronium or 3-hydroxypancuronium were being of a pancuronium neuromuscular blockade and for antagonized ( ρ < 0 05) (Fig 2) antagonism of its metabolites differed significantly (F « 24 6 ρ < 0 001) (Fig 1) The ED» (dose of neostig­ mine which caused a 50% antagonism) was 16, 11, 29, Discussion and 26 μg/kg for pancuronium, 3-hydroxypancuro- As much as 20 to 30% of an injected dose of mum, 17-hydroxy pa neuroni urn, and 3,17-hydroxy- pancuronium is metabolized pnmanly to 3-hydroxy- pancuromum, respectively (Fig 1) Analysis of the pancuronium and to a lesser extent 17- and 3,17- dose-response curves in Fig 1 indicate that (1) More hydroxypancuromum 5 Because only 50 to 60% of the neostigmine was required to antagonize a neuromus­ administered pancuronium usually is recovered in the cular blockade from 17-or3,17-hydroxypancuronium urine and bile of most studies,5 a 10 the actual extent than was required to antagonize neuromuscular to which pancuronium is metabolized may, however, blockade produced by 3-hydroxypancuronium or be more than estimated 3-Hydroxypancuronium is a pancuronium itself ( ρ < 0 05) (2) Less neostigmine moderately potent neuromuscular blocking agent was required to antagonize a 3-hydroxypancuronium However, even if large amounts of this metabolite neuromuscular blockade than was required to antag­ were to accumulate after pancuronium, no problem onize a pancuronium block ( ρ < 0 05) (3) Neostig­ with antagonism should occur since less neostigmine mine dose-response curves were the same for 17- and is required to antagonize the neuromuscular blockade

49. JOH Pc I70H Pc 3,I70H Pc droxypancuromum neuromuscular blockade than that required for pancuronium, but the duration of antagonism was significantly shorter (Fig 2) Perhaps Onsei P. Time . ^; 17- or 3,17-hydroxypancuronium has a greater affin­ ι mr ) f-\t- ity for the cholinergic receptor at the postjunctional '-г и ι membrane than does pancuronium Although an at­ ι, I • tractive hypothesis, binding constants for the metab­ 1 ¡ι Ι' ' Ι Η Ι ι ι olites have not been determined Perhaps these me­ Λ,Λί i .11 ILI Ι i tabolites inhibit acetylchohnerase This would result in more acetylcholine at the synaptic cleft, requiring more muscle relaxant to compete for the receptor Aclion (mm) before neuromuscular blockade is produced This ! I Í explanation would explain the lesser potency of the I 17- or 3,17-hvdroxypancuronium metabolite Also, _ ML n- ι inhibition of acetylcholinesterase may limit the ability сгою с югокю of added neostigmine to further inhibit acetylcholin­ Meosiigmioe {pq'bq} esterase Obviously, the ability of these metabolites FIG 2 Comparison of onset time (lime from neostigmine admin­ istration to peak effect) and duration of action (time from neo­ to inhibit acetylcholinesterase needs to be determined stigmine administration to 50% restoration of the pancuronium in order to explore this hypothesis further Lastly, the depressed twilch tension) Bars represent means ± 1 SE Val­ less potent muscle relaxants obviously require higher ues m parentheses represent N plasma concentrations to produce a given level of neuromuscular blockade Do the increased numbers from this metabolite than from pancuronium itself of muscle relaxant molecules interfere in some way (Fig 1) Even though more neostigmine is required to with the kinetics of neostigmine inhibition of acetyl antagonize the neuromuscular blockade from the Cholinesterase7 In any event, our observations" sug­ other two metabolites, this should not represent a gest an inverse relationship between potency of mus­ significant clinical problem since these metabolites 6 cle relaxants and amount of neostigmine required for are not very potent as neuromuscular blockers and antagonism of neuromuscular blockade Additional are not present in concentrations as great as those studies are required to confirm the validity and mech­ observed with 3-hydroxypancuronium However, anisms of the possible relationship these conclusions should be viewed with some cau­ tion We determined the ability of neostigmine to In summary, significantly more neostigmine is re­ antagonize the neuromuscular blockade produced by quired to antagonize the neuromuscular blockade these metabolites when administered singly With produced by 17- and 3,17-hydroxypancuronium me­ prolonged administration of pancuronium, the neu­ tabolites than is required to antagonize the blockade romuscular blockade probably is a result of the com­ produced by pancuronium or its 3-hydroxypancuro- bined effects of pancuronium and all 3 of its metab­ mum metabolites Since the 17 and 3,17-hydroxy- olites We assume that since the neuromuscular pancuromum metabolites are not potent as muscle blockade from pancuronium or one of its metabolites relaxants and apparently do not appear in large is easily antagonized, a blockade from a mixture of amounts, no clinical problems should result insofar these compounds would also be easily antagonized as reversal of pancuromum-induced neuromuscular by neostigmine blockade is concerned Our present data suggest that muscle relaxants that are not very potent required significantly more neo­ REFERENCES stigmine to antagonize the resultant neuromuscular 1 BelafskyMA Klawans HL Prolonged ncuromimuldr blockade blockade For example, 17- and 3,17-hydroxypancu- with pancuronium bromide in a young healthy woman Anes­ thesiology 40 295-296 1974 6 ronium are less potent than pancuronium but require 2 Abrams RE Hornbein TF Inability to reverse pancuronium more neostigmine for antagonism of their neuromus­ blockade in a patient with renal and hepatic disease Ane^the cular blockade Similarly, gallamine which is less siologv 42 Э62-Э64 1975 3 Duvaldestm Ρ Agoston S Menzel Ό et al Pancuronium phar potent than pancuronium or d-tubocuranne also re­ macokinetics in palienls with liver cirrhosis Br J Anaesth 50 quires more neostigmine for antagonism of its neu­ 1131 1136 1978 romuscular blockade " Not only was more neostig­ 4 McLeod К Watson MI Rawlins MD Plasma concentration of pancuronium bromide in patients isith normal and impaired mine required for antagonism of a 17- or 3,17-hy- renal function Br J Anaeslh 48 341-M5 1975

so. 5 Agoston S, Vermeer CA, Kersten LW, et dl Ihe Ые of Homcwood IL RD Irwin Co, 1974, pp 160-165 6B9-703 436- pancuronium bromide in man Acta Arwiesthesiol Scand 17 45C 4Μ-4β2 267-275 1473 9 Buzeìlo W Der Stoffwechsel von Pancuronium Beim 6 Miller RD Aposton 5, Booip LHDJ, et al Tbc comparativo Menschen Anaesthesisl 24 13-18 1975 potency and pharmacokinetics of pancuronium and its metab­ 10 Somogvi AA, Shanks С A Triggs CI Disposition kinetics of olites in anesthetized man | Pharma tel Exp Thcr 207 539-543 paniuronium bromide in patients with total biliary obstruction 1

51. 6.4. Analysis of drug combinations When two drugs are studied for their possible interaction, prob­ lems often exist in analysis and graphic presentation of the results. To overcome this problem, the concept of isoboles and isobolograms was developed (Loewe 1953, 1959). With this method, discrimination between synergistic, antagonistic, and summative (additive) inter­ action is possible. Isoboles are principally connecting lines bet­ ween equipotent doses of compounds A and В and their combinations. To explain the theoretical fundamentals of this method, we will con­ sider 3 extreme interactions. First a pure summation, second a pure antagonism, and third a pure synergism.

Pure Summation Dose response curves for equipotent compounds A and В are drawn m a rectangular system (fig. 1). Suppose they interact purely sum- matively. Then two molecules of A plus two molecules of В will have the same effect as four molecules of either A or В and so on. The dose response curves for these combinations will thus form a sail of lines that are in one plane (fig. 2). Equipotent combinations can be connected by a straight line. This line can be drawn as an isobole in an isobologram (fig. 3).

•DA Figure 1. Dose response curves for compounds A (OA) and В (OB) in one rectangular system.

52. •од Figure 2. Dose response curves for compounds A (OA) and В (OB) and their combinations when purely summative. Equipotent combinations can be connected by a straight line (CD).

Figure 3. Isobologram for dose re­ sponse curves from fig. 2.

53. Pure Antagor.isrr When a theoretically extreme pure antagonism exists between the two compounds, their combinations will behave differently. The individual drugs will give dose response curves as in fig. 1. Suppose they are equipotent, i.e. one molecule of drug A has the same effect as one molecule of drug B, and further, that one mole­ cule of A can antagonize one molecule of В completely, and vice versa. When equimolar doses are administered, the dose response curve for the combination will then be in the plane of zero effect (fig. 4).

For simplicity, we consider the other combinations to be in two flat sails. Only in the zero effect plane will all the dose response curves have a common isobole. Plotted in an isobologram, it will be presented as a line to the far end (fig. 5). At other levels, only part of the combinations can be connected by isoboles which are in the isobologram rectangular to the X and Y axes. When the antagonism is not that extreme, i.e. the combination dose response curves are not presented in two rectangular sails and the equimolar combinations are not in the zero effect plane, they form a curved sail (fig. 6).

The isoboles are then curved lines in the isobologram (fig. 7)

ligure 4. Theoretical dose response curves for extreme antagonistic-acting Compounds A and В and their combinations.

54. Figure 5. Isobologram for fig. 4 when equi- potent antagonistic doses are considered in the zero effect plane.

Figure 6. Dose response curves for compounds A and В «hen an antagonistic interaction is present. Equipotent points are connected by an isobole (CD).

55. Figure 7. Isobologratn for dose response curves from fig. 6. Pure Synergism With theoretically extreme synergistic drugs, the combination of the smallest amount of compound A with the smallest amount of compound B, maximum effect already exists. In this case, the ex­ treme dose response curve is in fact the Effect axis (fig. 8). The isobole for all theoretical combinations, then, is in the plane of compound A and B.

Figure 8. Theoretical dose response curves for compound A and В when an extremely synergistic interaction exists. Equipotent points are con­ nected by an isobole (CD).

56. Plotted in an isobologram, it will be the X and Y axes (fig. 9)

CD* •DA Figure 9. Isobologram for fig. 8. When theoretically less synergistic combinations are used, the dose response curves for the combinations will form a curved sail (fig. 10) with a concomitant isobole that can be plotted in an isobologram (fig. 11).

Figure 10. Dose response curves for compounds A and В and their synergistically interacting combinations, with an isobole (CD).

57. Ό

•DA Figure 11. ibobologram for fig. 10.

In conclusion, we ca η say that the less antagonistic or the less synergistic the inte raction of two compounds, the more their isobolograms approach the straight line that exists at pure summa­ tion (fig. 12). Presenta tion of experimental results in isobolo- grams, therefore, clearly demonstrates the characteristics of inter- action between two drugs. Since most of the time not all possible combinations are studied, but only a few, isobolograms will contain only one point, as is the case in our following studies. In the re- raaining chapters, we used the isobologram technique to express the results of our interactio η studies.

Figure 12. Isobolograns for ant agonis Lie, synergistic, and summative interacting combinations of compound Λ and B.

58. Referenses

Loewe S. (1953): The problem of synergism and antagonism of combined drugs. Arzneimittel Forschung 3, 285-290. Loewe S. (1959): Rand bemerkungen zu quantitativen Pharmakologie der Kombinationen. Arzneimittel Forschung 9, 449-456.

59. J. Pharm. Pharmac. 1978, 30, 699-702. s Potentiation of neostigmine and pyridostigmine by 4-aminopyridine in the rat

RONALD D MILLER*, PETER A F DENNISSEN, FRANCIEN VAN DER POL, SANDOR AGOSTON, LEO HD! BOOII AND JAN F CRUL Institute of Anaesrhesiolngy, Catholic Uimersiry, hijnieçen, The Netherlands, and Institute of Clinical Experimental Anaesthesia and Clinical Pharmacology, State Unnerut) of Groningen, Groningen, The Netherlands

The interaction between 4-aniinop>riJine and neostigmine or pyndosligminc was studied in vivo in ihe rat sciatic nerve-anterior tibialis preparation using the constant infusion of paiiLuronium ІесЬпісціс The ED50 (dose of drug which produced a 50" antagonism) of neostigmine, pyridostigmine and 4-aniinopvridine were 18, 49 and 440 /ig kg ' rcspcctivelv Thi a Idi'ioT of 10) (ig kg ' of 4-a'Tiinopyridiiie, which prodjwcd no antagonism b> itself, decreased the neostigmine LD^O to 7 4/ig kg ' The idditionof 200 / « kg ' of ¿l-aminopynd- ine, which produced a 10 ^ an'agomsm bv itself decicased the FD50 of pjndosligmine to II /ig kg ' We conclude that both neostigmine and pyridostigmine interact with 4-amino- pyrine svnergistically

Neostigmine and pyridostigmine arc used clinically left tibialis anterior muscle was freed, sectioned and to antagonize a neuromuscular blockade produced connected to a Grass FI01 force displacement trans­ by pancuronium or ( ) lubocurarme (Miller, 1976) ducer The resting tension was adjusted to 20 g Neostigmine and pyridostigmine act primarily by Stimuli of 0 2 ms duration and of strength sufficient inhibition of acetylcholinesterase Recently, 4- to produce a mavimal twitch were applied to the aminopyridme has been used clinically as an antagon­ sciatic nerve through a bipolar electrode at a frequency ist of ( Vtubocurarme (Sloyano\, Vulehcv & others, of 0 1 Hz Tu itch tension was recorded on a poly­ 1976). However, the mechanism of this antagonism graph Ree al and muscle temperatures were main- differs from that of neosticmmc or pyndostismme tan.ed between 37 and 38° 4-Ammopyridine docs not inhibit accly(Cholin­ Pincuromum was administered indavcnously by esterase activity (Molgo,Lemeignan & Leehat 1977), continuous infusion to achieve and maintain 90% but increases the amount of ncctvlehohnc released depression of twitch tension When the mlusion rate by the motor nerve terminal in response to a nerve required to maintain 90 V, dcpicssion of twitch ten­ impulse (Bowman, Harvey &. Marshall, 1977, Molgo sion was constant for at least 15 mm, neostigmine, & otheis, (977) Since neostigmine or pyrido­ pyridostigmine or 4 aminopyridme or a combination stigmine antagonize a pancuromum-induced neuro­ of these urugs was given as an intravenous bolus muscular blockade b> a dnTcrent mechanism from Except when 4-aminopvridinc was given alone, (hat of 4-aminop) ridine, и с propose that these agents atropine 17 /ig kg ' w ,s given intravenously before may not act in an additive manner We tested this administration of drugs (see Miller, Van Nyhuis & possibility in the following study other,, 1974 for technique) Time from antagonist administration to peak effect (onset), magnitude of METHODS antagonism, and time from antagonist administra­ Eighty-five rats, 260 to 430 g were anaesthetized with tion to 50% return to the paneuronium-depressed pentobarbitone, 40 mg kg-1 and urethane, 500 mg twitch tension (duration) were calculated Only one kg-1, intrapentoncally Both jugular veins were dose of antagonist or combination of antagonists was cannulated and all drugs were administered through studied with each animal these cannulae A cannula in the caroud artery per­ Initially, dose-response curves were determined mitted transduction of arterial blood pressure A for neostigmine, pyridostigmine and 4-aminopyridine tracheostomy was performed and ventilation was alone Then neostigmine or pyridostigmine was controlled with a Braun air pump The tendon of the combined with various doses of 4-ammopyridine to * Correspondence Department of Anesthesia Uni­ determine whether these drugs interact with each versity of California, San Francisco, California 94143, other in an additive or synergistic manner Specifi­ USA cally, dose response curves were determined for neo­ •Work performed while Doctor Miller was a visiting -1 professor at ihe above institutions stigmine with 4-aminopyridine 100 /¿g kg and for

60. pyridostigmine with 4-dminopyridine 200 μg kg ' kg-1 of 4-aminopyridine decreased the ED50 of Dose response curves were determined for 4-amino- pyridostigmine Trom 49 to 11 μg kg-1 (big 3) pyndine with neostigmine 3 5 and 7 S jag kg ' and Addition of 3 5 and 7 5 μβ kg-1 of neostigmine with pyridostigmine 25 ^g kg-1 The dose response decreased the ED50 of 4-ammopyridine from 440 to curves were compared by anjK sis of variance

RFSULTS uu- The ED50 (dose of antagonist which produced a 50% antagonism of the pancuromum-depressed Τ ν twitch tension) of neostigmine, pyidosligmine and 4-aminopyridinewcre 18,49and440¿igkg '(Fig 1) 50-

100 1 /

50 10 25 50 100 Fie, 3 The effect of pyridostigmine alone (-) and given with 4 aminopyndine (200 ^g kg-1, ) on % antagonism (ordinate) of the pancuromum-depressed 100 200 ¿00 ΘΟ0 twitch The lines represent analysis of linear regression 75 30 The vertical lines represent the mean ± s e of four rats FIG 1 Correlation between dose (^g kg ') (abscissa) of Abscissa Pyridostigmine (ng kg ') neostigmine (NS), pyridostigmine (PS) and 4-amino- 0 pyridme (4-AP) on 0 antagonism (ordinate) of the pancuromum-depressed twiich The lines represent 220 μg kg ' and to 82 μg kg ' respectively (Fig 4) analysis of linear regression The vertical lines represent The ED50 of 4-aminopyridine also was reduced the mean it s e in four rats from 440 μg kg ' to 76 g kg-1 by pyridostigmine, 25 μ kg · (Fig 5) The dose response curves did not significantly deviate 6 -1 The ED50 values of the various combinations of from parallelism The 1200μg kg dose of 4-ammo- 4-aminopyridinc plus neostigmine or pyridostigmine pyndme caused generalized twitching of all muscles were plotted against an isobologram constructed which resembled convulsive activity in spite of the from the hD50 values of the drugs alone Either animal being anaesthetized with pentobarbitone combination produced an effect greater than simple This dose was not included in the calculations addition (Fig 6) because it caused no more antagonism than did the 800 μg kg"1 dose Addition of 100 με kg"1 of 4- When equivalent levels of antagonism are com­ aminopyndme decreased the ED50 of neostigmine pared, 4-aminopyridmc increased the onset times but from 18 to 7 4 μg kg ' (Fig 2) Addition of 200 μg not the duration of action of neostigmine (P <0 05)

uu- 100η t/

1 50- 50 r / / i"í

35 75 15 20 30 70 100 200 250 400 Θ00

FIG 2 ThecfTectorncostigniinealonei— -)andgivcn FIG 4 TheefTect οΓ4 aminopyndine alonc(-)andgiven with 4-aminopyridine (100 ^g kg 1 ) on 0n with neostigmine ( 3 5 MS kg ' - - - 7 5 ng antagonism (ordinate) of the pincuromum-deprcssed kg ') on " , antagonism (ordinate) of the pancuromum- twitch The lines represent amlysis of linear regression. depressed twitch I he lines represent analysis of linear The vertical lines represent the mean l sc of four rats rccression The vertical lines represent the mean £ se Abscissa Neostigmine (/ig kg ') ot lour cats Abscissa 4-aminopyridinc (^g kg ')

61. -.00 Ί

50

50 100 200 400 800

HG 5 The cITcLt οΓ 4 aminopvndmc ahme ( ) and given with р\гкІоч1і£тіпе (2^ /'g kg ' -) on '( oO andtagonism (onhnaie) of the pain-jronuini-deprLsseil twitLh The lines repiesent anaKsis o\ linear reuression I'/: The vcrtiLdl lines represent the mean se lor lour rais Abseissa 4 Λιηιηορ\ridine (^g kg ')

(Fig 7A) In contrast, 4-jminop>riclinc had no 0 :o fco effect on cilhcr the onset times or duration of Fu· 7 Correlanon helween time (mm) (abscissa) and pyridostigmine action with one execption (Ι ι с 7B) ' antagonism (oidmate) ol the pancuionium-dcprcssed The pyridostigmine 10 /tg kg ', pliis4-ammop\ridine twiteh \wili neostigirme (NS) (λ) or pyridostigmine 200 /¿g kg-1 combination had a loniiu onset lime (PS) ([)) alone and wiih 4-amin ip\ridine (4 ЧР) Ihe mnnbers beside Ihe drug abbreviations are the doses in than pyridostigmine alone (Ρ OOM (| ig 7H) /ridine DISCbSSlON antagonizes a neuromuscular blockade by iiKreasing 4-Aminopvridinc potentiated the effect of neostig­ both evoked and spontaneous release ot acetylcholine mine or p\ridostigmine (F ig fi) That is. (he effects from the motor nerve terminal rather than by of the neostigmine or pyridostigmine and 4 amino- inhibition of acetyleholinesterase (Bowman & pyndine combinations were greater than would have others, 1977) been predicted from the effects of the drugs given alone An effect other than simple addition might Neostigmine and pyridostigmine are drugs usually have been predicted from the fact that 4-amiiio- used to ant igom/ea non-depolari/mg neuromuscular blockade (Miller. I976) However, 4-атіпоругкІіпе mav offer some advantages The apparent absence of muscarinic cITccls eliminates the need for con­ comitant administialion ol atropine as is required with neostigmine and pyridostigmine (Fogdall & Miller. 197Я) In fact, recently, 4-aminopyridinc has been shown to have some svmpatbetic activity It potentiated responses of the adrenergicallv innerv­ ated rabbit vas tlelerens io transmural stimulation by causing mei eased noradrenaline release (Johns, Golko •"RGISM ι SYNrSGISM & others, I97fi) Also, 4-aminopyridine increased i„ 1 both evoked and spontaneous release of adrenaline 100 200 300 100 200 300 Irom the portal vein (1 cander, Arncr & Johansson, FIG 6 Correlation of the FD^Ofdose which produced a 1977) Despite this evidence for sympathetic stimula­ 50'n antagonism of the pancuionmni depressed tvMieh) tion, the blood pressure in this study was not altered ol 4-атіпор гкІіпс, neostigmine and pyridost gmine alone Irom which the line of identnv was lornicd The by administration of 4-aminopyridine dots represen! the \ ΙΉΟ of апоиь combinations ofthe Anoilwi advantage ol 4-aminopyridine is that it abo\c drugs Ordinate μ% kg ' AbiCissa 4-Amino- pyndmc (μ% kg ') will antagoni/c an antibiotic neuromuscular blockade

62. where neostigmine and pyridostigmine arc often sufficicnlly to attenuate or eliminate the need for ineffective (Bikhazi, Burked & others, 1977) With atropine to prevent muscarinic stimulation. Also, these advantages, perhaps 4-aminopyridinc should perhaps the combination of neostigmine or pyrido­ be used alone rather than in combination with neo­ stigmine plus 4-aminopyridine can be used to stigmine or pyridosugmmc Ho\ve\er, 4-ammo- antagom/c the anlibioiie-induced ncuiomuscular pyndinc readily crosses the blood brain barrier blockades where neostigmine or pyridostigmine Doses of "t-jminopyridme which completely antagon­ alone often fail (Miller, 1976) ize a non-depolan/ing neuromuscular blockade 7 he above advantages of the drug combination arc cause central nervous system stimulation which we specularne and need further studs That 4-amino- observed with the 1200 /¿g kg ' dose, the precise p>ridine markedh enhances the ahilnv of neostig­ relation between dose and central nervous system mine oi pyridostigmine to antagom/c a pancuron­ stimulation needs to be defined Perhaps the ium neuromuscular blockade needs confirmation in combination of neostigmine or pyridostigmine plas other species including man 4-aminopyridine will attenuate or eliminate the dis­ advantages of these drugs indiviuually That is, the Ack now It dut ment s dose of 4-aminopyridine will be low enough not to The authors gratefully acknowledge the editorial cause central nervous system stimulation, yet may advice of Dr Edmond I Fgcr, II and Dorothy reduce the doses of neostigmine or pjridostigmme Urban

RrFbRFNCFS BIKIIA/I G B.BLRKirr L KFLIY Ρ WIRTA, M & J-OLDLS, h (1977) Abstracts of Scienlnic Papers, American Society of Anestheìiotogittì, pp 461-464 BOWMAN W С Hutviv.A L & MARSIIAI I I G (1977) Naunui ЧтіеасЬегі·* Arch Phannac 297,99-101 FOGDAIL, R Ρ & MILLI R.R D (1971), Aneulu uofoçi, 39 504 W JOHNS A Goi ко, D S , LASZON Ρ А Л PVION D M (1976) tur J Phannac , 38, 71-78 LLANDLR, S , Акчік A & JOHANSSON В (19771 //ж/, 46 351-361 MlLLLR R II (1476) Amsllieuolo"! 44 1ІЯ-12Ч MILLLR, R D.VANNYIIIIS L S l GIR, 1 I II, VITFZ, Τ S & WAY, W L (1974) Ibid,41.Π 33. MOLGO, J , 1 ΐΜίκ,ΝΛΝ, M & I к НАГ Ρ (1977) J РІыппас i \р Vier , 203 651 661 STOYANOV.F VLLCHIV, Ρ , SiirCRiravA, M & MARINOVA, M (1976) Aiuiesth Reuu Inlen Ther , 4, 139-142

63. ^NFSTHLSIOLOCY, Vol 50, \o 5, Mny 1979 6.6 4-Aminopyridine Potentiates Neostigmine and Pyridostigmine in Man

Ronald D Miller, M D ,' Leo H D J Βοοη, M D ,t Sandor Agoston, M D . Ph D ,% Jan F Cru! M D . Rh D §

To eluiidate the interaction of 4*aimnop\ridine with neoslig- p\r ¡dosi igni mt Pul un mai \ obsei \.itions in man'and mine and pvndosligmine. the authors studied 57 anesthetized sludies in animals'1 ' show no mustannit eiled ol 4 surgical patients using a technique of constant infusion of pan­ curonium to quantitate antagonist ac(i\it\ •1-Λπιιπιιρ\ ridine. 0 15 ainiii(ip\ ι idmi In lau 4 ашшормкіше lias leieniK orO 35 mg kg. produced no antagonism while 0 5 mg kg produced ht m shttun io ha\i sMiipalheiii .iiti\H\ li fMHentiated a mean 24 * 6 per cent (peak) antagonism I he dose that produced iis|)onsis o( ibi adii пет gic all ν ι η nei \atid lahhil 50 per ceni antagonism (t D ,) of neostigmine alone nas 22 μ% kg, \as di It it us to nansimnal sinnulalion hv tatising with 0 35 mg'kg -4-am ι пору ridine, к nas 7 Mg kg The tDj of nou pini pin nu iikasi * and mo east d bol h ivoked pyridostigmine alone was I IO Mg kg, with 0 35 mg kg 4- aminopyndine, it was 27 Mg'kg 4-Aminopvridine prolonged the and spon l.nu ous к li asi- of noi ерик pilline (toni the onset times of both neostigmine and pyridostigmine, but pottal \iin'' \lso 4 aininopvi idme aniagoni/es ihc prolonged the duration of action of neostigmine only At a neumimist ul.n blockades pioduted hv most andbi- given level of antagonism of pam uron1 mg/kg) also the amounts of atropine needed to prevent a change in heart ratebv 68 and 70 per cent respectively The authors conclude that 4- stimulate ι IR CI tu κι I nei \ ous s\siem and cause aminopyndine potentiates antagonism of a pancuronium-mduced posiopt ι at ivi iistkssniss and conlusion ^unpublished neuromuscular blockade by neostigmine or pyridostigmine Also, dat.ι S \gi>ston) less atropine is needed to prevent cardiac muscarinic stimulation when 4-arninopyridinc is used with either neostigmine or We leieniK obser\ed s\nergisni between Ί- pyridostigmine (Key words Antagonists, neuromuscular relax­ auitnopv ι idine and neostigiiunc οι pvi idostigtinnc ants 4-aminopvridine, neostigmine, pyridostigmine Muscle in iaisH Suib svneigism m man could dei ι case the relaxants pancuronium Parasympathetic nervous system atro­ dose ol neosligmini oi p\ridosligitiinc ncccssdr\ (or pine ) aniagonism and thetebv atienuaie oi even eliminate iht need (oi concomitam administiation oí atropine I lit л( l· ι \ ι ( MOI IM si h RASH IM ti IH ι ION pioctuccd In In tht (Usctibcd heiein stud) we deinonstraied that псоміцтіпс .nul psi idoMiginiiH h.is heen u^i'd io svneigisni dois ociut and that the icqunement for .tniagoni/t' ппшк'роЬп i/mg ni ummust ul.tt Mix к- ali opini is lonioniuanth deireased íide-s lm Μ'ΜΊ.ΙΙ \e.irs I wo \ lliest disadvantages ol ntnstigmme аіні was induced with thiopental. 1 τ to 2 0 mg/kg.gamma- hvdtowhuivi к and 50 mg/kg. and lentanvl, I ~> μψ,Ι • І'іоІ(чч,и liiMiMiH ·Λ ЧІНМІНМОІС.Ц^ ( .іііімім Ι іммічіч kg i\ ( ontrolled venidaiion kepi end-tidal carbon Niliiicgiii I hi Nt ι ht il.ituU .uni IiiMimu ni ( Іиш il t\pt > mu nt.il dioxide comenitation Ыч ееп 4 and г) pet tent Aiusihtsi.i Si iu lm\t.)Mt\ ol (•іііпіііці и (.нпппщп [ lu I hiougb 2[ï-gauge needle elei nodes, we delivered NuhiiluuK 1 Sud ІШІПІК.1 Insimm ol \iitsih(sml(i^\ ( .uhulic l Г>І <ІМІ\ suptamaxinial st|uare-wa\e i)ipolar pulses of 0 1 rnset N.imtgin duratioti at 0 I H/ to the ulnar nerve at the wrist I he + VIIIOI Ktse.nch Xssmi.ilt Insinui« ni \ІКМІІІМОІОЦ ( .ilholu resultant lout of thumb adduition was i|uantitated l iirit ism \І|ІІКЦ( ii and Ir^imm ni ( Ιιηκ.ιΙ \\\H ιπηιηι,ιΙ with a force-displaiement transduier and reiorded Λ ικ si hi -LI Si ne I imt ι sit ν ni (ипшпцш «)n a pokgraph ή Finli\i .nul ( h,m in.in Insili uu ni \m sihtMnl(ig\ ( ЛІІПІК I lintlSIIV Nl|HKg(ll A teihnicjue of tonstanl infusion of pancuronium to Uoik ptilniintd ν,ΚιΙι Di Militi ws ,ι \ ixting Pinltssoi ici 1 hi Nuhulinds Attt plrd Ini puMitaiinn \ицичі 14 l'I/H r Bikha?! (.B Burkiu 1 Kilk 1' dal I hi iniciatnon of ¿mi Adilicss upiint iLipJisis in pitstni add г ^^^ Di Milki л his hint us I mbiKiii.ii nil and -1 anunnpviidiiu' at the nruroimiii ular l)t pili tini ni di ЧпічЬіма l шм t чіі\ ni ( alilniiii.i S.in Н.тг ып juiuuon Abst rails Annual Mctun^ nf tin Anuí к an StKielv nf ( .ildnini.i Ή 14 1 XnisilitsmlngiMs |Ч7(і pp 464-404

64. hü i ( ОІКІІП.Ш l>. IU..N ІНМЧІІІ-ІИІІК <\Ч) икі рмкііі-ііціміп« іГМ iluiu nul PSatore unii 1 mHiiopwiiliiK 0 \ > іііц кц ι I \f>) -¡сJ 50 tMih [» TU in іщ ni ρ IIK iituiiiuui iixliu« cl 1 l(Ч l)K\Sli>l1 l ill НИК II κ π«. ι.«..ι»/..I I he (Min Miih^ »Ι Im цмччпіі I idi (li>( m \ к 4.I11S ІІЦ nu.in - ] Sh di \ iluts Idi ilii Mi К Hls

20 75 120 200 Dose ÍAjg/kg)

í|uanin.ue ли.immisi асіімп was used loi all siiidios '' of liutai logiossion I ho (inves ueie comp.ued ίοι Alici a holtis ι η jet non ni pam ninnili m, !20 ^g'kg. η, paiallclisin and sitili In analvsis ol tovanante I ho paiKuinnium, 200 ¿ig/inl. v\as mlusi'd contmuoiisK oust i limes and il in at ion ol ad ion wen· ι от pai od In iiom .ι pump in ptodute a (onsiant 40 poi ceni mg'kg (η = S) 4-aniino|)vi idine ant.igoni/ed stigmine, pvi idosijgnune, and 'l-aininnpvt id me alone the pant u ionium* indue ed depiession ol twitt h Dose-1 espouse ( unes wei e (hen dolci mined lor lension, hut 0 5 mg/kg (n = 4) pioduted 21 ± β pei neostigmine and pviidosligmine in the píeseme ol cent antagtimsm I he tloses ol neosiigmmo and pvi­ 4-anunopv ι idine, 0 'УУ mg/kg Dose-iesponse tuives idosligmine ι hal pioduted ΊΟ pei teni antagonism ol also were deteimined loi 4-ammopvtidine m ihe the paniutonium-induted dopi essimi ol luitth (l· DV)) presence oí neostigmine 7 "i ¿¿g'kg, oi pviidostignune weie 22 and 1 K^g'kg lospettivolv (hg 1) Iheadch- 40 Mg/kg Oulv one dose ol aiuagomsi o? aniagoiusi tion ol 4-aiiunopv urline, 0 45 tng/kg, detreased ι he mixture was studied in eath patient 1 he dose of l· І) ,ч of neosiigmine and pvridosiigmine io 7 and 27 antagonist thai prod IH ed "lO pei tent antagonism ¿tg/lsg. lespet uveU (P < 0 01) (hg 1) I he cui ves did (t D^D) was dei ived hom turvesdetonninetl by analvsis not deviale fmiu paiallelism

65. — NS alone

-•NS*4 AP Χ 'О

hi, J ΓΙ lume ΙΙΜΙ|Η leni ie, ..Ι ριη,ϋΗ,ηπιη „,,Ι,ι,,.Ι , !.!>., ччі.т ,,Ι Ι,ΜΙ,ΙΙ , π,,,,η ,ш,и /,,Ι

11» II II llnis ll|>IISIIII lllisl Mil MC ,ΜΙΙ,Ι NIH \S, III „Чк,. Ih. des. ,,Ι Ι ,.ιι. норм li Im. IS.,' , IS ,1 1 , III, кц 1 II, ,ΐι,κ II,I III „k,ls,,|,l,s, 111,11, IIIS I SI III.,, IMI s ,.,,, sl.nll,,! ,1 , „Il ,1ns,

50 70

Time Irrwi)

hi. I l'Ini ni Inn nid ιηι.ιιιιιμι ,,Ι

«nsinn ,πΗ.ι^ηηι/.,Ι III. luniili.is κρι, .m .Ins. s ,,Ι pMi.lnsiiiíJiiin. (I'S ι m μι, кц III. lins, ni -Ι ιιιιιιι,,ρ.ι idilli US.,Ι» IS II Γ> пц ki; I lu tlnis ,ιι.,Ι In i.k.ls i.|>iistin in ins · I SI III... pili.nls „in siudud ll . ι. h .Ins,

66. /

.''' •'"X hl. -1 ( niriliiH.N Іч lui ui (І.ім "1 I ітміп|)\ mlirn Il >Mtliiil]i(i iitns іціііпк (NM 7 ι дц кц мі рмні. м^ PS ІШІН (PS 1(1 μμ, кц Ulli] ρ<ΙΙΜΙΙΙΚ< "I ршішпм и и •''A Illdlli ( (I rl( |) ( SMOI1 il ЦЯІι I Ц I Mill ini lL,(HM/t il I hi il \/ /^ Іітч κ |н,ч< m HIIKMNMI Inn И К^ИЧМОП tnliitui ""^NS οι \ примни ιΐκ ηιι ιιι · I St м| ν duo IMI limi S * /\/ pint nis s" x¿ / s y\ 'S

0 15 0 35 0 50 4 Amnopyridne (mg/kg)

Лі .і|>рн>\un.!it К t i|ii)|mlt ni dost ч 1 .uninn атторм itlint had Innati oust ι mms th.in p\ rido pv τ uh ικ int it jsid oust ι nines of IH ОЧІІ^ІПІІН МІЦІШІН 20(1 дц кц dont \ 0 0"і)(|ц« S) Рмніо- diid рмніомі^пшк' ,tiid iht (ІІІІЛІНШ ol ікочііціппи чпцпши 120 дц кц ііоін had a lo m» ti diiiaiion ol cll<4t fxttpi in (IK мпаііім dtisis K'^*11 '''K -' ai don (han ііні 20 μμ кц \ЧІІІ I aininopv tidint (/* I here was nr> thf U it tuo in mms u> oust ι DI • 0 0")) I lu st doM s pi od ut ι d si m ila ι ι \U nis oí ,ηι- din allons ol ai non ol п<о>пцпппі. Hi μι> кц .Иоін іацошмп ( oint ist l\ ibi dui allons ol anion ol and neoslignmu "> μμ k^ plus 1 аішпормкііік Π î> p\ ι кіочііцтіиі 200 μι^ кц alom and p\ ι кіочіцпиги Τ") μ^ кц Willi J-а шторм idi IH did not dit ft ι siculi С otnparison of onstt tuut s anti dui al ums ol ta ni К 1- \iiimop\ iidini с и ha tut d ашацоп^ш h\ at lion ol pv ι idosti^nniK alom and p\ ι кіичііцішт m ОЧІІЦШІІК anti p\ t кіочііцііиіи in л dost -dt jKMidt ni plus f atuiimp\ ι idint itwalsau iiuonsistt ni p.iiitin inalimi (Ііц I) ( ompai ічоп ol p\i ИІОЧПЦІПІІН 20 мц'кц plus 1- I tss аііоріік и as nt tissai ν ι ο pi ем ni hi ad мак lia ammopM idint' uiih p\i itlosii^rumt 10-120 дц'кц uhi η I aininopv ι idim was (otnhimd with neo alone shous no dilltiente m onstt iinus How­ чііцтмн οι ])\i кіочііцтин ai a цімп кмі of аіиац ever, p\ridnsugniine ΊΟ and l'i дк'кк with 4- οι usui (lig "Ό W lu η IH osi іц шин and p\t idosiiginiue

ï 100 ¿LS

\\i '·> ( unti κιοιι IH lütt и dose uf il πιμιπι ικ ( t *.ч и \ m puvtni ι t h тц* in hi πι t ui ui тип than 10 IH its nun h uni щи міцтпн l ) οι [)\r κΙυΜίμιηιπι (Χ) unii nul vMihoiii I пиши pHidiiH nul ))іиіііііці ol |1 in (uioniiini inducid dipussion и' ihiuli HiisiMii ,ιηι.ιμοιιι/ι d

0 5 05 07 Atropine ( mg / 70 hg )

67. ut н if IM η in (lost s ih.ii i.iiisi'tl ΊΟ ¡κ ι tt πι .uil іц (di di.ι w ι> si ціі] Ін,и и К (U( it.ist d li\ iht .iddiiion ol 1 omstn aliopnu 11 70 m μ 70 kg (г 0 87) ,ιικί 0 b!2 ,ι m ι порч ι к h ut' (lit; Ί) ( .udì к .и ι h\ I linn.is ,ιικί mg'7(lkg(i = 0 H">) ІІЧ|Н(ІІМК wciv піччікі lo pit In polt пчіои ott.ision.ilU oit ui uhi η пкыщпшн οι м ni .ι ι hangt in heul tait W ht η ">0 pti tint p\ ι КІО-ІІЦІПІПІ IN ЦІЯ Π unii aitopint loi апмцошмп atUiigomsm \%iis ai Int \td unii 1 aminopMicliiu 0 Vi ol m ummusuilai hloik.idi '" ' W ι piisumt ili.u an tii^'^K л,к' niosligniiiH oi p\ ι idostiginnu iht dost s ani.іцошм ι hai 11 (|uiit s It ss an opini u ill ht .ISMи iati d ol ai topnu ntt t ss.n \ m pu \t ni at Ііапці in IHM 11 ι aio unii It ss чіц indiani t u diu\as< ul u pi olili ms 11 uni all\ (kiitast'tl lo 0 2-1 mg'70 kg (i = 0 81 ) and 0 '20 mg 70 I his asMinipiion tan IH dot unu uu d onlv hv lui ι ht ι кц (!=0НН) itspt4ii\tl\ (Ρ <-{)()[) (hg ">) Hic t liiutal ni ils W ι IHIUM iht кКапіаці s ol a It sst ι tuivtstlid nol (Uviatt hom paialltlism \o hlood апории шцпипнш and possihlv a pitditiahk pitssurt' <>i Inai t tait t hangt it suilt d lioin atlmi nis- aiii.igoiustu oi an ani il »mi и nul lut d m m oinust niai iratmn of 1 aminopM idilli aloni at am dost tistd hloi kadt au suifuuni louan uu luilhti siudits \ll hough ι hi t lot ι lot nu pliaiogiair uas nol имчі ut* (lid nol obstt\c am sign ol ttniial 'KIMIUS svsitni ι ],» ш luns ^imlilK nkn-.uUdm dn i

Discussion References

Xniagouisiti ol a pani uioimnn ιηιίικι d mino | ) n^( ц K|i Μ,Κ, , κΐ) \ιι іц suini / тіюм IM nul mustul.U hint kadt h\ IH OSI Iglll Ι lit Uld p\ Ι idosllg )• mini mi m lumi IH пмипим uhi l)l· (к idi ν In рмкіо mint is рои пиан d h\ 1 ашіпормкіин I hal is 1 чщтпк '" m m \м чиимш.п \ \ч .ІИ- »ич ІЧТІ aininop\ ι iduH 0 \'ì mg kg uhith |)t(>dii(id no Millo Kl> \ιιι ιι,,ι insu) ni muinniiiMiiln M.xknk \MS I I 4*i U ' 1 ITI» aniagonism hv Usili dttttastd ilu Ι Π,,,Ν ol uto f Si..^ m« t I \ il.lio M Mnitthm ι M и il ( Imi. H U lo. мч I m | l'h и in п..Ι (M 71 74 ΙΌ, it It isi ol an i\ It höhnt (ют iht ninmi nt ι \t , ц ,„, x xl \| ,^ι, .ц ц Η ι mutai ialini ihan l)\ inhihition ol aiinltholui [niidnns un ili. «link им un ι iii\nis ним li lui | tsltiast ' riuimnol 11 K\ì\ іич |чТ7 Ulootlpitssuit and ht ut ι alt ut и imi t hangt dh\ " u m,il,s Nn'UlN x l-b·»-»"» ми.ім.і i шшн.рщНин

, , ' , t , . , . и МІІІІШІІ utivin uni nu, nlunilm. и It IM m ilit

Mit dost s ol i-.iniino])\ ι ниш idmunsit u tl in nus . t . „. . ( ^_ siiitK uhith isioiisisitni unii pion.us uponsol uo 7 ц,,,,, mi), \ыі,,кі>і „ι μ \,іЫиі, „,, uni Ι п.шю inilStailNIt atmin ' ' \nolhtl athaillagl ol 1 |i\iili.«kiil< шшш \іи%ііі \ιι ІІЦ ι( U и ) »7 ili> lìloikadis niodund In aiitihiolus uhin ut ostigmmi ,' , 11 -. 11 Ц Х1|||Ч KD IHliniSMIl Ì'W 1111 lili l'ili У .t il l'un ІІІМІІОМ and рм idosiig in uu ai ι от η nu I li t nu I lu st , . ^ , ^ ι . . aiKauiagtssiiggist du им ol 1-, mpwiduii ialini ,ι,, , „ j ι>ιη,,η l'h um иоікі -HU \ч7н than m osiigmmi 01 p\ 1 idosiigitiuu lloutuí dosis ч Mill· ι Kl) \ щ \\liui-is К«1 N H (пшритм nnus ol I aiuinopn idiiu git alt 1 ihan 1 hitst usid in om 1.. |» ik duo nul il <ніч .>i «поп ni томц, ( и··! siiidMM чти than 1 пш kg) taust ti 1111 al m 1 unis рмі.і .міи πικ WiMms,,.,.^ 11J--U |ч7 1 іі, l'i ( li^n 1 s\ l-i|inlil lllii \\ \iiiiiiii)ulitH ihn ш\ in svsit m c\tualioti usili ssnt ss and toniusion 1posi . . . ' , 1 и isi 4 ι mi » 1 сітці i])h ι ili im,!·, ι unm, и я is il ol optT.iini'U (uiipul)lislitd data S \gosioii) mu „ тмі I и lil ик »U \1nsii1 \ii Лц (UMI »7 7"-«ι I lu lonihiuaiion ol ut osi igni uu 01 p\ ι к lost igni mi 1 17^ unii I unmojniHluu 111 ι\ ittiiuiatt 01 tluninait tlu ,l ,',,ч«1 |M Kl' ^11"11 Kl) \iii іц ними ni / iui>n »пш m.lm.d ті.м.іітмііі.і Ι.Ι...Ι. ..It. ι» ,» мі., ι , и .. , ιι , ι Mil, mut 111 η in \MMiiiMiiifii ì Ι" .01- .1)4 ]Ч71 un ι\ idualh \\ ι ohst ι ud по t \idt ntt о и пи и , , ., , и » ι ч( . •> IJ l'ili! )Щ (H \ll ІЦШІГМЧ (1 lllllsi ι idisillls Ml sill Κι Ut I Ullis S\ Sit 111 sil ІШ1І Jl UH 1 mila Ol poslopi I iliu 1\ i\ in Ν I 1] («1 li\ holilis hl l'In uli liilii 1 l· \ ΙΪΙ\ΙΝ filili Ι ht anioiini ol aliopim nttdtd io pttunt hi id\ |>|> itin- ι μι

68. 6.7. Comparative reversal of pancuronium by neostigmine, edrophonium, 4-aminopyridine and their combinations in cats Leo H.D.J. Booi], M.D., Ronald D. Miller, M.D., and Yung J. Sohn, M.D. Submitted to Anesthesia and Analgesia

Introdw^ion Since the introduction of neuromuscular blocking agents m anesthesia, problems concerning their reversibility have existed (Miller 1976). For reversibility of nondepolarizing neuromuscular blocking agents, the cholinesterase-mhibiting drugs neostigmine and pyridostigmine are adequate. However, these drugs show unde­ sirable muscarinic side effects, and can usually not reverse anti­ biotic or antibiotic-nondepolarizer-combined blockades. The ability of 4-aminopyridine to antagonize neuromuscular blockade was des­ cribed m a recent study (Stoyanov et al. 1976). In a previous study we showed that with dosages that can safely be used in patients, 4-aminopyridine does not reverse deeper neuromuscular blockades (Miller et al. 1979). However, these low dosages of 4-aminopyridine potentiate neostigmine or pyridostigmine in a synergistic manner (Miller et al. 1978; Miller et al. 1979). A lower dose of neostig­ mine or pyridostigmine is thus needed to adequately reverse the pancuronium blockade. Less severe muscarinic side effects occur, as measured by the atropine requirement (Miller et al. 1979). In vitro studies showed that 4-aminopyridine can antagonize neuromuscu­ lar blockades induced or augmented by antibiotics (Burkett et al. 1979; Singh et al. 1978), as confirmed in man for lincomycm (Booi] et al. 1978), and in cats for polymyxine В (Lee et al. 1978); The onset and duration of action of neostigmine in combination with 4- ammopyndine are prolonged compared to the onset and duration of action of neostigmine alone (Miller et al. 1978; Miller et al.1979).

Since edrophonium is known to have a rapid onset of action, we investigated whether its combination with 4-aminopyridine or neo­ stigmine would be preferable to the combination of neostigmine and 4-aminopyridine. Edrophonium shortens the time of onset, while 4- ammopyndine prolongs the duration of action. The interaction between neostigmine and edrophonium is also included in this study.

Me thods Cats of either sex weighing between 2.5 and 4.2 kg were anes­ thetized with a-chloralose, 60 mg/kg and urethane, 250 mg/kg, intra- pentoneally. Both external jugular veins were cannulated for ad­ ministration of all drugs. A cannula in the left carotid artery permitted continuous registration of the arterial pressure. A tracheostomy was performed, and ventilation was controlled with room air by a Harvard respiration pump. The temperature was kept constant at between 37 and 380C. The tendon of the left tibialis anterior muscle was freed, sectioned, and attached to a force-dis­ placement transducer. Supramaximal square wave stimuli were applied to the ischiadic nerve at 0.15 Hz and a duration of 0.2 msec. TWitch tension and arterial pressure were recorded continuously on a poly­ graph. Pancuronium was administered as a bolus, followed by a continuous infusion, in order to keep the twitch depression at a

69. constant level of 90%. When twitch depression and infusion rate were constant for at least 30 minutes, a dose of neostigmine, edrophonium, 4-aminopyridine, or one of their combinations was administered. Magnitude of recovery, onset time (time from injec­ tion until peak effect) and duration of action (time from injection until 50% return of the original twitch depression) were calculated. When the twitch depression had returned to its original level for at least 45 minutes, the next dose of the same drug or combination was administered. The sequence of dosages was randomized. Linear regression analysis of the data points resulted in dose response curves, and a general linear test approach for testing the inequali­ ty of neostigmine and its combination with 4-aminopyridine was per­ formed. An analysis of covariance was done to demonstrate differ­ ences between edrophonium and its combinations. Analyses of variance for both onset time and duration of action with equipotent doses were done, followed by a multiple comparison method (Neter et al. 1974). In an additional group of cats, combinations of equipotent doses of edrophonium, neostigmine, and 4-aminopyridine were administered (EDgo's) to confirm the validity of the results.

Results A total of 35 cats were studied (table 1), from which dose response curves were obtained for neostigmine, 4-aminopyridine, and neostigmine plus 4-aminopyridine 15 yg/icg (fig. 1), and for edrophon­ ium, edrophonium plus neostigmine 10 |jg/kg, and edrophonium plus 4-amino- pyndine, 150 yg/kg (fig. 2). Neostigmine was significantly differ­ ent from neostimine plus 4-aminopyridine (p<0.001), but the dose response curves did not deviate from parallelism. The combinations of edrophonium plus neostigmine and edrophonium plus 4-aminopyridine were not different from edrophonium alone (p<0.867). The response of edrophonium showed a ceiling effect. 4-aminopyridine interacted synergistically with neostigmine (fig. 3), resulting in a decrease of the ED50 for neostigmine from 15.9 pg/kg to 5.0 pg/kg. The inter­ action between edrophonium and both neostigmine and 4-aminopyridine was antagonistic (fig. 3). When equipotent doses were considered, the onset time of neostigmine plus 4-aminopyridine was not different from the onset time of neostigmine alone (p<0.96). The onset time of the combination of edrophonium and 4-aminopyridine was, however,

Table 1. Number of cats studied in each group.

number of cats drug administered 5 neostigmine 4 edrophonium 4 4-aminopyridine 6 neostigmine + 4-aminopyridine 150 ug/kg 4 edrophonium + 4-aminopyridine 150 ug/kg 6 edrophonium + neostigmine 10 yg/kg 3 edrophonium 150 yg/kg + neostigmine 20 yg/kg 3 edrophonium 150 yg/kg + 4-aminopyridine 300 yg/kg

70. 100 -ι

75 E w aс I 50 га

25

—ι—ι—ι— τ—ι—ι—ι 10 30 50 100 300 500 dose (iug/kg) Figure 1. Dose response curves for neostigmine, 4-aminopyridine, and neostigmine plus 4-aminopyridine 150 yg/kg in cats under a-chloralose-urethane anesthesia (·-·: neostigmine; X—X: 4-araino- pyridine; о—o: neostigmine plus 4-aminopyridine).

100

75 - E to с о en ¿гаS 50 с га

25

25 50 100 200 500 800 dose (/ug/kg) Figure 2. Dose response curves for edrophonium, edrophonium plus neostigmine IO pg/kg, and edrophon­ ium plus 4-aminopyridine 150 Ug/kg in cats under a-chloralose-urethane anesthesia Ç— ì edrophonium; ·—·: edrophonium plus neostigmine; X—x: edrophonium plus 4-aminopyridine). 71. 5 10 20 50 100 150 50 100 150 Neosigmre (Ajg kg) Edrotoniwr ( ^g hg) Edro'onur- tg kq>

Figure 3. Isobolograms for the ED^Q of neostigmine, edrophonium, 4-amino- pyndine, and their combinations.

significantly prolonged compared to the onset time of edrophonium and the combination of edrophonium and neostigmine (p<0.003) (fig. 4). The duration of the neostigmine plus 4-aminopyridine combina­ tion was significantly prolonged in comparison to neostigmine alone (p<0.005). Also, the duration of action of edrophonium in combin­ ation with 4-aminopyridine was longer than edrophonium alone (p<0.002), while there was no difference in the combination of edrophonium with neostigmine (p<0.233). When neostigmine alone or m combination with edrophonium was administered, atropine was required to keep the arterial pressure unchanged, with neostigmine alone, 8 yg atro­ pine per 100 vg neostigmine was needed, wmle with the combination 4 \ig atropine per 100 yg, neostigmine plus edrophonium was required. When neostigmine was administered in combination with 4-aminopyri- dine, no atropine was required at all, as was the case with edro­ phonium alone or m combination with 4-aminopyridine. Disaussion The synergistic interaction between neostigmine and 4-ainino- pyndine is in accordance with our earlier studies in both rats and man (Miller et al. 1978; Miller et al. 1979). It results in all these species in a decrease of about 65% of the ED50 for neostigmine. A possible explanation for this synergistic interaction is that 4- aminopyndine increases the acetylcholine release (Bowman et al. 1977; Molgo et al. 1977), while neostigmine inhibits the Cholines­ terase activity. These together result m more acetylcholine being available than with either drug alone. If the main effect of edro­ phonium were an increase in acetylcholine release, as has been pro­ posed in the past (Blaber et al. 1959; Blaber 1972), additive inter­ action would be expected in the combination with 4-aminopyridine, and a synergistic interaction in combination with neostigmine. If its main effect were the same as neostigmine (Smith et al. 1952; Nastuk et al. 1958), i.e. Cholinesterase inhibition, an additive interaction with neostigmine and a synergistic interaction with 4-aminopyridine would be expected. However, if its main effect is

72. (3) 15 Τ

(4) 10 Τ (6) onset (mm) (4) Τ (4) (4) 2 3 0 1 6

150

100 Τ durationι (mm) 150

τ- τ 1 2 3 r=n Ι | 6

Figure ^. Time to peak effect and duration of action until 50% return of the depressed twitch tension for equipotent doses of neostigmine, edrophonium, ¿t-amino- pyndine, and their combinations (1: neostigmine 20 Pg/kg; 2: neostigmine 5 yg/kg plus 4-aminopyridine 150 pg/kg; 3: 4-aminopyridine 300 ug/kg; 4: edrophon­ ium 150 jg/kg; 5: edrophonium 150 yg/kg plus neostig­ mine 10 ug/kg; 6: edrophonium 150 Ug/kg plus 4-amino- pyridine 150 ug/kg). displacement of a blocking agent from post-functional receptors (Artusio et al. 1951; Randall 1951), additive interaction with both neostigmine and 4-aminopyridine is to be expected. Due to the antagonistic interaction we found, it must be concluded that edro­ phonium possesses a different, not yet explained, mechanism of action while antagonizing a pancuronium blockade. The ceiling effect m the dose response curve for edrophonium which we demon­ strated (although we only studied a few high doses of edrophonium), is in accordance with results found by others. Several authors found edrophonium to be inadequate in reversing deeper d-tubocurar- ine blockade both in man and in animals (Hunter 1952; Nastuk et al.

73. 1954; Kupperman et al. 1966; Katz 1967). Although with the combin­ ation of neostigmine and 4-aminopyridine no atropine was required in this study, some atropine was necessary to keep arterial pressure and/or heart rate constant in man (Miller et al. 1979). This indi­ cates that it is impossible to absolutely extrapolate the results found in cats to man.

In summary, we believe that, in contrast to the combination of neostigmine with 4-aminopyridine, the combination of edrophonium and 4-aminopyridine is clinically of no value for the antagonism of a nondepolarizing neuromuscular blockade.

References

Artusio J.F. J., Marburg B.E., and Crews M.A. (1951): A quantitive study of d-tubocurarine,tri-(di-ethyl-aminoethyxy) 1,2,3 Benzeen (Flaxedil) and a series of tri-methyl and di-raethyl-ethyl ammonium compounds in anesthetized man. Ann. N.Y. Acad. Sci. 54, 512-529. Blaber I.C. (1972): The mechanism of the facilitatory action of edrophonium in cat skeletal muscle. Br. J. Pharmacol. 46, 498-507. Blaber I.C, and Bowman W.C. (1959): A comparison between the effects of edro­ phonium and choline in the skeletal muscles of the cat. Br. J. Pharmacol. 14, 456-466. Booij L.H.D.J., Miller R.D., and Crul J.F. (1978): Neostigmine and 4-aminopyri­ dine antagonism of lincomycin-pancuronium neuromuscular blockade in man. Anesth. Analg. 57, 316-321. Bowman W.C, Harvey A.L., and Marshall I.G. (1977): The actions of aminopyridines on avian muscle. Naunyn Schmiedeberg1 s Arch. Pharmac. 297, 99-103. Burkett L., ВікЬагі СВ., Thomas K.C Jr., Rosenthal D.A., Wirta M.C, and Foldes F.F. (1979): Mutual potentiation of the neuromuscular effects of antibiotics and relaxants. Anesth. Analg. 58, 107-115. Hunter A.R. (1952): Tensilon: A new anti-curare agent. Br. J. Anaesth. 29, 175-186. Katz R.L. (1967): Neuromuscular effects of d-tubocurarine, edrophonium and neo­ stigmine in man. Anesthesiology 28, 327-336. Kupperman A.S., and Okamoto M. (1966): A comparison between the effects of tetra ethylammonium and triethyl (3-hydroxyphenyl) ammonium on frog neuromuscular transmission. Br. J. Pharmacol. Chemother. 26, 218-228. Lee C, de Silva A.Y.D., and Katz R.L. (1978): Antagonism of polymyxine β induced neuromuscular and cardiovascular depression by 4-aminopyridine in the anesthe­ tized cat. Anesthesiology 49, 256-259. Miller R.D. (1976): Antagonisms of neuromuscular blockade. Anesthesiology 44, 318-329. Miller R.D., Booij L.H.D.J., Agoston S., and Crul J.F. (1979): 4-aminopyridine potentiates neostigmine and pyridostigmine in man. Anesthesiology 50, 416- 420. Miller R.D., Dennissen F.A.F., van der Pol F., Agoston S., Booij L.H.D.J., and Crul J.F. (1978): Potentiation of neostigmine and pyridostigmine by 4-amino­ pyridine in the rat. J. Pharm. Pharmac. 30, 699-702. Nastuk W.L., and Alexander J.T. (1954): The action of 3-hydroxyphenyldimethyl-

74. ethylararaonium (Tensilon) on neuromuscular transmission in the frog. J. Pharma­ col. Exp. Ther. II, 302-328. Nastuk W.L., and Alving B.O. (1958): Further studies of 3-hydroxyphenyldimethyl- ethyl ammonium (edrophonium) and its closely related analogues with respect to activity at the neuromuscular junction. Biochem. Pharmacol. 1, 307-322. Neter Y., and Wasserman W. (1974): Applied linear statistical models. Homewood, Illinois: Richard D. Irwin Inc. pp. 160-165, 436-450, 480-482, 689-703. Randall L.O. (1951): Synthetic curare-like agents and their antagonists. Ann. N.Y. Acad. Sci. 54, 460-479. Singh Y.N., Marshall I.G., and Harvey A.L. (1978): Reversal of antibiotic induced muscle paralysis of 3,4 diaminopyridine. J. Pharm. Pharmac. 30, 249-250. Smith СМ., Cohen H.L., Pellikan E.W., and Unna K.R. (1952): Mode of action of antagonists to curare. J. Pharmacol. Exp. Ther. 105, 391-399. Stoyanov E., Vulchev P., Sthurbova Μ., and Marinova M. (1976): Clinical electro- myomechanographic and electromyographic studies in decurarization with pymadine. Anaesth. Res. Intens. Ther. 4, 139-142.

75. Br J Anaesth (1980), 52, 1097

6·8 DO NEOSTIGMINE AND 4-AMINOPYRIDINE INHIBIT THE ANTIBACTERIAL ACTIVITY OF ANTIBIOTICS?

L H D J BOOIJ, G С J VAN DER PLOEG, J F CRUL AND H L MUYTJENS

SUMMARY Neostigmine and 4-aminopyridine are used to antagonize the neuromuscular blockade induced by antibiotics or an antibiotic-non-depolanzing blocker combination I hey may also counteract the antibacterial activity of antibiotics It uas found that neostigmine and 4-aminop\ ridine do not interfere with antibacterial acti\ it> οι antibiotics in bacterial cultures using an agar solution method It is concluded that neostigmine and í-ammop^ridmc ma> be used to antagonize ncuromusLular blockade induced bv antibiotics alone or in combination with non depolarizing agents

The neuromuscular blocking effects of antibiotics I he antibiotics, 4-aminopyridine lOOngml ' and their interaction w ith neuromuscular blocking and neostigmine 3 pg ml ' were mixed in the agar agents arc well known (Timmerman, Long and Fresh Mueller-Hinton agar supplemented with Pittinger, 1959, Pittinger and Adamson, 1972, 5% sheep blood or Schaedler agar, or both, were Singh, Harvcv and Marshall, 1978) The resulting used for culture of aerobic and facultative blockade is reversible in some cases by Cholines­ anaerobic species Drug solutions were added in a terase inhibitors or calcium vVan Nijhuis, Miller ratio of one part of drug solution to nine parts of and Fogdall, 1976, Booii, Miller and Crul, 1978, medium, a control plate without drugs was in­ Singh, Harvey and Marshall, 1978), depending on cluded The bacterial suspensions used were 6-h the antibiotic administered It has been demon­ cultures of the individual strains tested in tryptone strated that in most circumstances 4-aminopvri- so\a broth diluted to a MacFarland 0 5 turbidity dine can reverse the neuromuscular blockade standard The agar plates were inoculated using a induced by antibiotic-myoneural blocker combi­ Denlev A 400 Multipoint Inoculator with ap­ nation íBooij, Miller and Crul, 1978, Burkett et proximately 1 3x10* colony forming units on al , 1979) Antibiotics are administered more each spot Each agar plate was inoculated with frequently in the operative period than previously three reference strains (A FCC or NCTC) and and it is important to know whether reversing a with freshly isolated strains from patients Alter blockade, induced or prolonged by antibiotics, 17 h incubation at 37 С the MIC was obtained with Cholinesterase inhibitors and 4-aminopv ri­ 'Barry, 1976) dine, influences the antibacterial effects of anti­ biotics In this studv we determined the effect of RESULTS neostigmine and 4-aminopvridine on antibiotic- I he bacterial strains on which the MIC was treated bacterial cultures determined are summarized in table I METHOD Addition of 4-aminopyridine or neostigmine, or The minimal inhibitory concentration 'MIC) of both, in concentrations of lOOngml ' and antibiotics with and without addition of 4-amino- 3 μg ml ' respectiveh, which are suggested to be pyridine or neostigmine or both, was determined peak plasma concentrations Cronnelly et al , with an agar dilution method The antibiotics 1979, S Agoston, personal communication) did studied were netilmvcin, gcntamicin, tobramycin, not alter MIC significantly (tables II, III sisomycin, neomycin, lincomycin and clinda­ mycin DISCUSSION Addition of neostigmine and 4-aminopyndinc to 1 FO Η Π J HOOIJ, M D , CTEHAHD ( J \AN DFR PlOEO, 4 D , Schaedler and Mueller-Hinton agar to produce PH D , JAN F С RLI , м η , HARRY 1 MUYTJENS, M D concentrations equal to the peak plasma concen­ t Departments of Anesthesiology and Medical Microhiolog\, tration after ι ν administration 'Cronnellv et al , Catholic Lniversity, Nijmegen I he Netherlands 1979, S Agoston, personal communication) did (. orrespondencc to Dr L H D J BOOIJ, Department of Anesthesiology, С atholic Univcrsitv, M Radboud Hospital, not influence the antibacterial activitv of anti­ Nijmegen, I he Netherlands biotics tested The administration of neostigmine

76. I ABIfc 1 HUL ten al lulium, un J üuuhiotus tutbd + = tested - noi ι ested

Antibiotic

С u II и іч Nail (».пи I obra biso Nto L into ( linda

1 PsíudoniOfiJ". аігиі^іПіпа ЛІС( 2785Î 2 i \iht.Tuhiu mit Л I ( С 25M22 Ъ Siuptnhnonu^ aurtu·· А К ( 25<)23 4 S r rep toi ок. t4S рпеитотші clin isol 2к 5 S Auri.u\ clin isol )x 6 / toh clin isol 2x 7 Kíebueüa spp clin isol )x 8 1* acritf>ino%a clin isol ÍK 4 l nur >büit<.r spp clin isol 2x 10 Proteus mirabilis clin isol 11 Inüol positiv». Proteus ърр с Im isol 12 ¡i at ter aides fr ü^ihs N( I С 9343 \ 1 С С òb ) 3 14 I шегш monücMogLtiet clin isol 15 ( Uniridìum perjringens clin isol 4x 16 lì fraglia subbp /гихіІі\ clin isol 2x 17 H fragili^ subsp tiiLtaioiaomuron clin isol 18 Hai tiroidei 7 ulgatus clin isol 2x 19 liuLttroidei meianinogeniLtis dsüLihurolvtii.ii·' clin isol 20 lìacttroida disrasoms clin isol 21 PiptOUrtptOLOLLlH tinuiTOblU*· clin isol 22 / u\obüLti.rnnn mortijírum clin isol 2Î Strip г indura clin isol 24 \lrip іичаііч clin isol

I ABLfc II MU mg ¡ure ' for the атшокіхючіаи wiihout and with the addition of 4-aminopyndine 100 ng ml {A t neostigmine 1 yig ml ' li , and both ( l or ι. uit urt,-. vit tubU l (hil\ г aliw* „huh iJiangt. ar^giicn Dm itepoj diluttoti {hal) or double the original Al К ' iï infuri the rangt oj ih*, muhod

( ulturc No MU ABC MIC L С MIC MIC

0 5 0 5 0 5 0 5

i 0 25 0 25 4 2 4 2 2 •^8 4\ 2-> Я 4 8 5 0 25 0 25 0 25 0 5 0 25 (1 25 0 25 (1 1 0 5 0 5 0 5 0 25 1 0 5 2 6 0 5 0 5 1 1 0 5 05 0 5 2 7 0 5 0 5 1 05 0 5 0 5 0 5 05 05 0 25 τ 7 0 5 0 5 1 0 5 0 5 0 5 0 5 1 7 0 5 0 5 05 0 5 0 5 0 25 256 N Iti 1 2-> 4 4 4 2 16 Я 4 1 1 12 H 111 2 4 4 4 2 16 9 0 25 0 5 0 5 2 9 0 5 0 5 0 5 0 5 0 5 0 25 τ in 1 0 5 0 25 1 0 5 0 5 0 5 0 25 4 11 0 5 0 25 0 25 0 5 0 5 0 5 0 5 0 5 0 5 0 25

77. 'I ABl bill /Vf /С mg lurt 1 ) (or hrmwivtw and * ImdamvLin wilhout and Zìith 4- ammopvndvic i00 ng mi ' (A , neosiigmwe î \ig mi ' \.B)or both ,(^ hunuitures •ice tabU' I On¡\ laluL·, whtíh ürt ι handed ari gn LU OHI ^up of dilution half or double iht ongtnaf .VÍ/í," is iti thin th< rangt of tht method

l incomjun CIlindam\cin

Culturi: no

2 0 25 О 25 О 125 0 125 Η О 125 І2 2 2 I I -I 0 25 0 25 0 125 0 25 1 0U6 0 06 0 06 0 125 8 2 256 256 ; О 125 16 4 О 125 16 1 0 06 17 16 1 IH 2 0 06 14 0 25 О 25 20 В 0 5 21 0 25 0 06 0 06 0 06 0 03 22 0 125 0 03 21 0 5 0 06 24 І2 Ì2

and 4-aminopyndine may be beneficial where C.ronnell\,R .Stanski.D R ,Miller.R D .Shcmer.I. В ,and neuromuscular blockade is caused by antibiotics Sohn, V J ІЧ7Ч) Renal function and the pharmacokinetics or ihcir interactions with non-depolan/.ing ol neostigmine in anesthetized man Лм*'(гЛі*м/<ікі, SI, 222 Miller, R 1) , НооцЛ. H L) J , Agoston, S , and Crul, J I·' neuromuscular blockers. Neostigmine and prob­ I1)?·}' l-Aminop\ ridine potentiates neostigmine and p\nd- ably pyridostigmine enhance the blockade pro­ osligmine in man .ÎNi\t/iL,ii>/ calcium and neostig­ nervous system eifects occur with doses of 4- mine Aru ,ΓΛΙ-ΪΙΟΛΙ^Λ , 48, 4 IH aminopyndine up to 0.35 mg kg '. Higher doses, Timmerman, J С , Long, J Ρ , and Piumgcr, ( Β I9594 however, cause agitation and disorientation of the Neuromuscular blocking properties ol various antibiotic patient (Miller et al., 1979) and should not be used. agents li>\inìl Appi Phúrmüuil , 1, 299 Van Niihius, L S, , Miller, R 1) , and Fogdall, R Ρ '1976, It is concluded that neostigmine and 4-amino- 'I he interaction of d-lubocurarme, pancuronium, polv- pyndine can be used to antagonize antibiotic- msxine II and neostigmine in neuromuscular function induccd neuromuscular blockage without inter- Amslh Anjlg Cine ,55,244 fenng with antibacterial activity.

ACKNCm I EDOI-MtNT LA NHOS'I IGMINI· ET L'AMINOPYRimNE-4 INHIBlNI-hLLhS LAC I IVITf·. AN I 1- The authors thank Mrs Joke ν d Ros for technical assistance BAC lERIENNT. Dl-S ANFIBIOTIQUliS'

RfcFEHEStCFS Barry, A I- <}97ЬЛ I'he АштпсгчЬч. Suscepnbilili Test,ρ 76 On utilise la neostigmine ct raminopvridme-4 pour antago- Philadelphia Lea & bebigcr Boon, 1 Η [) J , Miller, R D, and Crul, J V (1978- niser le blocage ncuromusculairc provoque par les anti­ Neostigmine and 4-dminop> ridine antagonism of biotiques ou par un melange bloquant antibiotique non de- lincomvun-pancuronium neuromuscular blockade in man polansant hiles peuvent également reagir contre 1 activité anti- Anctrh Anulg 'CUvt , 57, ilb bactenenne des antibiotiques On s'est aperçu que la neo­ Burkcll.l. ,Bikha/i,C; B,Kell\,( I ,l)csiree,A K.Wirla, stigmine et Гатіпоругк1іпе-4 ne troublent pas l'activité anti- M ti , and holdes, Ъ F ; ІЧІЧ) Mutual potentiation of the bactenenne des antibiotiques dans des cultures bactériennes neuromuscular effects ol antibiotics and relaxants Aneslh pour lesquelles un a utilise la methode de la solution d'agar- Ariulg '(.fai , 58, 107 agar On en conclut qu'il est possible d'utiliser la neostigmine et

78. 1 aminopvndine-4 pour antagoniscr Ie blixage neuromus- Bltxkade verwendt werden können, die durch Antibiotika— mlairc cause par les antibiotiques seuls on en melange avec deb entweder allein oder in einer Kombination mit nicht-de- agents non depolarisants polarisierendcn Mitteln—hervorgerufen wurde

BAKTLRIZIDL WIRKUNG VON ANIIBIOIIKA INHIBÍ N I А ЛС FIVIDAD AN IIBAC Π RIA! Db DURC H M Ob I ICïMINl UND 4-AMINOPYRIDIM I OS AN I IBIO I К Ob Ι Α ΝΚ)-Ι S IIGMINA Y LA Gl НЬММІ5 A4INOPIRIDINA-4>

/LSAMMENfAiSLMj SUMARIO Neobngmine und 4-Aminopvridine werden zur Bckampíung Se usan la neo-estigmina ν la aminopindina'4 para antagoni7ar der neuromuskulären Blockade verwendet, die durch el bloqueo neuromuscular inducido por los antibióticos о рог Antibiotika oder eine Kombination von diesen mit mcht-de- una mezcla bloqucadora antibiotica no-üepolarizanre Pueden polansierendcn Blockierungsmitteln hervorgerufen wird bs también reaccionar en contra de la actividad antibacterial de los ist auch möglich, dass sie der bakteriziden latigkcit von antibióticos Se comprobo que la neo-estigmina y la Antibiotika entgegenwirken I s wurde festgesteJit, dass aminopiridina-4 no imerhercn con la actividad aniibactcnal de Neostigmine und 4 Aminp\ndine keinen störenden Fintìuss los antibióticos en cultivos bacteriales cuando se uso el metodo auf die bakterizide I atigkeit von Antibiotika in de la solución de agar-agar Se llega a la conclusion de que se Baktenenkulturcn ausüben, wenn eine Agarlosungsmethode pueden unli/ar la neo-tstigmina y la aminopiridma-4 para verwendet wird bs wird geschlossen, dass Neosiigmini. und 4 antjgum¿ar el bloqueo neuromuscular inducido por los anti­ Ami nop\ ridine /UT Bekämpfung einer neuromuskulären bióticos solos o en combinación con agentes no-depolanzantes

79. "The design of panauvonium was an important step forward in medicinal chemistry and pharmacology, since it is one of few examples of success in the rational approach to drug design."

- William C. Bowman (1980) Professor and Chairman of Physiology and Pharmacology, University of Strathclyde, Glasgow, Scotland.

80. CHAPTER 7 CONCLUSIONS AND CLINICAL IMPLICATIONS Muscle relaxation during general anesthesia and intensive care treatment is requested to either facilitate intubation, facilitate artificial ventilation, or make surgery possible. At present, mus­ cle relaxation is achieved by the administration of specific muscle relaxing drugs. To date, pancuronium bromide is the most frequently- used relaxant of the nondepolarizing type. Although it shows less severe side effects than the other nondepolanzers currently used, prolonged muscle relaxation and problems in reversibility of pancur­ onium were reported in the literature (Belafsky et al. 1974; Adams et al. 1975; Geha et al. 1976). Some of these adverse reactions could be explained from interaction with concomitantly administered drugs like anesthetics and antibiotics (chapter lì. Others could be explained from changes in the pharmacodynamic and pharmacokinetic behavior due to hypothermia, acid-base balance disturbances, and renal and/or hepatic failure (chapters 1 and 2). For some cases, no explanation was available. Since pancuronium is partially meta­ bolized in the liver, it was suggested that the metabolites are responsible for the remainder of the side effects. The metabolic pathways appeared to be deacetylation at the C3 and C17 atoms of the pancuronium molecule, resulting in 3-OH-pancur- onium, 17-OH-pancuronium, and 3,17-diOH-pancuronium derivatives. If these metabolites possess neuromuscular transmission blocking prop­ erties, they may contribute to the pancuronium-induced blockade. In circumstances of changed metabolism, they may therefore influence the degree, duration, and reversibility of such blockades as compared with normal circumstances, and hence could be the origin of prolonged blockade and irreversibility. Changes in body temperature, induction or inhibition of liver enzymes, or hepatic diseases may, as occurs with many other drugs, cause a changed metabolism. We demonstrated (chapters 3, 4, and 5) that all metabolites possess neuromuscular blocking properties, pancuronium being twice as potent as 3-OH-pan- curonium, and 40 to 50 times as potent as 17-OH-pancuronium or 3,17- diOH-pancuronium.

Due to this potency and the estimated amount produced, only 3-OH-pancuronium can be considered as contributing to the pancuronium blockade to a clinically important extent (chapter 4). This contri­ bution may be even larger if 3-OH-pancuronium were to interact syner- gistically with pancuronium or behave pharmacodynamically and pharma- cokinetically in a different way. Because of their individual po­ tencies and the amount to which they are produced, 17-OH-pancuronium and 3,17-diOH-pancuronium would only be of clinical importance if they interacted synergistically with pancuronium. We demonstrated that the metabolites in man do not behave differently from pancuron­ ium either pharmacodynamically or pharmacokinetically (chapter 4). In the in vitro and in vivo animal studies we performed, a synergis­ tic interaction could not be demonstrated. In the in vitro experi­ ments only summative (additive) interaction was present, whereas in the in vivo experiments only an antagonistic effect of the metabol­ ites on pancuronium was seen (chapter 5). An explanation for this discrepancy could not be proposed. As the interaction seems to be antagonistic, this cannot therefore cause a prolongation of the pan­ curonium blockade.

81. In view of significant species differences, it is impossible to predict the pharmacodynamic behavior of nondepolarizing relaxants in man from animal studies. Since an indication of the potency and the duration of action is necessary before studies in man can safely be performed in patients, we looked for a method to obtain such an indication. The isolated arm technique (chapter 3) proved to be a reliable method to predict the potency and duration of action (i.e. the pharmacodynamics) of relaxants in volunteers without causing interference with respiration and psychomotor functions of the volunteer (chapter 3). The pharmacodynamic results we obtained for pancuronium and its metabolites with the isolated arm technique were substantiated by the results obtained in patients with the same compounds (chapter 3.2). As mentioned above, significant differences between pancuronium and its metabolites in onset of action, duration, and recovery could not be demonstrated, with the exception that 17- OH-pancuronium had a shorter duration of action in patients (chapter 4) . We therefore conclude that the pancuronium metabolites do not pharmacodynamically behave differently from pancuronium. It has been shown by others that the degree of the existing blockade correlates well with the plasma concentration of pancuronium after intravenous administration (Agoston et al. 1979; Shanks et al. 1979) . This indicates a clear correlation between pharmacodynamics and pharmacokinetics. It is therefore not surprising that the pharma­ cokinetics of the metabolites of pancuronium do not differ, either (chapter 4). It must be concluded that prolonged duration of action is more likely due to absolute or relative overdose of pancuronium, disturb­ ances in liver uptake or renal excretion, or possible interaction with concomitantly administered drugs (antibiotics and/or anesthe­ tics) . The metabolites of pancuronium do not play a significant role. Problems are also described with reversibility of pancuronium blockade. In some cases the blockade cannot be reversed, while m other cases reversal is accompanied by many muscarinic side effects (chapter 6.1). Especially when other factors like acidosis, hypo­ thermia, and drug interactions are excluded, the pancuronium meta­ bolites were blamed. We therefore undertook to determine whether neostigmine can equally reverse blockade by pancuronium and its metabolites. 3-OH-pancuronium needed less neostigmine than pancur­ onium, whereas 17-OH-pancuronium and 3,17-diOH-pancuronium needed more neostigmine (chapter 6.3), and blockade by any of these could be completely reversed. Since 17-OH-pancuronium and 3,17-diOH-pan- curonium are only present in small amounts, and are not very potent, a significant degree of blockade due to these compounds will not occur. Thus, dosages of neostigmine that normally reverse the pure pancuronium part of the blockade will also be sufficient to reverse the metabolite-induced partial blockades, such that they do occur. Failure of reversal of pancuronium blockades therefore remain the result of other factors as mentioned above, including the adminis­ tration of insufficient amounts of Cholinesterase inhibitors. Large amounts of Cholinesterase inhibitors cause severe mus­ carinic side effects. Administration of high dosages of pyndostig-

82. mine or neostigmine is thus undesirable. Since it is thought that the main effect of these compounds m reversing curanform blockade is the availability of increased amounts of acetylcholine at the motor end-plate, other drugs resulting in increased synaptic acetyl­ choline production may be useful. The combined administration of neostigmine or pyridostigmine with such a drug may be advantageous and diminish the muscarinic side effects. For this reason, 4-amino- pyridine, an acetylcholine release-facilitating compound, was com­ bined with neostigmine or pyridostigmine. A synergistic interaction was demonstrated (chapter 6.5 and 6.6). This has led to a consider­ able decrease in the amount of neostigmine or pyridostigmine needed (chapter 6.6), and, indeed, resulted in less severe or even absent muscarinic side effects. From our studies it can also be concluded that edrophonium has a mechanism of action different from neostig­ mine or pyridostigmine because it is antagonized by both neostigmine and 4-aminopyridine (chapter 6.7). Concomitant or subsequent admin­ istration of edrophonium with 4-aminopyridine or neostigmine should therefore be avoided.

When antibiotics enhance nondepolarizing neuromuscular blockades or cause blockades by themselves, neostigmine and/or 4-aminopyridine are frequently administered in an attempt to reverse such blockades. When reversal is successful, the antibacterial activity of the anti­ biotic may also be antagonized, which is, of course, undesirable. In bacterial cultures we therefore studied the possibility of such an antagonism by determining minimal inhibiting concentrations of antibiotics with and without neostigmine and/or 4-aminopyridine. Neither neostigmine nor 4-aminopyridine antagonized the antibacterial activity of the antibiotics we studied (chapter 6.8).

Due to its many effects (chapter 6.2), it is not recommended that 4-aminopyridine be administered alone, although its combined administration with neostigmine or pyridostigmine can perhaps be advantageous.

In conclusion, it can be stated that the role of the metabolites of pancuronium is of minor importance m the determination of the degreee, duration, and antagonism of a pancuronium neuromuscular blockade.

Re f ρ re η с e s

Adams R.H., and Hombein T.F. (1975): Inability to reverse pancuronium blockade in a patient with renal and hepatic diseases. Anesthesiology 42, 362-364. Agoston S., Feldman S.A., and Miller R.D. (1979): Plasma pancuronium neuromuscular blockade after injection into the isolated arm, bolus injection, and continuous infusion. Anesthesiology 51, 119-122. Belafsky M.A., and Klawans H.L. (1974): Prolonged neuromuscular blockade with pancuronium bromide in a young healthy woman. Anesthesiology 40, 295-296. Geha D.G., Blitt, CD., and Moon B.J. (1976): Prolonged neuromuscular blockade with pancuronium in the presence of acute renal failure: A case report. Anesth. Analg. 55, 343-345. Shanks C.A., Somogyi A.A., and Triggs E.J. (1979): Dose-response and plasma con­ centration-response relationship of pancuronium in man. Anesthesiology 51, 1111-1118.

83. "It should be remembered, hauever, that the use of relaxants constitutes a deliberate encroachment on one of the most important physiological mechanisms, respiration. Every instance of their application should be looked upon as an experiment in applied pharmacology."

- Francis F. Foldes (1967) Emeritus Professor of Anesthesia, Montefiore Hospital and Albert Einstein University, New York, U.S.A.

84. CHAPTER 8 SUMMARY Pancuronium is currently the most frequently-used nondepolar­ izing muscle relaxant. Compared to the other clinically available nondepolarizers, its side effects are less severe. It is metabol­ ized in the liver, and excreted mainly m the urine. The rate and extent of metabolism are not precisely known, due to the present lack of a specific quantitative method of determina­ tion in biological fluids. Based on aspecific semiquantitative methods, the metabolism is assumed to be 30 to 45 % of the dose administered. 25 to 35 % is transformed into 3-OH-pancuronium, and 50 to 10% into 17-OH-pancuronium and 3,17-diOH-pancuronium together. As demonstrated in chapters 3, 4, 5, and 6, all derivatives result in neuromuscular blocking activity, and may thus contribute to a pancuronium neuromuscular blockade. Their potency ratios compared to pancuronium are: only 1/2 for 3-OH-pancuronium, and 1/40 to 1/50 for 17-OH-pancuronium and 3,17-diOH-pancuronium. Based on the assumed extent of metabolism and their potencies, only 3-OH-pancur- onium is capable of contributing to a pancuronium neuromuscular blockade, provided the other derivatives do not interact in a poten­ tiating manner. As demonstrated in chapter 6, such an interaction does not exist. Since 3-OH-pancuronium, like the other metabolites, does not behave pharmacokmetically differtly from pancuronium, this derivative also seems to be of no significant clinical impor­ tance. Only if a large amount of 3-OH-pancuronium, or a significant quantity of pancuronium, accumulates in the body (renal failure), can a prolonged duration of action be expected. In the case of hepatic disease, metabolism of pancuronium may be diminished, and the plasma level hence elevated for a longer period of time. These are the only currently available explanations for the prolonged duration of action, aside from interaction with other drugs (chapter 1). The metabolites of pancuronium by themselves are thus clinical­ ly insignificant. Antagonism of pancuronium may be necessary. When the anti­ cholinesterases are used, major muscarinic side effects can be seen, depending on the dose administered. Small amounts of 4-aminopyri- dine synergistically potentiate neostigmine and pyridostigmine, thereby decreasing the amount needed (chapter 7). Edrophonium is different, however. It is antagonized by both neostigmine and 4- aminopyndine. A different mechanism of action for edrophonium compared to neostigmine and pyridostigmine seems to exist. Neo­ stigmine can antagonize the action of both pancuronium and its metabolites completely. Moreover, less neostigmine is needed to antagonize a 3-OH-pancuronium blockade (chapter 6). If this com­ pound would contribute to the pancuronium blockade, no difficulties with reversing the paralysis would be expected. 17-OH-pancuronium and 3,17-diOH-pancuronium require more neostigmine, but are pro­ duced in such small quantities that when sufficient amounts of antagonists are administered to reverse pancuronium, no problems exist. Incomplete reversal is thus not due to the metabolites of pancuronium. Especially 4-aminopyridine is active when an anti-

85. biotic-induced neuromuscular blockade exists. It is therefore pre­ ferable in combined antibiotic-nondepolarizer blockades. The anti­ bacterial activity of the antibiotic is then not affected by 4- aminopyridine or neostigmine (chapter 6).

86. SAMENVATTING Momenteel is pancuronium het meest gebruikte niet-depolanzerende spierrelaxans. Vergeleken met andere klinisch gebruikte niet- depolanzerende relaxantia heeft het de minst ernstige bijwerkingen. Het wordt in de lever gemetaboliseerd en voornamelijk via de urine uitgescheiden. Snelheid en mate van metabolisme zijn niet exact bekend omdat een specifieke kwantitatieve bepalingsmethode m bio­ logische vloeistoffen ontbreekt. Gebaseerd op aspecifieke semi- kwantitatieve methoden wordt verondersteld dat het metabolisme 30 tot 45 procent van de toegediende hoeveelheid bedraagt. Daarbij wordt 25 tot 35 procent omgezet in 3-OH-pancuronium en 5 tot 10 procent in 17-OH-pancuronium en 3,17-diOH-pancuronium tesamen. Zoals in de hoofdstukken 3,4,5 en 6 wordt aangetoond hebben alle derivaten neuromusculaire overdracht blokkerende eigenschappen, en kunnen derhalve bijdragen aan een door pancuronium geïnduceerde blokkade. De effect ratios zijn vergeleken met pancuronium: slechts k voor 3-OH-pancuroniam en 1/40 tot 1/50 voor 17-OH-pancuronium en 3,17-diOH-pancuronium. Gebaseerd op de veronderstelde mate van metabolisme en de effectiviteiten wordt alleen van 3-OH-pnacuronium aangenomen dat het een bijdrage aan een door pancuronium veroor­ zaakte neuromusculaire blokkade kan leveren. Hierbij wordt aan­ genomen dat er geen potentierende wisselwerking tussen de meta- bolieten bestaat. Zoals wordt aangetoond in hoofdstuk 6 treedt een dergelijke wisselwerking niet op. Omdat 3-OH-pancuronium zich, evenals de andere metabolieten, farmacokinetisch niet verschillend gedraagt ten opzichte van pancuronium, lijkt ook deze afgeleidde niet van klinische betekenis te zijn. Alleen als een grote hoeveelheid 3-OH-pancuronium of een significante hoeveelheid pancu­ ronium in het lichaam ophoopt (niennsufficientie) kan een verlengde werkingsduur worden verwacht. In het geval van lever- ziekten zou de afbraak van pancuronium verminderd kunnen zijn, en de plasma concentratie dus voor een langere tijd hoog blijven. Op dit moment vormen dit de enige verklaringen voor verlengde werkinsduur naast interactie met andere farmaca (Hoofdstuk 1). Klinisch zijn de metabolieten van pancuronium daarom van geen belang. Soms is het nodig om pancuronium te antagoneren. Indien daarvoor acetylcholinesterase remmers worden gebruikt, dan kunnen, afhanke­ lijk van de toegediende hoeveelheden, ernstige muscarine bijwerk­ ingen optreden. Kleine hoeveelheden 4-aminopyridine versterken de werking van neostigmine en pyridostigmine. Hierdoor wordt de benodigde hoeveelheid verminderd (Hoofdstuk 6). Edrophonium gedraagt zich echter anders. Het wordt zowel door neostigmine als door 4-aminopyridine tegengewerkt. Hierdoor lijkt de aanname van een ander werkingsmechanisme voor edrophonium in vergelijking met neostigmine en pyridostigmine gerecitvaardigd. Neostigmine is in staat de werking van zowel pancuronium als haar metabolieten op te heffen. Om een 3-OH-pancuronium blokkade op te heffen is zelfs minder neostigmine nodig. Indien deze stof mocht bijdragen aan een pancuronium blok, zijn er geen problemen bij het antagon­ eren van de verslapping te verwachten. 17-OH-pancuronium en 3,17- diOH-pancuromum behoeven meer neostigmine, maar worden in dusdanig kleine hoeveelheden gevormd dat indien voldoende grote hoeveelheden antagonist worden toegediend om de pancuronium te antagoneren, geen problemen zullen ontstaan. Onvolledig antagonisme is derhalve niet

87. het gevolg van de metabolieten van pancuronium. Vooral 4-aminopyri- dxne is ook actief indien een door antibiotica geïnduceerde neuro- musculaire blokkade bestaat. Het verdient dan ook de voorkeur bi] een gecombineerde antibiotica - niet depolanzend relaxans blokkade. De anti-bacteriele werking van de antibiotica wordt daar- bi] noch door 4-aminopyridine noch door neostigmine aangetast (Hoofdstuk 6).

88. 89. 90. CURRICULUM VITAE Leonardus Henricus Domitianus Joseph Booij born in Dordrecht on October 27, 1946 1959 - 1964 HBS.В at Titus Brandsma College, Dordrecht 1964 - 1973 Medical school and internships Catholic University Nijmegen 1967 - 1973 Assistant, Department of Physiology Catholic University Nijmegen 1973 - 1977 Resident in Anesthesia Department of Anesthesiology Catholic University Nijmegen since 1977 Licensed specialist of anesthesiology since 1977 Staff member Department of Anesthesiology Catholic University Nijmegen 1978 - 1979 Visiting professor of Anesthesiology Department of Anesthesia University of California San Francisco, U.S.A. since 197Θ Affiliate member of the American Society of Anesthesiologists 1970 - 1978 Member of the Town Council of Beuningen

91.

STELLINGEN

I Scientific development of anesthesia in The Netherlands is inhibited by emphasis on clinical work over research in the academic anesthesia departments.

De wetenschappelijke ontwikkeling van de anesthesiologie in Nederland wordt geremd door een te sterke overheersing van het klinische werk in de academische anesthesie afdelingen.

II That nondepolarizing muscle relaxants can act specifically on the motor end-plate has been demonstrated by the development of Norcuron

Dat niet depolarizerende spier relaxantia zeer specifiek op de motor-eind- plaat kunnen werken is gedemonstreerd aan de ontwikkeling van Norcuron. I G Marshall, S Agoston, L H D J Booij, N N Durant and F F Foldes Br J Anaesth 52 11-20S, 1980

III Hypothermia exerts an effect on nondepolarizing neuromuscular blockade opposite to the effect usually described in the literature.

Hypothermie heeft een effect op de met depolarizerende zenuwspier blok­ kade welke tegenover gesteld is aan die welke in de literatuur meestal wordt gevonden. W С Bowman pharmacology of neuromuscular function pp 108-109, Wright & Sons Ltd Bristol 1980

IV Increased calcium release in skeletal muscle cells is not the origin, but a result, of malignant hyperthermia.

Toegenomen calcium vrijmaking in skeletspiercellen is niet de oorzaak, maar een gevolg van maligne hyperthermic. Τ E Nelson and E Η Flewellen Texas reports on Biol and Med 38 105-120, 1979 ν The development of clean, short-acting anesthetic drugs is obligatory to make total intravenous anesthesia safe and acceptable.

De ontwikkeling van kort werkende anesthesie farmaca zonder bijwerkingen is voorwaarde om een volledige intraveneuze anesthesie techniek veilig en aanvaardbaar te maken. T. M. Savege, M. A. E. Ramsay, J. P. J. Curran, J. Cottes, P. T. Walling and В. R. Simpson. Anaesthesia 30: 757-764, 1975.

VI From a scientific viewpoint, routine epidural application of morphino- mimetics for pain relief is premature.

Uit wetenschappelijk oogpunt is de routine-matige epidurale toediening voor pijn bestrijding nog voorbarig. F. Magora, D. Olshwang, D. Eimerl, J. Shorr, R. Katrenelson, S. Cotev and J. T. Davidson. Br. J. Anaesth. 52: 247-252, 1980.

VII Regionalization and concentration of traumatology and major emergency care is necessary for reasons of treatment efficiency and economy.

Regionalisatie en concentratie van de traumatologie en grotere eerste hulp is noodzakelijk om redenen van effectiviteit van behandeling en kosten bewaking.

VIII The clinical use of 4-aminopyridine will be limited by the multitude of pharmacological effects.

Het klinisch gebruik van 4-aminopyridine zal beperkt zijn door de veelheid van haar farmacologische effecten. This Thesis, Chapter 6.2.

IX When a new drug with less severe as well as fewer side effects is registered, its predecessors with more side effects should be withdrawn from the market.

Als een nieuw geneesmiddel met minder en geringere bijwerkingen is ge­ registreerd, zouden zijn voorgangers met meer bijwerkingen uit de markt moeten worden genomen. χ The application of stricter ethical norms will decrease the cost of the present "sophisticated advanced" medical care.

De toepassing van strengere ethische normen zal de kosten van de huidige "sophisticated advanced" medical care doen dalen.

XI The problem of the empty stomach in anesthesia needs a sober approach.

Het probleem van de nuchterheid in de anesthesie behoeft een nuchtere aan­ pak. C. D. Blitt, H. L. Gutman, D. D. Cohen, H. Weisman and J. B. Dillon, Anesth. Anaig. 49: 707-713, 1970.

XII On many occasions disputes between surgeons and anesthetics are induced and solved by the plasma potassium level of the patient.

Vaak worden meningsverschillen tussen chirurgen en anesthesisten op­ geroepen of opgelost door een afwijkende plasma kalium concentratie van de patient.

XIII The verdict "Everyone knows better, although no one really knows it well" is reflected in the many committees existing today.

Het gezegde "Iedereen weet het beter, doch niemand weet het goed" wordt weerspiegeld in de vele commissies welke thans bestaan.