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Comparative effects of velnacrine, tacrine and physostigmine on the twitch responses in the rat phrenic-hemidiaphragm preparation
Gen. Pharmac. Vol. 24, No. 5, pp. 1101-1105, 1993 Printed in Great Britain. All rights reserved
0306-3623/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd
COMPARATIVE EFFECTS OF VELNACRINE, TACRINE A N D PHYSOSTIGMINE ON THE TWITCH RESPONSES IN THE RAT PHRENIC-HEMIDIAPHRAGM PREPARATION FI~LIX BOSCH, MIGUEL MORALES, ALBERT BADIA and JOSEP-ELADI BAglOS* Divisi6 de Farmacologia, Departament de Farmacologia i Psiquiatria, Facultat Medicina, Universitat Aut6noma de Barcelona, 08193-Bellaterra, Spain [Tel. (3) 581 19 64 (3) 581 19 52; Fax (3) 581 20 04] (Received 1 March 1993) Abstract--l. Cholinesterase inhibitors potentiated twitch responses induced by nerve stimulation, with physostigrnine more potent than tacrine and velnacrine. However, at higher concentrations, tacrine decreased twitch responses in a concentration-dependent manner. 2. Tacrine strongly depressed directly-induced twitch responses, whereas the other drugs had minimal effects. 3. Physostigmine was the most potent drug in reversal of tubocurarine-induced blockade, but its antagonism index was similar to those obtained with tacrine and velnacrine at higher concentrations. When evaluating their ability to reverse neomycin-induced blockade, all drugs exhibited a similar effect. 4. It is concluded that tacrine and velnacrine are less potent than physostigmine in potentiating skeletal neuromuscular transmission. Additionally, the blocking effects of tacrine could limit its therapeutic efficacy.
INTRODUCTION In 1986, Summers et al. described the improving effects of tacrine (THA) in patients with Alzheimer's disease. This communication was followed by several studies that have essentially confirmed that tacrine could ameliorate some of the symptoms of these patients although liver toxicity has been an important factor in limiting its widespread use (Gamzu et al., 1990). Following these studies, considerable effort has been devoted to the synthesis of new cholinesterase inhibitors, including tacrine derivatives with the same clinical efficacy but devoid of the adverse effects. Velnacrine (l-hydroxytacrine, HP-029) is a tacrine derivative that has reached the stage of clinical development after it has been shown to be less toxic in experimental models (Shutske et aL, 1989). However, its pharmacological profile at cholinergic synapses has not been extensively studied and the articles describing its properties are limited (Bradley et al., 1988; Braga et al., 1991; Bosch et al., 1992a). Although the effects of velnacrine are similar to those exhibited by tacrine (Bradley et al., 1988; Braga et al., 1991), several differences suggest that velnacrine is somewhat different in some aspects. Tacrine, but not velnacrine, increases the frequency of giant M E P P and has blocking properties on sodium current in motoneurones (Braga et al., 1991).
*To whom all correspondence should be addressed.
The effects of both drugs were also different when considering their effects in tetanic fade and tension (Bosch et al., 1992a). These differences warrant a further evaluation of the effects of these compounds in cholinergic synapses. The present study was undertaken to analyse the comparative effects of velnacrine, tacrine and physostigrnine (as cholinesterase inhibitors of reference) on the rat neuromuscular junction in basal twitch responses and in the presence of neuromuscular blockade.
MATERIALS AND METHODS Right and left phrenic nerve-hemidiaphragm muscles removed from male Sprague-Dawley rats (250--300 g) were used. Details of experimental procedures have been described previously (Bafios et al., 1988). Briefly, rats were lightly anaesthetized with ether and decapitated. After quick dissection, each phrenic-hemidiaphragm was suspended in organ baths of 75 ml volume with Krebs-Henseleit solution of the following composition (mmol): NaCl 118, KC! 4.7, CaC12 2.5, KPO 4 H2 1.2, MgSO4 1.2, NaHCO3 25 and glucose 11.1. The preparation was bubbled with 5% CO2 in oxygen and the temperature was maintained at 25 + l°C. Several types of studies to assess drug effect on the neuromuscular junction of the rat were performed. Twitch responses were evaluated after indirectly- (through the nerve) and directly-induced (on the muscle surface) stimulation. Afterwards, studies were done to examine the ability of each drug to reverse the neuromuscular block induced by neomycin or tubocurarine on indirectly-elicited twitch responses. The changes on baseline tension of the phrenichemidiaphragm preparation were also evaluated in indirectly-elicited twitches.
1101
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F~LIX Bosch et al.
Stimulation parameters Indirectly-induced twitch responses were obtained by stimulating the phrenic nerve with square pulses of 0.5 msec duration at 0.2 Hz and supramaximal voltage. Muscular stimulation was performed with square pulses of 5msec at the same frequency and voltage and adding tubocurarine (5#mol) to prevent the potential indirect component of direct stimulation (Foldes et al., 1969). Reversal of neuromuscular blockade The ability of each drug to revert a previous neuromuscular blockade on indirectly-elicited twitch responses was analysed. Postsynaptic blockade was obtained with the addition of tubocurarine (1-1.5 #mol), whereas neomycin (1-1.5mmol) produced a preferentially presynaptic inhibition. Anticholinesterase drugs were added when a reduction of twitch response to 70-80% of control values was obtained. The effect of each drug was evaluated during 15min of exposure. To avoid the possible carry-over effects, only one drug concentration was tested on each preparation.
However, this effect was slightly lower t h a n obtained with velnacrine (maximal effect = 1 1 4 . 4 + 1.8% at 5 min with I pmol). Additionally, twitch responses were clearly inhibited with 3 0 # m o l or higher concentrations until reaching a total blockade with 1 m m o l (data not shown). Figure I(C) summarizes the results o b t a i n e d with physostigmine. This drug maximally potentiated twitch responses with 1 l~mol at 5 min (126.9___ 5.3%), and lower responses t h a n control were o b t a i n e d with c o n c e n t r a t i o n s equal or higher t h a n 1 0 0 p m o l (83.5 ___6.1% at 15 min) these results being very similar to those obtained with tacrine. This inhibition was evident after 15 min of exposition to 700/zmol of physostigmine (20.9 + 7.1%).
15min
Evaluation of drug effects In the experiments evaluating indirectly-elicited twitch responses at the baseline, changes were evaluated as potentiation or depression of control responses (100%) and quantified accordingly as a percentage change. To evaluate the effect on neuromuscular blockade, the antagonism index (AI or% of antagonism) described by Riesz et al. (1986) was calculated. Drug effects on direct stimulation were evaluatc~d as percentage of inhibition of control responses after 15 min of exposure and expressed as negative values. All results were expressed as mean __+standard error of the mean (SEM).
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Drugs Tacrine hydrochloride was purchased from Janssen Chimica (Belgium) and tubocurarine HCI, physostigmine and neomycin sulphate from Sigma (U.S.A.). Velnacrine maleate was synthesized by the Departament de Farmacologia i Quimica Terap6utica, Universitat de Barcelona. Drugs were diluted to final concentrations with distilled water. Physostigmine was additionally diluted with ethyl alcohol ( < 0.1% in the final dilution).
.
[--] lOmin [ ~ ] 1 5 m i n
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B
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C
RESULTS
Effects o f drugs on indirectly-elicited baseline tension Figure 1 summarizes the effects o f each drug at different times o f evaluation a n d shows t h a t twitch responses along the time differed with each drug. All o f them increased the baseline tension in a concent r a t i o n - d e p e n d e n t m a n n e r , a l t h o u g h some differences were evident when they were compared. Velnacrine p o t e n t i a t e d twitch responses between 1 a n d 3 0 0 g m o l [Fig. I(A)] the m a x i m a l effect being reached 5 m i n after 1 0 0 # m o l ( 1 2 4 . 0 + 7.8%). A t 3 0 0 # m o l [concentration n o t s h o w n in Fig. I(A)], a slight depression was observed at 10 m i n ( 9 7 . 6 _ 3.0%) a n d at 15 (91.5 _ 8.1%). N o further inhibition o f control values were observed with 7 0 0 # m o l or l m m o i of velnacrine. Figure I(B) shows t h a t tacrine p o t e n t i a t e d twitch responses when c o n c e n t r a t i o n s from 0.7 to 10/~mol were used.
0.3
0.7
1
10
100
700
DRUG C O N C E N T R A T I O N (I.tmol) Fig. 1. Effects of velnacrine (A), tacrine (B) and physostigmine (C) on indirectly-elicited twitch responses after 5, 10 and 15min of their addition to the bath. Phrenic nerves were stimulated supramaximally with square waves pulses of 0.5 msec duration at 0.2 Hz. The baseline tension was considered as control response (100%) and higher values were represented as potentiation and lower values as inhibition. Values are expressed as the mean of 6-9 experiments and range bars indicate the SEM. Concentrations higher than 700 # mol of velnacrine were not tested because of difficulties in preparing solutions. Several intermediate concentrations were also obtained but are not represented in order to simplify the figure.
Velnacrine, tacrine and physostigmine Z O
Although velnacrine (10/~mol) obtained the maximal value (AI = 27.1 _+ 7.3%), this value was not different to those obtained with physostigmine or tacrine. These drugs presented a similar concentration-effect - * - VEL range, although the former was slightly more potent - o - THA in reversing neomycin-induced blockade.
100
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o L~ < [., Z L) ¢h
1103
60
- A - PHY DISCUSSION
20 0 .... 0.01
± 0.1
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DRUG CONCENTRATION (mmol) Fig. 2. Concentration-response curves for velnacrine (VEL), tacrine (THA) and physostigmine(PHY) on directly-elicited twitch responses. Only inhibitory responses of drugs were observed in this experimental procedure. Phrenic nerves were stimulated supramaximally with square waves pulses of 5 msec duration at 0.2 Hz. The indirect component was previously blocked with tubocurarine (5/~mol). Values are expressed as the mean of 6-8 experiments and range bars indicate the SEM.
Effects on directly-elicited twitch responses Tacrine caused a marked inhibition of directlyelicited twitches whereas the effects of the others were only apparent when 1 mmol was used (Fig. 2). A concentration-dependent depression of twitch response was observed with concentrations of tacrine ranging from 0.1 to 1.3 mmol. A high inhibition was observed when the latter concentration was tested ( - 9 1 . 8 + 3 . 6 % ) . Maximal twitch depression with both velnacrine and physostigmine was obtained at 3 mmol ( - 13.3 + 11.6 and - 10.7 + 9.6%, respectively).
Effects of drugs on drug-induced neuromuscular blockade The three drugs reversed the tubocurarine-induced blockade [Fig. 3(A)]. The most potent drug was physostigmine and the least velnacrine, whereas tacrine had an intermediate potency [Fig. 3(A)]. After the maximal effect was achieved, a decrease of the effects of all drugs was observed. Maximal effect was obtained with physostigmine at 3/~mol (AI = 89.8 + 3.3%). The reversal effect of physostigmine began at lower concentrations (0.07 #mol) than tacrine and velnacrine. The later drug required higher concentrations than physostigmine and tacrine for reversing tubocurarine-induced blockade, although its maximal effect (AI = 79.1 + 11.2% at 100/zmol) was close to that obtained with physostigmine and indeed higher than the maximal reversal effect reached with tacrine (AI = 71.7 _ 5.5 at 3/zmol). The ability of all drugs tested in reversing neomycin-induced blockade was lower than that obtained with tubocurarine inhibition [Fig. 3(B)].
The present study confirms and extends the evidence that the pharmacological profile of tacrine and velnacrine has some differences (Bradley et aL, 1988; Braga et aL, 1991; Bosch et al., 1992a). Such differences suggest that, besides their anticholinesterase activity, these drugs could exert some actions at other sites at cholinergic synapses. When evaluating their effects on indirectly-elicited twitch responses, all drugs have a similar maximal potentiating effect, although the concentration range needed to reach it varied among the compounds. In this respect, physostigmine was slightly more potent than tacrine. However, the concentration of velnacrine needed to obtain a maximal potentiation was ten times greater than that required by tacrine. This result might be correlated with the anti~holinesterase activity of both compounds, as their ICs0 has been 100
A
80
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10
100
1000
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-
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1 10 100 DRUG CONCBNTRATION (~mol)
- * - VBL
- o - THA
- ' - PHY
Fig. 3. Reversal of the depressant effect of tubocurarine (A) and neomycin (B) on the indirectly-elicited responses by velnacrine (VEL), tacrine (THA) and physostigmine(PHY). Each drug was added when an inhibition of 70-80% of control twitch response was obtained with tubocurarine (I-I .5 #tool) or neomycin (1-1.5 mmol). The reversal effect measured by the antagonism index was evaluated after 15 rain of exposure of each anticholinesterase drug.
1104
F~LIXBOSCHet al.
described as 0.31 and 4.8/~mol for tacrine and velnacrine, respectively (Shutske et al., 1988). After the maximal effect had been reached, a progressive decrease of twitch responses was observed with all compounds, probably by the desensitization that follows the exposure of nicotinic receptors to high concentrations of acetylcholine (Bowman, 1990). The lower anticholinesterase activity of velnacrine could account for its minimal inhibitory effect. A clear differential effect was observed when the effects of drugs were evaluated in directly-elicited twitch responses. Here, tacrine showed a significant depressing effect over 100#mol until reaching the absolute blockade in the highest concentration tested. By contrast neither physostigmine nor velnacrine had significant actions in the usual concentration range, suggesting that this blockade of directly-elicited twitch is probably independent from anticholinesterase activity. Although this effect has been described previously (Karis et al., 1966), there is little information about its precise mechanism. Tacrine has several actions independent of cholinesterase inhibition (see Freeman and Dawson, 1991 for a review). Regarding its effects on neurotransmitter release, it has been shown that tacrine may block potassium channels in different nerve preparations (Freeman and Dawson, 1991), including motoneurones (Harvey and Rowan, 1988; Braga et al., 1991), although this action probably does not account for the described blocking effect. However, it has also been reported that tacrine may reduce sodium currents by interfering with the channel. This effect has been reported in M y x i c o l a giant axons (Schauf and Sattin, 1987), hippocampal neurons (Rogawski, 1987), Xenopus laevis axons (Elinder et al., 1989) and indeed mammalian motoneurones (Harvey and Rowan, 1988; Braga et al., 1991). This local anaesthetic-like effect may contribute to the muscular blockade observed in the present experiments. Although not tested, this action could at least in part, be responsible for the depressing effects of tacrine on indirectly-elicited twitch responses, besides its desensitiziting action on nicotinic receptors. The fact that effects of velnacrine and physostigmine in directly-elicited twitches were only apparent at very high concentrations suggests that these drugs are practically devoid of this action on sodium channels. A differential effect of anticholinesterase drugs on pre- and postsynaptic blockade was observed. All drugs reversed tubocurarine-induced blockade confirming previous results (Karis et al., 1966; Bradley et al., 1988; Braga et al., 1991) but showed differences in both concentration range and maximal effect. Physostigmine was again the most potent as its effect began at 0.1 and was maximal at 3#mol.
Effects of tacrine began at 0.3 and were the highest at 3 #mol. By contract velnacrine began to be active at 3 and full effect was obtained at 100#mol. Basically, these effects were similar to those observed in baseline twitch responses but concentration-response curves were slightly shifted to the right. However, the maximal effect achieved with tacrine in reversing neuromuscular blockade was lower than those observed with the other drugs. The fact that tacrine is a more potent cholinesterase inhibitor than veinacrine suggest that its effect might be limited by other collateral actions, such as blockade of postsynaptic receptors (Thesleff et al., 1990; Braga et aL, 1991; Bosch et aL, 1992a) or the already commented effects on sodium currents. The cholinesterase inhibitors used in this study were less potent in reverting the blockade induced by neomycin. This drug has the common property of aminoglycosides of decreasing calcium entry into motor nerve terminals (Singh et al., 1982; Bourret and Mallart, 1989; Bafios et al., 1989). It has been described that velnacrine and tacrine are unable to completely revert the neuromuscular depression induced by low calcium and high magnesium solutions (Bradley et al., 1988; Braga et al., 1991). Thereby, the small effects of these drugs on neomycin-induced blockade could be reasonably expected on these grounds. These results have additional interest in suggesting that the action of tacrine on potassium channels would contribute little to its final effects, as a potassium blocker, i.e. aminopyridines or guanidine, has a strong reversal action on aminoglycoside-induced blockade (Molg6 et al., 1979; Bosch et aL, 1992b). In summary, tacrine and velnacrine are less potent than physostigmine in potentiating skeletal neuromuscular transmission. However, velnacrine is devoid of some unspecific actions of tacrine that can limit its final action.
SUMMARY
After the description that tacrine could improve patients with Alzheimer's disease, considerable effort has been devoted to the synthesis of new cholinesterase inhibitors. Velnacrine (1-hydroxytacrine, HP-029) is a tacrine derivative which has exhibited a similar pharmacological activity to the parent drug with lesser toxicity. However, several differential effects warrants a further evaluation of its actions in cholinergic synapses. The present study analysed the effects of tacrine, velnacrine and physostigmine at the rat hemidiaphragm preparation. All drugs potentiated twitch responses induced by nerve stimulation, physostigmine being more potent than tacrine and
Velnacrine, tacrine and physostigmine velnacrine. Tacrine strongly depressed directlyinduced twitch responses, whereas the o t h e r drugs h a d lesser effects. Physostigmine was the most p o t e n t drug in reversing t u b o c u r a r i n e - i n d u c e d blockade, but its a n t a g o n i s m index was similar to those o b t a i n e d with velnacrine at higher concentrations. W h e n evaluating their ability to reverse neomycin-induced blockade, all drugs exhibited a similar effect. It is concluded t h a t tacrine a n d velnacrine are less p o t e n t t h a n physostigmine in p o t e n t i a t i n g skeletal neuromuscular transmission. Additionally, some effects exhibited by tacrine but not by velnacrine could limit its therapeutic efficacy. Acknowledgements--The authors want to thank to Drs M. Sim6n and D. Pujol from the Departament de Farmacologia i Quimica Terap6utica, Universitat de Barcelona for the synthesis of velnacrine maleate. FB was supported in part by a grant of the program Ajuts a Projectes de Recerca per a Investigadors Joves de la CIRIT (AR89, Generalitat de Catalunya, Spain). REFERENCES
Bafios J. E., Badia A. and Jan6 F. (1988) Facilitatory action of adrenergic drugs on muscle twitch evoked by nerve stimulation in the curarized rat phrenic hemidiaphragm. Archs Int. Pharmacodyn. Ther. 293, 219-227. Bafios J. E., Bosch F. and Badia A. (1989) The neuromuscular blocking effect of dibekacin and its reversal by Ca 2+ and drugs in the isolated rat phrenic-hemidiaphragm preparation. Gen. Pharmac. 20, 507-511. Bosch F., Morales M., Badia A. and Bafios J. E. (1992a) Effects of velnacrine, tacrine and physostigmine on tetanic twitch responses at the rat neuromuscular junction. Eur. J. Pharmac. 222, 163-166. Bosch F., Morales A., Badia A. and Bafios J. E. (1992b) Influence of extracellular calcium on the actions of guanidine at the rat neuromuscular junction. Archs Int. Pharmacodyn. Ther. 315, 110-119. Bourret C. and Mallart A. (1989) Depression of calcium currents at mouse motor nerve endings by polycationic antibiotics. Brain Res. 478, 403-406. Bowman W. C. (1990) Pharmacology of Neuromuscular Junction, pp. 126-133. Wright, London. Bradley R. J., Edge M. T., Moran S. G. and Freeman A. M. (1988) Effects of cholinergic drugs used in Alzheimer therapy at the mammalian neuromuscular junction. In Current Research in Alzheimer Disease (Edited by Giacobini E. and Becker R.), pp. 199-209. Taylor & Francis, New York. Braga M. F. M., Harvey A. L. and Rowan E. G. (1991) Effects of tacrine, velnacrine (HP029), suronacrine (HP128), and 3,4-diaminopyridine on skeletal neuromuscular transmission in vitro. Br. J. Pharmac. 102, 909-915.
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Elinder F., Mohammed A. K., Winblad B. and Arhem P. (1989) Effects of THA on ionic currents in myelinated axons of Xenopus laevis. Eur. J. Pharmac. 164, 599-602. Foldes F. F., Brodman E. B., Kranzler H, N., Underwood P. S. and Hermsworth B. A. (1969) The effects of germine diacetate on the rat phrenic nerve diaphragm preparation. Anesthesiology 31, 522-53 I. Freeman S. E. and Dawson R. M. (1991) Tacrine: a pharmacological review. Prog. Neurobiol. 36, 257-277. Gamzu E. R., Thal L. J. and Davis K, L. (1990) Therapeutic trials using tacrine and other cholinesterase inhibitors. In Advances in Neurology (Edited by Wurtman R. J. et al.), pp. 241-245, Vol. 51. Raven Press, New York. Harvey A. L. and Rowan E. G. (1988) Actions of THA, 3,4-diaminopyridine, physostigmine and galanthamine on neuronal K + currents at a cholinergic nerve terminal. In Current Research in Alzheimer Disease (Edited by Giacobini E. and Becker R.), pp. 191-197. Taylor & Francis, New York. Karis J. H., Nastuk W. L. and Katz R. L. (1966) The action of tacrine on neuromuscular transmission: a comparison with hexafluorenium. Br. J. Anaesth. 38, 762-774. Molg6 J., Lemeignan M., Uchiyama T. and Lechat P. (1979) Inhibitory effect of kanamycin on evoked transmitter release. Reversal by 3,4-diaminopyridine. Eur. J. Pharmac. 57, 93-97. Riesz M., Kapati E. and Szporny L. (1986) Antagonism of non-depolarizing neuromuscular blockade by aminopyridines in cats. J. Pharm. Pharmac. 38, 15~158. Rogawski M. A. (1987) Tetrahydroaminoacridine blocks voltage-dependent ion channels in hippocampal neurons. Eur. J. Pharmac. 142, 169-172. Schauf C. L. and Sattin A. (1987) Tetrahydroaminoacridine blocks potassium channels and inhibits sodium inactivation in Myxicola. J. Pharmac. Exp. Ther. 243, 609-613. Shutske G. M., Pierrat F. A., Cornfeldt M. L., Szewczak M. R., Huger F. P., Bores G. M., Haroutunian V. and Davis K. L. (1988) (+)-9-Amino-l,2,3,4-tetrahydroacridin-l-ol. A potential Alzheimer's disease therapeutic of low toxicity. J. Med. Chem. 31, 1278-1279. Shutske G. M., Pierrat F. A., Kapples K. J., Cornfeldt M. L., Szewczak R. R., Huger F. P., Bores G. M., Haroutunian V. and Davis K. L. (1989) 9-Amino-l,2,3,4-tetrahydroacridin-l-ols: synthesis and evaluation as potential Alzheimer's Disease therapeutics. J. Med. Chem. 32, 1805 1813. Singh Y. N., Marshall I. G. and Harvey A. L. (1982) Preand postjunctional blocking effects of aminoglycoside, polymixin, tetracicline and lincosamide antibiotics. Br. J. Anaesth. 59, 226231. Summers W. K., Majovski L. V., Marsh G. M., Tachiki K. and Kling A. (1986) Oral tetrahydroaminoacridine in long-term treatment of senile dementia, Alzheimer type. New Engl. J. Med. 315, 1241-1245. Thesleff S., Sellin L. C. and Tagerud S. (1990) Tetrahydroaminoacridine (tacrine) stimulates neurosecretion at mammalian motor endplates. Br. J. Pharmac. 100, 487-490.
0306-3623/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd
COMPARATIVE EFFECTS OF VELNACRINE, TACRINE A N D PHYSOSTIGMINE ON THE TWITCH RESPONSES IN THE RAT PHRENIC-HEMIDIAPHRAGM PREPARATION FI~LIX BOSCH, MIGUEL MORALES, ALBERT BADIA and JOSEP-ELADI BAglOS* Divisi6 de Farmacologia, Departament de Farmacologia i Psiquiatria, Facultat Medicina, Universitat Aut6noma de Barcelona, 08193-Bellaterra, Spain [Tel. (3) 581 19 64 (3) 581 19 52; Fax (3) 581 20 04] (Received 1 March 1993) Abstract--l. Cholinesterase inhibitors potentiated twitch responses induced by nerve stimulation, with physostigrnine more potent than tacrine and velnacrine. However, at higher concentrations, tacrine decreased twitch responses in a concentration-dependent manner. 2. Tacrine strongly depressed directly-induced twitch responses, whereas the other drugs had minimal effects. 3. Physostigmine was the most potent drug in reversal of tubocurarine-induced blockade, but its antagonism index was similar to those obtained with tacrine and velnacrine at higher concentrations. When evaluating their ability to reverse neomycin-induced blockade, all drugs exhibited a similar effect. 4. It is concluded that tacrine and velnacrine are less potent than physostigmine in potentiating skeletal neuromuscular transmission. Additionally, the blocking effects of tacrine could limit its therapeutic efficacy.
INTRODUCTION In 1986, Summers et al. described the improving effects of tacrine (THA) in patients with Alzheimer's disease. This communication was followed by several studies that have essentially confirmed that tacrine could ameliorate some of the symptoms of these patients although liver toxicity has been an important factor in limiting its widespread use (Gamzu et al., 1990). Following these studies, considerable effort has been devoted to the synthesis of new cholinesterase inhibitors, including tacrine derivatives with the same clinical efficacy but devoid of the adverse effects. Velnacrine (l-hydroxytacrine, HP-029) is a tacrine derivative that has reached the stage of clinical development after it has been shown to be less toxic in experimental models (Shutske et aL, 1989). However, its pharmacological profile at cholinergic synapses has not been extensively studied and the articles describing its properties are limited (Bradley et al., 1988; Braga et al., 1991; Bosch et al., 1992a). Although the effects of velnacrine are similar to those exhibited by tacrine (Bradley et al., 1988; Braga et al., 1991), several differences suggest that velnacrine is somewhat different in some aspects. Tacrine, but not velnacrine, increases the frequency of giant M E P P and has blocking properties on sodium current in motoneurones (Braga et al., 1991).
*To whom all correspondence should be addressed.
The effects of both drugs were also different when considering their effects in tetanic fade and tension (Bosch et al., 1992a). These differences warrant a further evaluation of the effects of these compounds in cholinergic synapses. The present study was undertaken to analyse the comparative effects of velnacrine, tacrine and physostigrnine (as cholinesterase inhibitors of reference) on the rat neuromuscular junction in basal twitch responses and in the presence of neuromuscular blockade.
MATERIALS AND METHODS Right and left phrenic nerve-hemidiaphragm muscles removed from male Sprague-Dawley rats (250--300 g) were used. Details of experimental procedures have been described previously (Bafios et al., 1988). Briefly, rats were lightly anaesthetized with ether and decapitated. After quick dissection, each phrenic-hemidiaphragm was suspended in organ baths of 75 ml volume with Krebs-Henseleit solution of the following composition (mmol): NaCl 118, KC! 4.7, CaC12 2.5, KPO 4 H2 1.2, MgSO4 1.2, NaHCO3 25 and glucose 11.1. The preparation was bubbled with 5% CO2 in oxygen and the temperature was maintained at 25 + l°C. Several types of studies to assess drug effect on the neuromuscular junction of the rat were performed. Twitch responses were evaluated after indirectly- (through the nerve) and directly-induced (on the muscle surface) stimulation. Afterwards, studies were done to examine the ability of each drug to reverse the neuromuscular block induced by neomycin or tubocurarine on indirectly-elicited twitch responses. The changes on baseline tension of the phrenichemidiaphragm preparation were also evaluated in indirectly-elicited twitches.
1101
1102
F~LIX Bosch et al.
Stimulation parameters Indirectly-induced twitch responses were obtained by stimulating the phrenic nerve with square pulses of 0.5 msec duration at 0.2 Hz and supramaximal voltage. Muscular stimulation was performed with square pulses of 5msec at the same frequency and voltage and adding tubocurarine (5#mol) to prevent the potential indirect component of direct stimulation (Foldes et al., 1969). Reversal of neuromuscular blockade The ability of each drug to revert a previous neuromuscular blockade on indirectly-elicited twitch responses was analysed. Postsynaptic blockade was obtained with the addition of tubocurarine (1-1.5 #mol), whereas neomycin (1-1.5mmol) produced a preferentially presynaptic inhibition. Anticholinesterase drugs were added when a reduction of twitch response to 70-80% of control values was obtained. The effect of each drug was evaluated during 15min of exposure. To avoid the possible carry-over effects, only one drug concentration was tested on each preparation.
However, this effect was slightly lower t h a n obtained with velnacrine (maximal effect = 1 1 4 . 4 + 1.8% at 5 min with I pmol). Additionally, twitch responses were clearly inhibited with 3 0 # m o l or higher concentrations until reaching a total blockade with 1 m m o l (data not shown). Figure I(C) summarizes the results o b t a i n e d with physostigmine. This drug maximally potentiated twitch responses with 1 l~mol at 5 min (126.9___ 5.3%), and lower responses t h a n control were o b t a i n e d with c o n c e n t r a t i o n s equal or higher t h a n 1 0 0 p m o l (83.5 ___6.1% at 15 min) these results being very similar to those obtained with tacrine. This inhibition was evident after 15 min of exposition to 700/zmol of physostigmine (20.9 + 7.1%).
15min
Evaluation of drug effects In the experiments evaluating indirectly-elicited twitch responses at the baseline, changes were evaluated as potentiation or depression of control responses (100%) and quantified accordingly as a percentage change. To evaluate the effect on neuromuscular blockade, the antagonism index (AI or% of antagonism) described by Riesz et al. (1986) was calculated. Drug effects on direct stimulation were evaluatc~d as percentage of inhibition of control responses after 15 min of exposure and expressed as negative values. All results were expressed as mean __+standard error of the mean (SEM).
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Drugs Tacrine hydrochloride was purchased from Janssen Chimica (Belgium) and tubocurarine HCI, physostigmine and neomycin sulphate from Sigma (U.S.A.). Velnacrine maleate was synthesized by the Departament de Farmacologia i Quimica Terap6utica, Universitat de Barcelona. Drugs were diluted to final concentrations with distilled water. Physostigmine was additionally diluted with ethyl alcohol ( < 0.1% in the final dilution).
.
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C
RESULTS
Effects o f drugs on indirectly-elicited baseline tension Figure 1 summarizes the effects o f each drug at different times o f evaluation a n d shows t h a t twitch responses along the time differed with each drug. All o f them increased the baseline tension in a concent r a t i o n - d e p e n d e n t m a n n e r , a l t h o u g h some differences were evident when they were compared. Velnacrine p o t e n t i a t e d twitch responses between 1 a n d 3 0 0 g m o l [Fig. I(A)] the m a x i m a l effect being reached 5 m i n after 1 0 0 # m o l ( 1 2 4 . 0 + 7.8%). A t 3 0 0 # m o l [concentration n o t s h o w n in Fig. I(A)], a slight depression was observed at 10 m i n ( 9 7 . 6 _ 3.0%) a n d at 15 (91.5 _ 8.1%). N o further inhibition o f control values were observed with 7 0 0 # m o l or l m m o i of velnacrine. Figure I(B) shows t h a t tacrine p o t e n t i a t e d twitch responses when c o n c e n t r a t i o n s from 0.7 to 10/~mol were used.
0.3
0.7
1
10
100
700
DRUG C O N C E N T R A T I O N (I.tmol) Fig. 1. Effects of velnacrine (A), tacrine (B) and physostigmine (C) on indirectly-elicited twitch responses after 5, 10 and 15min of their addition to the bath. Phrenic nerves were stimulated supramaximally with square waves pulses of 0.5 msec duration at 0.2 Hz. The baseline tension was considered as control response (100%) and higher values were represented as potentiation and lower values as inhibition. Values are expressed as the mean of 6-9 experiments and range bars indicate the SEM. Concentrations higher than 700 # mol of velnacrine were not tested because of difficulties in preparing solutions. Several intermediate concentrations were also obtained but are not represented in order to simplify the figure.
Velnacrine, tacrine and physostigmine Z O
Although velnacrine (10/~mol) obtained the maximal value (AI = 27.1 _+ 7.3%), this value was not different to those obtained with physostigmine or tacrine. These drugs presented a similar concentration-effect - * - VEL range, although the former was slightly more potent - o - THA in reversing neomycin-induced blockade.
100
z [.h
o L~ < [., Z L) ¢h
1103
60
- A - PHY DISCUSSION
20 0 .... 0.01
± 0.1
1
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DRUG CONCENTRATION (mmol) Fig. 2. Concentration-response curves for velnacrine (VEL), tacrine (THA) and physostigmine(PHY) on directly-elicited twitch responses. Only inhibitory responses of drugs were observed in this experimental procedure. Phrenic nerves were stimulated supramaximally with square waves pulses of 5 msec duration at 0.2 Hz. The indirect component was previously blocked with tubocurarine (5/~mol). Values are expressed as the mean of 6-8 experiments and range bars indicate the SEM.
Effects on directly-elicited twitch responses Tacrine caused a marked inhibition of directlyelicited twitches whereas the effects of the others were only apparent when 1 mmol was used (Fig. 2). A concentration-dependent depression of twitch response was observed with concentrations of tacrine ranging from 0.1 to 1.3 mmol. A high inhibition was observed when the latter concentration was tested ( - 9 1 . 8 + 3 . 6 % ) . Maximal twitch depression with both velnacrine and physostigmine was obtained at 3 mmol ( - 13.3 + 11.6 and - 10.7 + 9.6%, respectively).
Effects of drugs on drug-induced neuromuscular blockade The three drugs reversed the tubocurarine-induced blockade [Fig. 3(A)]. The most potent drug was physostigmine and the least velnacrine, whereas tacrine had an intermediate potency [Fig. 3(A)]. After the maximal effect was achieved, a decrease of the effects of all drugs was observed. Maximal effect was obtained with physostigmine at 3/~mol (AI = 89.8 + 3.3%). The reversal effect of physostigmine began at lower concentrations (0.07 #mol) than tacrine and velnacrine. The later drug required higher concentrations than physostigmine and tacrine for reversing tubocurarine-induced blockade, although its maximal effect (AI = 79.1 + 11.2% at 100/zmol) was close to that obtained with physostigmine and indeed higher than the maximal reversal effect reached with tacrine (AI = 71.7 _ 5.5 at 3/zmol). The ability of all drugs tested in reversing neomycin-induced blockade was lower than that obtained with tubocurarine inhibition [Fig. 3(B)].
The present study confirms and extends the evidence that the pharmacological profile of tacrine and velnacrine has some differences (Bradley et aL, 1988; Braga et aL, 1991; Bosch et al., 1992a). Such differences suggest that, besides their anticholinesterase activity, these drugs could exert some actions at other sites at cholinergic synapses. When evaluating their effects on indirectly-elicited twitch responses, all drugs have a similar maximal potentiating effect, although the concentration range needed to reach it varied among the compounds. In this respect, physostigmine was slightly more potent than tacrine. However, the concentration of velnacrine needed to obtain a maximal potentiation was ten times greater than that required by tacrine. This result might be correlated with the anti~holinesterase activity of both compounds, as their ICs0 has been 100
A
80
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60 4O 20 0 0.01
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0.1
1
.......................
10
100
1000
B 4O
0
-
0.1
-
-
- -,ll
1 10 100 DRUG CONCBNTRATION (~mol)
- * - VBL
- o - THA
- ' - PHY
Fig. 3. Reversal of the depressant effect of tubocurarine (A) and neomycin (B) on the indirectly-elicited responses by velnacrine (VEL), tacrine (THA) and physostigmine(PHY). Each drug was added when an inhibition of 70-80% of control twitch response was obtained with tubocurarine (I-I .5 #tool) or neomycin (1-1.5 mmol). The reversal effect measured by the antagonism index was evaluated after 15 rain of exposure of each anticholinesterase drug.
1104
F~LIXBOSCHet al.
described as 0.31 and 4.8/~mol for tacrine and velnacrine, respectively (Shutske et al., 1988). After the maximal effect had been reached, a progressive decrease of twitch responses was observed with all compounds, probably by the desensitization that follows the exposure of nicotinic receptors to high concentrations of acetylcholine (Bowman, 1990). The lower anticholinesterase activity of velnacrine could account for its minimal inhibitory effect. A clear differential effect was observed when the effects of drugs were evaluated in directly-elicited twitch responses. Here, tacrine showed a significant depressing effect over 100#mol until reaching the absolute blockade in the highest concentration tested. By contrast neither physostigmine nor velnacrine had significant actions in the usual concentration range, suggesting that this blockade of directly-elicited twitch is probably independent from anticholinesterase activity. Although this effect has been described previously (Karis et al., 1966), there is little information about its precise mechanism. Tacrine has several actions independent of cholinesterase inhibition (see Freeman and Dawson, 1991 for a review). Regarding its effects on neurotransmitter release, it has been shown that tacrine may block potassium channels in different nerve preparations (Freeman and Dawson, 1991), including motoneurones (Harvey and Rowan, 1988; Braga et al., 1991), although this action probably does not account for the described blocking effect. However, it has also been reported that tacrine may reduce sodium currents by interfering with the channel. This effect has been reported in M y x i c o l a giant axons (Schauf and Sattin, 1987), hippocampal neurons (Rogawski, 1987), Xenopus laevis axons (Elinder et al., 1989) and indeed mammalian motoneurones (Harvey and Rowan, 1988; Braga et al., 1991). This local anaesthetic-like effect may contribute to the muscular blockade observed in the present experiments. Although not tested, this action could at least in part, be responsible for the depressing effects of tacrine on indirectly-elicited twitch responses, besides its desensitiziting action on nicotinic receptors. The fact that effects of velnacrine and physostigmine in directly-elicited twitches were only apparent at very high concentrations suggests that these drugs are practically devoid of this action on sodium channels. A differential effect of anticholinesterase drugs on pre- and postsynaptic blockade was observed. All drugs reversed tubocurarine-induced blockade confirming previous results (Karis et al., 1966; Bradley et al., 1988; Braga et al., 1991) but showed differences in both concentration range and maximal effect. Physostigmine was again the most potent as its effect began at 0.1 and was maximal at 3#mol.
Effects of tacrine began at 0.3 and were the highest at 3 #mol. By contract velnacrine began to be active at 3 and full effect was obtained at 100#mol. Basically, these effects were similar to those observed in baseline twitch responses but concentration-response curves were slightly shifted to the right. However, the maximal effect achieved with tacrine in reversing neuromuscular blockade was lower than those observed with the other drugs. The fact that tacrine is a more potent cholinesterase inhibitor than veinacrine suggest that its effect might be limited by other collateral actions, such as blockade of postsynaptic receptors (Thesleff et al., 1990; Braga et aL, 1991; Bosch et aL, 1992a) or the already commented effects on sodium currents. The cholinesterase inhibitors used in this study were less potent in reverting the blockade induced by neomycin. This drug has the common property of aminoglycosides of decreasing calcium entry into motor nerve terminals (Singh et al., 1982; Bourret and Mallart, 1989; Bafios et al., 1989). It has been described that velnacrine and tacrine are unable to completely revert the neuromuscular depression induced by low calcium and high magnesium solutions (Bradley et al., 1988; Braga et al., 1991). Thereby, the small effects of these drugs on neomycin-induced blockade could be reasonably expected on these grounds. These results have additional interest in suggesting that the action of tacrine on potassium channels would contribute little to its final effects, as a potassium blocker, i.e. aminopyridines or guanidine, has a strong reversal action on aminoglycoside-induced blockade (Molg6 et al., 1979; Bosch et aL, 1992b). In summary, tacrine and velnacrine are less potent than physostigmine in potentiating skeletal neuromuscular transmission. However, velnacrine is devoid of some unspecific actions of tacrine that can limit its final action.
SUMMARY
After the description that tacrine could improve patients with Alzheimer's disease, considerable effort has been devoted to the synthesis of new cholinesterase inhibitors. Velnacrine (1-hydroxytacrine, HP-029) is a tacrine derivative which has exhibited a similar pharmacological activity to the parent drug with lesser toxicity. However, several differential effects warrants a further evaluation of its actions in cholinergic synapses. The present study analysed the effects of tacrine, velnacrine and physostigmine at the rat hemidiaphragm preparation. All drugs potentiated twitch responses induced by nerve stimulation, physostigmine being more potent than tacrine and
Velnacrine, tacrine and physostigmine velnacrine. Tacrine strongly depressed directlyinduced twitch responses, whereas the o t h e r drugs h a d lesser effects. Physostigmine was the most p o t e n t drug in reversing t u b o c u r a r i n e - i n d u c e d blockade, but its a n t a g o n i s m index was similar to those o b t a i n e d with velnacrine at higher concentrations. W h e n evaluating their ability to reverse neomycin-induced blockade, all drugs exhibited a similar effect. It is concluded t h a t tacrine a n d velnacrine are less p o t e n t t h a n physostigmine in p o t e n t i a t i n g skeletal neuromuscular transmission. Additionally, some effects exhibited by tacrine but not by velnacrine could limit its therapeutic efficacy. Acknowledgements--The authors want to thank to Drs M. Sim6n and D. Pujol from the Departament de Farmacologia i Quimica Terap6utica, Universitat de Barcelona for the synthesis of velnacrine maleate. FB was supported in part by a grant of the program Ajuts a Projectes de Recerca per a Investigadors Joves de la CIRIT (AR89, Generalitat de Catalunya, Spain). REFERENCES
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