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A comparison of the pharmacological actions of 4-aminopyridine and two of its derivatives in the monkey
European Journal of Pharmacology, 135 (1987) 155-159
155
Elsevier F_.JP00677
A comparison of the pharmacological actions of 4-aminopyridine and two of its derivatives in the monkey Pieter T . M . Biessels
1,,,
M a r t i n C. H o u w e r t j e s 1, S a n d o r A g o s t o n 2 a n d A l a n S. H o r n 3
l Department of Pharmacology and Clinical Pharmacology, Universityof Groningen, 2 Research Group of the Institutes of Anesthesiology and Clinical Pharmacology, Unioersityof Groningen, and 3 Department of Pharmacy, University of Groningen, Groningen, The Netherlands
Received 25 September 1986, revised MS received 14 November 1986, accepted 16 December 1986
The neuromuscular, cardiovascular and central nervous system stimulating effects of 4-aminopyridine (4-AP), 2,4-diaminopyridine (2,4-DAP) and LF-14 were investigated in the monkey. All these compounds were shown to reverse the stable neuromuscular blockade produced by the intravenous infusion of pancuronium bromide. The doses producing 50% antagonism (EDs0) of the pancuronium-induced neuromuscular block were 0.50, 0.54 and 0.71 mg/kg for LF-14, 2,4-DAP and 4-AP respectively. The compounds had only slight cardiovascular effects. In contrast to 4-AP, LF-14 and 2,4-DAP did not reduce the duration of ketamine/diazepam-induced anesthesia, suggesting minimal if any central nervous system effects of these two compounds. Aminopyridines; Muscle relaxants; Neuromuscular transmission I. I n t r o d u c t i o n
The clinical use of 4-aminopyridine (4-AP) as a reversal agent for non-depolarizing muscle relaxants (Agoston et al., 1982) is limited by its side-effects, especially due to its central nervous system stimulation. Side-effects are also frequently seen in other clinical applications of 4-AP, e.g. treatment of myasthenia gravis (Lundh et al., 1979) and the Eaton-Lambert syndrome (Agoston et al., 1978). Restlessness and excitement predominate (Murray and Newson-Davis, 1981) but convulsions have been reported in some cases following relatively large doses (Ball et al., 1979). These side-effects are due to the central action of 4-AP and it is therefore reasonable to assume that more polar derivatives of 4-AP might have fewer central * To whom all correspondence should be addressed: Dept. of Pharmacology/Clinical Pharmacology, University of Groningen, Bloemsingel 1, 9713 BZ Groningen, The Netherlands.
effects because they have more difficulty in passing the blood-brain barrier. Indeed, 3,4-diaminopyridine (3,4-DAP) is known to produce less central stimulation than 4-AP (Harvey and Marshall, 1977), because it is less able to pass into the brain (Lemeignan et al., 1982). It also has a stronger peripheral action in vitro (Harvey and Marshall, 1977) although its action in vivo was almost the same as that of 4-AP (Durant et al., 1982). We have therefore synthesized several more polar derivatives of 4-AP and tested them in the rat both in vitro and in vivo (Biessels et al., 1984). In these studies the two derivatives, LF-14 (3[(dimethylamino)carbonyl]amino,4-aminopyridine) and 2,4-DAP, appeared to be more potent antagonists of the neuromuscular blockade than 4-AP, showing no or only negligible CNS effects in the rat. We therefore decided to test both compounds and 4-AP in the monkey. The aim of this study was to compare the relative antagonistic potencies of LF-14, 2,4-DAP and 4-AP in reversing the pancuronium-induced
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
156 neuromuscular blockade, to compare their cardiovascular side-effects and to compare their CNS stimulating effects as judged by their effects on the duration of ketamine/diazepam-induced anaesthesia.
2. Materials and methods
2.1. Antagonism of neuromuscular blocking agents Five 'pig tail', Macaca nemestrina, monkeys weighing between 7-11 kg were used for the 48 experiments. The animals were given at least one week rest between two experiments. The animals were anaesthetized with ketamine (10 mg/kg) administered intramuscularly (i.m.). Anaesthesia was maintained with thiopental, which was given in small bolus injections through an infusion line in the hind leg vein. The animals were intubated and ventilated with room air enriched with oxygen (15 m l / k g per stroke) at a frequency of 30 strokes/ min. The muscle relaxant was administered through a second intravenous (i.v.) line. The indirectly evoked twitch response of the adductor pollicis muscle was recorded continously, with supramaximal stimuli of 0.2 ms duration applied to the ulnar nerve at a frequency of 0.1 Hz. Blood pressure was monitored continously via an intraarterial line in nine experiments only i.e. experiments in which the highest doses of antagonists were used, to avoid unnecessary damage to the blood vessels from repeated arterial punctures. Heart rate was recorded by ECG and measurements were made at the start of the infusion of the muscle relaxant, during the neuromuscular block at 10, 20 and 30 min and after administration of one of the antagonists at 0.5, 2, 5, 10, 20, 30, 45 and 60 rain. Twitch tension, ECG and blood pressure were recorded on a polygraph. After the twitch tension had been stable for at least 15 min, infusion of the muscle relaxant, pancuronium bromide, was started at a high rate. When the twitch height depression was approximately 90%, the infusion rate was adjusted to maintain a 95% block. When this had been steady for at least 30 rain one of the reversal agents was administered. Only one dose of one antagonist
was studied in each experiment. The percentage of antagonism was measured and also the starting time i.e. the time from injection to maximal antagonistic effect and the duration of action i.e. time from injection to 50% reappearance of the maximal effect. In order to exclude the spontaneous recovery of the neuromuscular blockade, the infusion of the muscle relaxant required to maintain a 95% block was kept constant throughout the experiment.
2.2. Reversal of ketamine/diazepam anaesthesia (CNS effects) Five monkeys, Macaca arctoides (three male and two female), were used, weighing between 7-12 kg. The monkeys were anaesthetized with ketamine (10 m g / k g i.m.) for transport and proper handling. One vein of the hind leg was cannulated for administration of the drugs. Bolus injections of ketamine (10 mg/kg), diazepam (0.2 mg/kg) and glycopyrrolate (0.04 mg/kg) were given at the start of the experiment. A ketamine infusion was then started at a speed of about 10 m g / k g per h. The infusion was stopped after 30 min and one of the compounds under investigation was given in a dose of 1 m g / k g (total volume 1 ml) or, in the control group, the same volume of saline. The time to nystagmus, first movements of head or leg and purposeful movements was measured. 'Full recovery' was considered as the time at which an additional dose of an anaesthetic agent (thiopental) had to be given for appropriate handling of the animals. The animals were given at least one week rest between the experiments.
2.3. Stat&tics All values are expressed as the means + S.D. and Student's t-test for unpaired data was used for analysis of the results. P values less than 0.05 were considered significant.
2.4. Materials The drugs used were ketamine (Parke Davis), thiopental (Nesdonal, Rhone Poulenc) pancuronium bromide (Organon Teknika) diazepam
157
(Hospital Pharmacy, Groningen) 4-aminopyridine (4-AP) (Janssen Chimica) 2,4-diaminopyridine (2,4-DAP), which was synthesized by us and 3[(dimethylamino)carbonyl]amino,4-aminopyridine (LF-14), which was kindly donated by L. Lalezari and F. Foldes. Montefiore Hospital, New York.
3. Results
lOOJ
A
0
I
I
I
I
0.2
dose mg/kg
3.1. Antagonism of the neuromuscular blockade In this study the contribution of the spontaneous recovery to the antagonistic activity of the drugs to be tested was eliminated by maintaining a steady neuromuscular block by continuous infusion of pancuronium bromide. The dose-response curves (as a percentage antagonism of the neuromuscular block) are shown in fig. 1B for LF-14 and 4-AP and fig. 1A for 2,4-DAP and 4-AP. The EDs0 and EDg0 values i.e. doses of drug which produced 50 and 90% antagonism respectively of the pancuronium depressed twitch tension were calculated from these curves. The EDs0 and ED90 values were: 0.50 + 0.05 and 1.1 + 0.15 mg/kg; 0.54 + 0.6 and 1.30 + 0.12 mg/kg; 0.70 + 0.05 mg/kg for LF-14, 2,4-DAP and 4-AP respectively. No ED9o value was determined for 4-AP because the highest dose (1.05 mg/kg) already caused slight convulsions. In one experiment we gave a dose of 1.45 mg/kg which did not produce more antagonism than the lower dose of 1.05 mg/kg but caused only more convulsions. The starting time and duration of action are shown in table 1. The starting time seemed dose-independent for 2,4-DAP and 4-AP but the
I
I
I
I
0.5 1.0 dose mg/kg
Fig. 1. (A and B) The antagonistic effects of LF-14 (A), 2,4-DAP (O) and 4-AP (e) on the twitch tension of the adductor pollicis muscle of the monkey. Pancuronium bromide was infused at a constant rate throughout the experiment. Results are expressed as a percentage of the antagonism of the pancuronium-depressed twitch. The vertical bars indicate the S.D. n = 4 for all points.
highest dose of LF-14 produced a significantly longer starting time. The effects on heart rate are shown in fig. 2. The values are given as percentage increase of the control values. The control values are the mean values of four measurements taken at the start of the infusion and during the 30 min steady state block before the administration of the antagonist. The duration of the effects on heart rate and blood pressure is shown in fig. 3A,B for the highest doses of the three antagonists. These effects were not of long duration and were back to the control values within 20 min in most cases. LF-14 induced a short-lasting decrease in the heart rate which, after some fluctuation, became a tachycardia that returned to control values after 20 min.
TABLE1 E f ~ c t s o n s t ~ f i n g t i m e a n d d u r a t i o n o f a c t i o n . Values are the meansof ~urexpefiments ±S.D. Dose
4-AP
(mg/kg)
Start (min)
Duration (min)
2,4-DAP Start (min)
Duration (min)
LF-14 Start (min)
Duration (mi~
0.3 0.5 0.75 1.~
5.5±1.0 8.0±1.1 7.5±1.5 8.0±1.5
30± 7 66± 8 85±14 1~±15
4.5±1.0 5.0±1.2 4.8±0.8 5.0±1.0
31± 8 47±10 1~±18 110±20
6.5±0.5 7.0±2.0 7.0±2.0 12 ±3.5
~±10 56±11 80±15 120±16
158 l
effect on heart rate 120O
~~
fdl recovery
t(min) SO-
100-
~0-
80-
30-
I I nystogmus
r
20I
02
I
I
1
0.S dose mg/kg
1.0 10-
Fig. 2. Effects of various doses of LF14 (A), 2,4-DAP ( © ) and 4-AP (O) on the heart rate. The results are expressed as a percentage of the control values.
3.2. Reversal of the ketamine-diazepam-induced anesthesia The times that elapsed from the end of the constant infusion of ketamine until the beginning of nystagmus and until full recovery are shown in fig. 4. 4-AP appeared to reduce significantly (P < 0.05) the recovery time from the ketamine/diazepam anesthesia, while LF-14 and 2,4-DAP had only a slight or no effect in this respect. In case of 4-AP all animals showed twitching of the legs and two animals clearly showed convulsions. Only one animal showed twitchings of the legs following the administration of LF-14 while 2,4-DAP exerted no convulsive activity whatsoever at this dose level. BP
o
100-~ /
c
HR
~
100
O
-"-~ ' --
~
•
80Y. I 2
I 5
t (rain)
I 10
I 15
Fig. 3. Effects of the highest doses (1 m g / k g ) of LF-14 (a), 2,4-DAP ( O ) and 4-AP (O) on the heart rate (lower graph) and m e a n arterial blood pressure (upper graph). The results are expressed as a percentage of the control values. * Significantly different from control value, P < 0.05.
0 control
/ , - AP
LF - 16
2.t,- DAP
Fig. 4. The effects of LF-14, 2,4-DAP and 4-AP (1 m g / k g ) on k e t a m i n e / d i a z e p a m anaesthesia in the monkey. Vertical bars indicate the recovery time plus S.E.M. and n = 1 0 for the control, and n = 5 for the test compounds. * Significantly different from control value, P < 0.05.
4. Discussion
The present results demonstrate that, in monkeys, LF-14, 2,4°DAP and 4-AP are very similar as to the nature of the neuromuscular actions they produce. All three reversed the pancuronium-induced neuromuscular block and the starting time and duration of their action were almost the same. In addition, their ED50 values were almost the same. The difference in antagonistic potency found in vivo and in vitro in the rat (Biessels et al., 1984) almost completely disappeared in the monkey. This could possibly be explained by a species-related difference, by sensitivity to the actions of these compounds or by pharmacokinetic factors. However, at higher doses, the potency of 4-AP seems less than the potency of the two other compounds in reversing a neuromuscular block, indicating that 4-AP could only partially reverse a neuromuscular block caused by non-depolarizing neuromuscular blocking agents. Therefore, and because of its central stimulating side effects, an ED9o value could not be determined for 4-AP. There were only slight differences in the cardiovascular effects of these three compounds. 4-AP produced only a small increase in blood pressure, as did 2,4-DAP and LF-14. The latter two compounds influenced the heart rate, dose dependently in the case of 2,4-DAP. However, an
159
increase in heart rate need not be considered as a disadvantage. As a potential clinical antagonist for neuromuscular blocking agents 2,4-DAP might be administered together with cholinesterase inhibitors. One of the advantages of such a combination would be that the dosage of both compounds could be reduced because they might act synergistically, as has been shown for 4-AP and neostigmine or pyridostigmine (Miller et al., 1978). We did not find a transient decrease in arterial blood pressure as was found in the cat and dog for 4-AP (Bowman et al., 1982) nor did we find any bradycardia as was seen in the cat for 4-AP and 3,4-DAP (Durant et al., 1982). In contrast to their peripheral actions, the central actions of these compounds were clearly different. 4-AP showed a definite reversal of ketamine/diazepam anesthesia, as was also found by Martinez-Aguirre and Crul (1979) and by Agoston et al. (1980) in humans. LF-14 seemed to have a slight effect on the recovery time from anesthesia, although the recovery time was not significantly different (P < 0.05) from the control value. 2,4-DAP seemed to have no effect on the CNS. LF-14 and 2,4-DAP are both more polar than 4-AP (Biessels et al., 1984), therefore it is assumed that they have more difficulty in passing the blood brain barrier. Consequently these two compounds are less likely to produce CNS effects or to have any convulsive activity, at least at clinical dose levels. In conclusion, there appears to be little difference between LF-14, 2,4-DAP and 4-AP in reversing the non-depolarizing neuromuscular blockade. Also their cardiovascular effects differ only slightly. However, their pronounced peripheral action gives 2,4-DAP and LF-14 a clear advantage over 4-AP in view of their potential clinical use as reversal agents of non-depolarizing neuromuscular blocking agents, or for use in the treatment of neuromuscular disorders.
References Agoston, S., P.J. Salt, W. Erdmann, T. Hilkemeyer, A. Bencini
and D. Langrehr, 1980, Antagonism of ketamine diazepam anaesthesia by 4-aminopyridine in human volunteers, Br. J. Anaesth. 52, 367. Agoston, S., D.R.A. Uges and R.L. Sia, 1982, Therapeutic applications of 4-aminopyridine in anaesthesia, in: Aminopyridines and Similarly Acting Drug, eds. P. Lechat, S. Thesleff and W.C. Bowman (Pergamon Press, Oxford) p. 303. Agoston, S., T. Van Weerden, P. Westra and P. Broekert, 1978, Effects of 4-aminopyridine in Eaton-Lambert syndrome, Br. J. Anaesth. 50, 383. Ball, A.P., R.B. Hopkinson and J.D. Farrel, 1979, Human botulism caused by clostridium botulinum. Type E. The Birmingham outbreak, Q.J. Med. 48, 473. Biessels, P.T.M., S. Agoston and A.S. Horn, 1984, A comparison of the pharmacological actions of some new 4-aminopyridine derivatives, European J. Pharmacol. 106, 319. Bowman, W.C., R.J. Marshall, J.W. Rodger and A.O. Savage, 1981, Actions of 4-aminopyridine on the cardiovascular systems of anaesthetized cats and dogs, Br. J. Anaesth. 53, 555. Durant, N.N., N. Nguyen, C. Lee and R.L. Katz, 1982, A comparison of 3,4-diaminopyridine and 4-aminopyridine in the anaesthetized cat, European J. Pharmacol. 84, 215. Harvey, A.H. and J.G. Marshall, 1977, The actions of three diaminopyridines or the chick biventer cervicis muscle, European J. Pharmacol. 44, 303. Lemeignan, M., H. Millart, D. Lamiable and P. Lechat, 1982, Evaluation of 4-aminopyridine and 3,4-diaminopyridine penetrability into cerebrospinal fluid in anesthetized rats, Brain Res. 304, 166. Lundh, H., D. Nilsson and J. Rosen, 1979, Effects of 4aminopyridine in myasthenia gravis, J. Neurol. Neurosurg. Psychiat. 42, 171. Martinez-Aguirre, E. and J.F. Crul, 1979, Effect of tetrahydroaminoacridine and 4-aminopyridine on recovery from ketamin-diazepam anaesthesic in the maccacus rhesus monkey, Acta Anaesth. Belg. 30, 231. Miller, R.D., P.A.F. Denissen, F. Van der Pol, S. Agoston, L.H.D. Booij and J.F. Crul, 1978, Potentiation of neostigmine and pyridostigmine by 4-AP in the rat, J. Pharm. Pharmacol. 30, 699. Murray, N.M.F. and J. Newson-Davis, 1981, Treatment with oral 4-aminopyridine in disorders of neuromuscular transmission, Neurology 31,265.
155
Elsevier F_.JP00677
A comparison of the pharmacological actions of 4-aminopyridine and two of its derivatives in the monkey Pieter T . M . Biessels
1,,,
M a r t i n C. H o u w e r t j e s 1, S a n d o r A g o s t o n 2 a n d A l a n S. H o r n 3
l Department of Pharmacology and Clinical Pharmacology, Universityof Groningen, 2 Research Group of the Institutes of Anesthesiology and Clinical Pharmacology, Unioersityof Groningen, and 3 Department of Pharmacy, University of Groningen, Groningen, The Netherlands
Received 25 September 1986, revised MS received 14 November 1986, accepted 16 December 1986
The neuromuscular, cardiovascular and central nervous system stimulating effects of 4-aminopyridine (4-AP), 2,4-diaminopyridine (2,4-DAP) and LF-14 were investigated in the monkey. All these compounds were shown to reverse the stable neuromuscular blockade produced by the intravenous infusion of pancuronium bromide. The doses producing 50% antagonism (EDs0) of the pancuronium-induced neuromuscular block were 0.50, 0.54 and 0.71 mg/kg for LF-14, 2,4-DAP and 4-AP respectively. The compounds had only slight cardiovascular effects. In contrast to 4-AP, LF-14 and 2,4-DAP did not reduce the duration of ketamine/diazepam-induced anesthesia, suggesting minimal if any central nervous system effects of these two compounds. Aminopyridines; Muscle relaxants; Neuromuscular transmission I. I n t r o d u c t i o n
The clinical use of 4-aminopyridine (4-AP) as a reversal agent for non-depolarizing muscle relaxants (Agoston et al., 1982) is limited by its side-effects, especially due to its central nervous system stimulation. Side-effects are also frequently seen in other clinical applications of 4-AP, e.g. treatment of myasthenia gravis (Lundh et al., 1979) and the Eaton-Lambert syndrome (Agoston et al., 1978). Restlessness and excitement predominate (Murray and Newson-Davis, 1981) but convulsions have been reported in some cases following relatively large doses (Ball et al., 1979). These side-effects are due to the central action of 4-AP and it is therefore reasonable to assume that more polar derivatives of 4-AP might have fewer central * To whom all correspondence should be addressed: Dept. of Pharmacology/Clinical Pharmacology, University of Groningen, Bloemsingel 1, 9713 BZ Groningen, The Netherlands.
effects because they have more difficulty in passing the blood-brain barrier. Indeed, 3,4-diaminopyridine (3,4-DAP) is known to produce less central stimulation than 4-AP (Harvey and Marshall, 1977), because it is less able to pass into the brain (Lemeignan et al., 1982). It also has a stronger peripheral action in vitro (Harvey and Marshall, 1977) although its action in vivo was almost the same as that of 4-AP (Durant et al., 1982). We have therefore synthesized several more polar derivatives of 4-AP and tested them in the rat both in vitro and in vivo (Biessels et al., 1984). In these studies the two derivatives, LF-14 (3[(dimethylamino)carbonyl]amino,4-aminopyridine) and 2,4-DAP, appeared to be more potent antagonists of the neuromuscular blockade than 4-AP, showing no or only negligible CNS effects in the rat. We therefore decided to test both compounds and 4-AP in the monkey. The aim of this study was to compare the relative antagonistic potencies of LF-14, 2,4-DAP and 4-AP in reversing the pancuronium-induced
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
156 neuromuscular blockade, to compare their cardiovascular side-effects and to compare their CNS stimulating effects as judged by their effects on the duration of ketamine/diazepam-induced anaesthesia.
2. Materials and methods
2.1. Antagonism of neuromuscular blocking agents Five 'pig tail', Macaca nemestrina, monkeys weighing between 7-11 kg were used for the 48 experiments. The animals were given at least one week rest between two experiments. The animals were anaesthetized with ketamine (10 mg/kg) administered intramuscularly (i.m.). Anaesthesia was maintained with thiopental, which was given in small bolus injections through an infusion line in the hind leg vein. The animals were intubated and ventilated with room air enriched with oxygen (15 m l / k g per stroke) at a frequency of 30 strokes/ min. The muscle relaxant was administered through a second intravenous (i.v.) line. The indirectly evoked twitch response of the adductor pollicis muscle was recorded continously, with supramaximal stimuli of 0.2 ms duration applied to the ulnar nerve at a frequency of 0.1 Hz. Blood pressure was monitored continously via an intraarterial line in nine experiments only i.e. experiments in which the highest doses of antagonists were used, to avoid unnecessary damage to the blood vessels from repeated arterial punctures. Heart rate was recorded by ECG and measurements were made at the start of the infusion of the muscle relaxant, during the neuromuscular block at 10, 20 and 30 min and after administration of one of the antagonists at 0.5, 2, 5, 10, 20, 30, 45 and 60 rain. Twitch tension, ECG and blood pressure were recorded on a polygraph. After the twitch tension had been stable for at least 15 min, infusion of the muscle relaxant, pancuronium bromide, was started at a high rate. When the twitch height depression was approximately 90%, the infusion rate was adjusted to maintain a 95% block. When this had been steady for at least 30 rain one of the reversal agents was administered. Only one dose of one antagonist
was studied in each experiment. The percentage of antagonism was measured and also the starting time i.e. the time from injection to maximal antagonistic effect and the duration of action i.e. time from injection to 50% reappearance of the maximal effect. In order to exclude the spontaneous recovery of the neuromuscular blockade, the infusion of the muscle relaxant required to maintain a 95% block was kept constant throughout the experiment.
2.2. Reversal of ketamine/diazepam anaesthesia (CNS effects) Five monkeys, Macaca arctoides (three male and two female), were used, weighing between 7-12 kg. The monkeys were anaesthetized with ketamine (10 m g / k g i.m.) for transport and proper handling. One vein of the hind leg was cannulated for administration of the drugs. Bolus injections of ketamine (10 mg/kg), diazepam (0.2 mg/kg) and glycopyrrolate (0.04 mg/kg) were given at the start of the experiment. A ketamine infusion was then started at a speed of about 10 m g / k g per h. The infusion was stopped after 30 min and one of the compounds under investigation was given in a dose of 1 m g / k g (total volume 1 ml) or, in the control group, the same volume of saline. The time to nystagmus, first movements of head or leg and purposeful movements was measured. 'Full recovery' was considered as the time at which an additional dose of an anaesthetic agent (thiopental) had to be given for appropriate handling of the animals. The animals were given at least one week rest between the experiments.
2.3. Stat&tics All values are expressed as the means + S.D. and Student's t-test for unpaired data was used for analysis of the results. P values less than 0.05 were considered significant.
2.4. Materials The drugs used were ketamine (Parke Davis), thiopental (Nesdonal, Rhone Poulenc) pancuronium bromide (Organon Teknika) diazepam
157
(Hospital Pharmacy, Groningen) 4-aminopyridine (4-AP) (Janssen Chimica) 2,4-diaminopyridine (2,4-DAP), which was synthesized by us and 3[(dimethylamino)carbonyl]amino,4-aminopyridine (LF-14), which was kindly donated by L. Lalezari and F. Foldes. Montefiore Hospital, New York.
3. Results
lOOJ
A
0
I
I
I
I
0.2
dose mg/kg
3.1. Antagonism of the neuromuscular blockade In this study the contribution of the spontaneous recovery to the antagonistic activity of the drugs to be tested was eliminated by maintaining a steady neuromuscular block by continuous infusion of pancuronium bromide. The dose-response curves (as a percentage antagonism of the neuromuscular block) are shown in fig. 1B for LF-14 and 4-AP and fig. 1A for 2,4-DAP and 4-AP. The EDs0 and EDg0 values i.e. doses of drug which produced 50 and 90% antagonism respectively of the pancuronium depressed twitch tension were calculated from these curves. The EDs0 and ED90 values were: 0.50 + 0.05 and 1.1 + 0.15 mg/kg; 0.54 + 0.6 and 1.30 + 0.12 mg/kg; 0.70 + 0.05 mg/kg for LF-14, 2,4-DAP and 4-AP respectively. No ED9o value was determined for 4-AP because the highest dose (1.05 mg/kg) already caused slight convulsions. In one experiment we gave a dose of 1.45 mg/kg which did not produce more antagonism than the lower dose of 1.05 mg/kg but caused only more convulsions. The starting time and duration of action are shown in table 1. The starting time seemed dose-independent for 2,4-DAP and 4-AP but the
I
I
I
I
0.5 1.0 dose mg/kg
Fig. 1. (A and B) The antagonistic effects of LF-14 (A), 2,4-DAP (O) and 4-AP (e) on the twitch tension of the adductor pollicis muscle of the monkey. Pancuronium bromide was infused at a constant rate throughout the experiment. Results are expressed as a percentage of the antagonism of the pancuronium-depressed twitch. The vertical bars indicate the S.D. n = 4 for all points.
highest dose of LF-14 produced a significantly longer starting time. The effects on heart rate are shown in fig. 2. The values are given as percentage increase of the control values. The control values are the mean values of four measurements taken at the start of the infusion and during the 30 min steady state block before the administration of the antagonist. The duration of the effects on heart rate and blood pressure is shown in fig. 3A,B for the highest doses of the three antagonists. These effects were not of long duration and were back to the control values within 20 min in most cases. LF-14 induced a short-lasting decrease in the heart rate which, after some fluctuation, became a tachycardia that returned to control values after 20 min.
TABLE1 E f ~ c t s o n s t ~ f i n g t i m e a n d d u r a t i o n o f a c t i o n . Values are the meansof ~urexpefiments ±S.D. Dose
4-AP
(mg/kg)
Start (min)
Duration (min)
2,4-DAP Start (min)
Duration (min)
LF-14 Start (min)
Duration (mi~
0.3 0.5 0.75 1.~
5.5±1.0 8.0±1.1 7.5±1.5 8.0±1.5
30± 7 66± 8 85±14 1~±15
4.5±1.0 5.0±1.2 4.8±0.8 5.0±1.0
31± 8 47±10 1~±18 110±20
6.5±0.5 7.0±2.0 7.0±2.0 12 ±3.5
~±10 56±11 80±15 120±16
158 l
effect on heart rate 120O
~~
fdl recovery
t(min) SO-
100-
~0-
80-
30-
I I nystogmus
r
20I
02
I
I
1
0.S dose mg/kg
1.0 10-
Fig. 2. Effects of various doses of LF14 (A), 2,4-DAP ( © ) and 4-AP (O) on the heart rate. The results are expressed as a percentage of the control values.
3.2. Reversal of the ketamine-diazepam-induced anesthesia The times that elapsed from the end of the constant infusion of ketamine until the beginning of nystagmus and until full recovery are shown in fig. 4. 4-AP appeared to reduce significantly (P < 0.05) the recovery time from the ketamine/diazepam anesthesia, while LF-14 and 2,4-DAP had only a slight or no effect in this respect. In case of 4-AP all animals showed twitching of the legs and two animals clearly showed convulsions. Only one animal showed twitchings of the legs following the administration of LF-14 while 2,4-DAP exerted no convulsive activity whatsoever at this dose level. BP
o
100-~ /
c
HR
~
100
O
-"-~ ' --
~
•
80Y. I 2
I 5
t (rain)
I 10
I 15
Fig. 3. Effects of the highest doses (1 m g / k g ) of LF-14 (a), 2,4-DAP ( O ) and 4-AP (O) on the heart rate (lower graph) and m e a n arterial blood pressure (upper graph). The results are expressed as a percentage of the control values. * Significantly different from control value, P < 0.05.
0 control
/ , - AP
LF - 16
2.t,- DAP
Fig. 4. The effects of LF-14, 2,4-DAP and 4-AP (1 m g / k g ) on k e t a m i n e / d i a z e p a m anaesthesia in the monkey. Vertical bars indicate the recovery time plus S.E.M. and n = 1 0 for the control, and n = 5 for the test compounds. * Significantly different from control value, P < 0.05.
4. Discussion
The present results demonstrate that, in monkeys, LF-14, 2,4°DAP and 4-AP are very similar as to the nature of the neuromuscular actions they produce. All three reversed the pancuronium-induced neuromuscular block and the starting time and duration of their action were almost the same. In addition, their ED50 values were almost the same. The difference in antagonistic potency found in vivo and in vitro in the rat (Biessels et al., 1984) almost completely disappeared in the monkey. This could possibly be explained by a species-related difference, by sensitivity to the actions of these compounds or by pharmacokinetic factors. However, at higher doses, the potency of 4-AP seems less than the potency of the two other compounds in reversing a neuromuscular block, indicating that 4-AP could only partially reverse a neuromuscular block caused by non-depolarizing neuromuscular blocking agents. Therefore, and because of its central stimulating side effects, an ED9o value could not be determined for 4-AP. There were only slight differences in the cardiovascular effects of these three compounds. 4-AP produced only a small increase in blood pressure, as did 2,4-DAP and LF-14. The latter two compounds influenced the heart rate, dose dependently in the case of 2,4-DAP. However, an
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increase in heart rate need not be considered as a disadvantage. As a potential clinical antagonist for neuromuscular blocking agents 2,4-DAP might be administered together with cholinesterase inhibitors. One of the advantages of such a combination would be that the dosage of both compounds could be reduced because they might act synergistically, as has been shown for 4-AP and neostigmine or pyridostigmine (Miller et al., 1978). We did not find a transient decrease in arterial blood pressure as was found in the cat and dog for 4-AP (Bowman et al., 1982) nor did we find any bradycardia as was seen in the cat for 4-AP and 3,4-DAP (Durant et al., 1982). In contrast to their peripheral actions, the central actions of these compounds were clearly different. 4-AP showed a definite reversal of ketamine/diazepam anesthesia, as was also found by Martinez-Aguirre and Crul (1979) and by Agoston et al. (1980) in humans. LF-14 seemed to have a slight effect on the recovery time from anesthesia, although the recovery time was not significantly different (P < 0.05) from the control value. 2,4-DAP seemed to have no effect on the CNS. LF-14 and 2,4-DAP are both more polar than 4-AP (Biessels et al., 1984), therefore it is assumed that they have more difficulty in passing the blood brain barrier. Consequently these two compounds are less likely to produce CNS effects or to have any convulsive activity, at least at clinical dose levels. In conclusion, there appears to be little difference between LF-14, 2,4-DAP and 4-AP in reversing the non-depolarizing neuromuscular blockade. Also their cardiovascular effects differ only slightly. However, their pronounced peripheral action gives 2,4-DAP and LF-14 a clear advantage over 4-AP in view of their potential clinical use as reversal agents of non-depolarizing neuromuscular blocking agents, or for use in the treatment of neuromuscular disorders.
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