EUROPEAN JOURNAL OF PHARMACOLOGY 11 (1970) 187-194. NORTH-HOLLANDPUBLISHINGCOMPANY
PRELIMINARY PHARMACOLOGICAL SCREENING OF STYRYLPYRIDINE CHOLINE ACETYLTRANSFERASE
INHIBITORS
B.A.HEMSWORTH * and F.F.FOLDES Departments o f Anesthesiology o f the Montefiore Hospttal and Medical Center and the Albert Einstein College o f Medicine, Bronx, New York, USA
Received 10 March 1970
Accepted 20 April 1970
B.A HEMSWORTH and F.F.FOLDES, Preliminary pharmacological screening o f styrylpyridine Choline acetyltransferase inhibitors, European J. Pharmacol. 11 (1970) 187-194. Preliminary pharmacological screening of twelve styrylpyridine analogues with specific and/or selechve in vitro choline acetyltransferase (ChAc) activity was undertaken. Observations were made on acute toxicity and the behavioral and neurological profile after sub-lethal doses in mice and rabbits. The neuromuscular effect of the compounds was investigated both in vivo and in vitro. The compounds caused a non-depolarization or a biphasic depolarization block. The block caused by bisquaternary or monoquaternary ChAc inhibitors in vitro could be readily reversed by repeated washing with fresh bathing solution; in contrast the block caused by the tertiary inhibitors, e.g. 4-(1-naphthylvinyl)-pyridine (compound liD, was more difficult or impossible to reverse. All compounds investigated decreased the twitch tension of the directly stimulated muscle. Neuromuscular blocking doses of compound III caused a transient but severe fall of blood pressure and slowing of the respiratory rates in cats On the basis of these preliminary studies the pharmacological effects of the compounds investigated cannot be attributed to their ChAc inhibitory effect alone. It seems probable that other yet unclarified effects of these compounds may partly or wholly be responsible for their pharmacological effects. Choline acetyltransferase inhibitors Tertiary and quaternary compounds
Neuromuscular effect Pharmacologtcal screening
1. INTRODUCTION A number of styrylpyridine analogues have recently been shown to inhibit choline acetyltransferase (ChAc; acetyl-CoA: choline O-acetyltransferase, EC 2.3.1.6.) in vitro (Smith et al., 1967; CavaUito et al., 1969, 1970). Some of these compounds were specific in their action on ChAc and were shown to inhibit the synthesis of acetylcholine (ACh) in vitro by a reversible non-competitive mechanism of action (Cavallito et al., 1969). Until this time no specific inhibitors of ChAc were known (Smith et al., 1967). Other compounds such as triethylcholine (TEC) and hemicholinium (HC-3) have been shown to inhibit ACh synthesis (Macintosh et al., 1956; Gardiner 1961; Bull and Hemsworth, 1965), by a completely * Present address: Dept. of Pharmacology, University of Rochester, Rochester N.Y. 14620.
different mechanism. Instead of inhibition of ChAc their action is thought to be due to competition with choline for a carrier mechanism necessary to transport extracellular choline to the intracellular sites at which it is acetylated (Macintosh et al, 1958; Macintosh, 1961; Bowman and Rand, 1961, Bowman and Hemsworth, 1965). TEC (Bowman et al., 1962, Bowman and Hemsworth, 1965) and HC-3 (Long and Reitzel, 1958; Reitzel and Long, 1959a) produce a slowly developing, frequency-dependant transmission failure in nerve muscle preparations of rabbits and cats. Transmission is restored by choline (Reitzel and Long, 1959b; Bowman et al., 1962). In the present study several of the styrylpyridine inhibitors were tested for their in vitro and in vwo neuromuscular activity. In addition the general phar-
188
B.A.Hemsworth, F F Foldes, Pharmacology of chohne acetyltransferase mhtbttors
macological properties of the most specific tertiary inhibitor, 4-(1-naphthylvlnyl)-pyrldine, were also observed m vtvo.
Muscle contractions were recorded by means of Grass model FT 0.03C force displacement transducers on a Grass model 5D polygraph.
2. METHODS
2.2.2. Rat phrenic nerve diaphragm In experiments on the isolated phrenic nervediaphragm preparation of the rat (Bulbring, 1946) both hemidiaphragms were mounted in McEwen's (1956) solution at 37°C The muscles were excited by supramaxlmal rectangular pulses of 0.1 msec duration applied to the phrenic nerves One nerve was stimulated 0.1/sec and the other 1/sec. The contractions were recorded by means of Grass force displacement transducers on a Grass polygraph.
2.1. Observations on conscious animals
2.1.1. Mice i) Tox~clty: Determinations of acute toxicity were made on male C.F.I.S. Stram (Carworth Farm) mice of 20 to 25 g body weight. ii) Behavioral profile The mice were observed for awareness by testing their alertness and response to manipulations The behavior of the mice was observed with regard to restlessness, lrntablhty and fear The spontaneous and pain-ehclted motor activity of the mice was also noted. The lrritabdity of the mice was evaluated by obserwng their response to noise and the presence or absence of tremors and convulsions. Gait and righting reflex was used to estimate motor coordination. Muscle tone was determined by testing the grap strength of the forepaw. 2.1.2. Rabbits After the administration of ChAc lnhibltors some ammals were exercised by turning them on their backs and repeating this procedure every time they regained their normal position. Other rabbits were restrained by placing them in a cage which allowed only hmited movements.
2.2.3. Observations on the isolated frog muscle The frog rectus abdominis muscle was suspended in frog-Ringer solution (NaC1 6.75, KC1 0.32, CaC12 0.09, and NaHCO3 0.12g/l). ACh was added at regular intervals in doses which caused submaxlmal contractions. The effect of the various ChAc inhibitors on the ACh induced contractions were observed. 2.3. Other observations on anesthetized animals 2.3.1. Cats and rabbits Respiration was recorded by means of a Grass volumetric pressure transducer and blood pressure was recorded from the carotid artery by means of a Statham model P 23AC pressure transducer 3. RESULTS
2.2. Observations on nerve-muscle preparations 2.2.1. Cat sciatic nerve-tlbiahs anterior muscle Cats were anesthetized by intraperltoneal injection of a mixture containing 80 mg/kg chloralose and 6 mg/kg pentobarbital sodium. Rabbits were anesthetized by the intravenous injection of 1 5g/kg urethane. Maximal twitches and tetani of the tlbiahs anterior muscles of cats and rabbits were elicited by stimulating the sciatic nerves with supramaximal rectangular pulses of 0.1 msec duration. In most experiments the contractions of both tibialis anterior muscles were recorded simultaneously with stimulation rates of 0.1/sec and 1/sec respectively. Direct stimulation of the muscle was carried out by means of two platinum wires inserted into the muscle, one at each end, with supramaximal stimuli of 1 msec duration.
ChAc lnhlbltors investigated are shown in table 1. The table also shows the molar concentration of the compounds which inhibit ChAc and acetylcholinesterase (ACHE) 50% and the ratios of the antxAChE and anti-ChAc activities. The greater this ratio the more specific is the compound for ChAc. 3.10bservattons on conscious animals 3.1.1. Mice i) Acute toxicity: The LDs0 of 4(l-naphthylvinyl)-pyndine (compound III, table 1) administered intravenously, subcutaneously or int~aperitoneally to mice are gwen in table 2. The LDs o and the 95% confidence hmits were determined by the method of Litchfield and Wilcoxon (1949). Death occurred in 15 to 30 min. Lethal doses of the ChAc inhibitor
B.A.Hemsworth, F.F.Foldes, Pharmacology of choline acetyltransferase mhibitors
189
Table 1 Structural formulae of the choline acetyltransferase mhlbitors investigated for pharmacological actwlty. (The [soChAc and Iso AChE were obtained from Smith et al, 1967 and Cavalhto et ai., 1969, 1970.)
- CH:CH -C N'- (CH2)6-N( '.; C
I
3
I50 ChAc
I50 Ache
R a t t o 150 Ache I50 ChAc
g x 10 -7
6 x tO "7
0 7
3 x L0-5
3 x I0 "7
I 0
2Br"
CH: CH
II
-(CH2J3-N (CH313 2Br"
IiI
~CH:
VIII
ix
x xi
xn
HCI
2 s ,, l0-s
sCH3
0-:--(3
~
:CH-~
LSxI0
I"
"C = C -.~N +- CH3
'-C = C - ~
~-c
HCI
25%at2x10
_=c -'~'-c"3 I -
~'-CH:CH -~N'
CH:CH
lxt0
- CH3
35x10
0 , "C 3 I"
caused respiratory depression and immediately prior to death convulsions wh,ch were probably asphyxlal in origin. Choline in concentrations ranging from 10 to 200 mg/kg did not protect the mice from the toxic effects o f compound III. ii) Effects on behavioral and neurological profile: Sublethal doses o f 100 mg/kg o f compound III in-
"6
-6
3 2 x t0 -6
O0
3x[O
"5
"5
2
I x t0"3
7 x t0 °6
I"
6t
~
5 x .0"6
+'cH3
~ CH3
2 x I0 "/~
6 x 10 -4
HCl
--CH :CH ~ N~÷ - CH3
~-CH
oo
3 3 x tO "6
I"
~
VI
vrI
-~
--CH : C H - ~
IV
V
CH
200
3 x LO"3
°4
~.t2xt0
gxl0
2ixlO
"4
-4
3 8 x 10 .4
500
"4
900
60
120
jected intraperitoneally into mice produced a tranquilizing effect. After 10 min the grip strength diminished and the ammals were unable to cling to a wire with their forepaws. The mice acquired a staggering gait and became more sensit]ve to noise. Maximum effects developed in 45 to 60 min and recovery commenced after 2 to 3 hr. Subsequently the mice
190
B.A.Hemsworth, F.F.Foldes, Pharmacology of choline acetyltransferase inhibitors
Table 2
type of breathing which lasted for 2 hr, 4 hr after the injection the animals were seemingly fully recovered. Choline chloride (Smg/kg) and neostigmine methylsulphate (0.15 mg/kg) injected intravenously during the muscular paralysis had no effect.
Toxicity of 4-(1-naphthylvinyl)-pyridine (compound III table 1) by various routes of injection in mice. LDso and 95% confidence limits in parentheses (mg/kg). Route of injection
LDso
Subcutaneous Intraperitoneal
150 (132-178) 300 (261- 332) 75 (61-99)
Intravenous
3.2. Observations on nerve-muscle preparations The compounds were investigated for pre- or post-junctional blocking action on the cat tibialis muscle, the rat hemi-diaphragm and the frog rectus abdomlnis muscle. The results obtained on these preparations are shown in tables 3, 4, and 5.
appeared normal for 24 hr; after 48 hr, however, they became ill and hypothermic and most succumbed in 3 to 4 days. There were no convulsions before death.
3.2.1. Rat phrenic nerve-diaphragm Table 3 shows the concentrations of the various compounds capable of producing complete block of the contractions of the indirectly stimulated rat hemi-diaphragm in vitro. Compounds I and II in concentrations of 0.04 /~moles/ml and 0.1 ~amoles/ml, respectively, produced rapidly developing postjunctional neuromuscular block which was antagonized 20% by edrophonium and 30% by neostigmine (see fig. 1).
3.1.2. Rabbits Four rabbits were injected intravenously with 100 mg/kg of compound III. Two rabbits were continuously exercised immediately after injection and these animals died after 8 and 11 min, respectively. The other two rabbits were restrained and these animals survived. However, 30 min after injection they did develop muscular weakness and a gasping
Table 3 Effects of compounds on the rat phrenic nerve diaphragm. Concentration producing Compound
100% block of muscle contractions
Effect of edrophonium on block
Effect of neostigmine on block
Effect of removing drug from bathing fluid
Partial reversal
Rapid reversal
~noles/ml I
0.04
0.62
Slight initial antagonism . . . . . No reversal . . . . ,, . . . .
II III IV V Vt VII VIII IX X XI Xll
0.10 0.87* 0.15
0.48
.
.
.
.
0.48
. . . .
. . . .
. . . .
. . . .
0.64
.
.
.
.
1.4"
0.63 0.22* 0.22
. Deepens block
Reversal only after 2-3 lax Reversal
,,
* These compounds were insoluble at concentrations greater than those indicated.
Reversal only after 2-3 hr Reversal Reversal only. after 2-3 hr Reversal
Reversal only after 2-3 hr
B.A.Hemsworth, F F.FoMes, Pharmacology of choline acetyltransferase inhibitors
191
Table 4 Actaon of compounds on the cat scmtic nerve-tlbialis muscle preparation Compound
Concentration producing 100% block of muscle contractions (/~moles/kg) Lv.
Effect of edophonmm on block
Effect of neostigmine on block
I II lIl IV
0.04 2.1 110 54
Partial antagonism Partial antagomsm No effect No effect
Partial antagomsm Partial antagonism Deepens block Deepens block
Compound III 0.87/amoles/ml produced a slowly developing block of muscle contractions when the nerve was stimulated at rates of 0.1 or 1/sec The block produced was not reversed by edrophonium or neostigmine. After removal of the drug from the organ bath and repeated washing, neuromuscular activity gradually returned to about 60% of control in 60 min in the slowly stimulated preparation. There was no return of activity after rapid stimulation The response of the muscle fibers to direct stimulation were also diminished by the same concentration of compound III. Compounds I V - X I I had similar actions to compound III.
Table 5 Effect of compounds on the frog rectus abdominis muscle
Compound
Concentration producing 100% block of ACh
Type of block
contractures (~moles/ml) I
0.019
No n-depolarizmg
II III IV V VI VII VIII IX X XI XII
0.051 0.54 0.67 1.4 1.9" 0.15 0.78 0.22 0.34 0.13 0.33
Non-competitive ,, ,, ,, ,, ,, ,, ,, ,, ,,
* 0,75 tanoles/ml caused a potentiation of the response to ACh.
3.2.2. Cat sciatic nerve-tibialis muscle The intravenous rejection of compound IlI into the cat under chloralose anesthesia caused a slowly developing block of the contractions of the nbialis anterior muscle when the nerve was stimulated at a rate of 1/sec but had no effect on the muscle stimulated at 0.1/sec (fig. 2). Compound III had a greater effect the higher the frequency of stimulation. After injecnon of 110/~moles/kg of compound III there was at first a slight potentiation of the contractions of the indirectly stimulated muscle. In the more slowly stimulated muscle where the muscle contractions were also potentiated after 110 /lmoles/kg of compound II1, no muscular block occurred and the contractions remamed potentiated throughout the experiment (see fig. 2). In the more rapidly stimulated muscle the block produced by compound III was shown to be irreversible ~nd no recovery was obtained even after ceasing nervous stimulation of the muscle and waiting for a period of 2hr. 110 /~moles/kg of compound III caused a slowly developing irreversible block of the directly stimulated muscle when the stimulation rate was 1/sec. 54 /~moles/kg compound IV produced similar effects on neuromuscular transmission as compound III. The block produced by 0.04/amoles/kg of compound I or by 2.1 /.lmoles/kg compound II was of a postjunctional non-depolarizing type, which was partially reversed by edrophomum and by neostigmme injected when the original twitch was decreased by 50%. 3.2.3. Frog rectus abdominus muscle Table 5 shows the concentrations of the compound capable of blocking by 50% the contractions elicited by 0.5 /ag/ml ACh on the frog rectus ab-
B.A Hemsworth, F.F.FoMes, Pharmacology of chohne acetyltransferase inhibttors
192 l/sec
t 0.1/sec
W
T
t
w
W
w
'
4 rain
Ftg. 1. Maxtmal twitches of isolated rat hemldiaphragm elicited indirectly by stimulation of the phremc nerve at frequencies of 1/sec and 0.1/sec. At the ftrst set of arrows 0 1 #moles/ml compound II added to the bath. At the second set of arrows 0.01 ~moles/ml edrophonmm added to the bath. At W the bath fluid was replaced with fresh McEwen's (1956) solution.
RESPIRATION
l BLOODPRESSURE ramsHg 140I1201"
7_
0.1/ sec l
. . . .
r ,rE ft /II llli/t tlill/iltr' l/see
I
~
I
I
r.......... f I, ......
,
,
lttllt lltlttttllIlItllliIltIl/litttt/l/Illftflit
t
4 rain
Fig. 2. ObservaUons of effect of compound lII on anestheUzed cat. Upper record, respttation; second record, blood pressure; third and fourth records, maximal twitches of right and left tlblalis anterior muscles elicited indirectly by stimulation of the sciatic nerve 1/10 sec and 1/sec, respectwely. At the arrows 110/~moles/kg compound III was reJected intravenously.
B.A.Hemsworth, F.F.Foldes, Pharmacology of choline acetyltransferase inhibitors
dominus muscle. All compounds caused a non-depolarization block and none by itself caused contracture of the muscle. The block produced by compounds III-XII was not antagonized by larger doses of ACh. The effects of larger doses of compounds III-XII were difficult to remove from the organ bath by repeated washings. Compound III 1.0/amole/ml produced an 80% block of the ACh contractions from which the muscle did not recover even after repeated washings. 0.75 /amoles/ml compound VI caused a potentiation of the ACh contractions which was possibly due to the anticholinesterase effect of this compound. No contracture of the frog muscle was observed after addition of either of the b~squaternaries (compounds I and II) to the bathing fluid in doses up to 1.0/amoles/ml. 3.3. Other observations on anesthetized cats and rabbits 3.3.1. Effects on respiration and blood pressure 110/amoles/kg compound III caused an immediate fall in the blood pressure of the anesthetized cat and a gasping type of respiration (see fig. 2). 50 gmoles/kg had the same effect in anesthetized rabbits. The blood pressure returned to normal within 10 min, however, the respiration did not return to normal until 40 min after the injection. The time course of these effects was different from the time course of the effect of the drug on neuromuscular transmission.
4. DISCUSSION The results presented indicate that the actions of the styrylpyridine compounds are different from those of TEC and HC-3. Our findings suggest that these compounds may have other actions which in vivo mask their in vitro specific ChAc inhibitory effect. Many drugs are known to affect ACh content and release in the CNS. Anesthetics cause an increase in brain ACh content (Crossland and Merrick, 1954) but cause a decrease in the release of ACh from the cortex (Mitchell, 1963). TEC and HC-3 have been shown to produce catatonia in rats and reduce the total amount of brain ACh (Slater, 1968). It is of interest that compound III (table 1) produces atran-
193
quilizing effect in mice. Brain ACh levels were not estimated after injection of compound III into mice. The drug, however, is a tertiary amine which should cross the blood brain barrier. The death of mice which occurs after 2 days could also be due to central effects. It is of interest that Goldberg and Ciofalo (1968) found that compound III augmented the stimulating effects of amphetamine in trained rats. The toxicity of the compound is clearly different from that of TEC and HC-3 where choline has a definite protective effect. The finding that the same dose of compound III that was lethal in continuously exercised rabbits was seemingly harmless in restrained animals could indicate that exercised animals succumbed because of the exhaustion of the ACh deposits. The actions of the compounds on motor nerveskeletal muscle preparahons are also different from the effects of TEC and HC-3. The effects of the styrylpyridlne compounds on the directly stimulated muscle suggests that inhibition of ChAc may not be the primary effect of the drug in vivo. On the other hand, with some compounds the concentrations capable of blocking neuromuscular transmission in the rat diaphragm (table 3) were similar to their Is o for ChAc. The neuromus:ular block caused by these compounds was only reversed after several hours of washing the preparation with fresh bathing soluhon Chohne added to the organ bath during this period did not assist in restoring muscle contractions as it does with HC-3 block in the rat diaphragm preparation (Hemsworth, 1965). The compounds are noncompetitive reversible inhlbitors of ChAc #z vttro (Cavallito et al., 1969), therefore it should be easy to remove them from the tissues with repeated washings. Since this ~s not the case it is probably that these compounds exert a direct action on the muscle fibers. In the cat and rabbit some of the compounds had a marked effect m vivo, on blood pressure and respiration. A reduced blood flow to skeletal muscles was not the cause of the neuromuscular paralysis. however, as the tLrne course of the return of blood pressure to normal did not follow the time course of the neuromuscular block. It ,.s most hkely that the effects of these compounds on respiration is central The mechamsm of the hypotensive effect of compound III was not studied but a marked pupdlary dilatation was observed immediately after m.lection of
194
B.A.Hemsworth, F.F.Foldes, Pharmacology of choline acetyltransferase inhibitors
the drug and it is likely that the fall in blood pressure is due to ganglionic block. The bis-quaternary compounds (I and II, table 1) have been studied previously (Cavallito et al., 1964) and were shown to cause a biphasic block preceded b y muscle fasciculations. We also found that the block caused b y compounds I and II could be reversed by edrophonium and neostigmine but no initial fasciculations were observed and the compounds did not produce contracture o f the frog rectus muscle. The compounds caused non-depolarizing block o f ACh contractions on the frog rectus muscle. It is conceivable that the large, planar, lipophilic moieties of the molecules may counteract the depolarizing effect o f bis-tnmethyl ammonium moieties o f compounds I and II.
ACKNOWLEDGEMENTS Supported m part by U.S. Pubhc Health Service Grant NB-08895-01.
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tlonal and electromc features of styrylpyndine analogs, J. Med. Chem. 12, 134 Cavalhto, C.J, H.S.Yun, T.Kalalan, J Crispin Smith and F.F Foldes, 1970, Chohne acetyltransferase lnhlbltors Dimensional and substltuent effects among styrylpyndme analogs, J. Med. Chem. 13,221. Crossland, J. and A J.Mernck, 1954, The effect of anesthesia on the acetylcholine content of brain, J. Physiol. (London) 125, 56 Gardiner, J.E., 1961, The mhlblhon of acetylchollne synthesis in brain by a hemlchohnium, Blochem. J. 81,297 Goldberg, M.E and V.B.Clofalo, 1969, Alteration of the behavioral effects of amphetamine by agents which modify cholinerglc function, Psychopharmacologla (Berhn) 14, 142. Hemsworth, B.A., 1965, Studies on the pharmacology of analogues of choline, Ph.D. Thesis, Umverslty of London. Lltchfleld, J.T. and F.Wdcoxon, 1949, A simphfled method of evaluating dose-response experiments, J. Pharmacol Exptl. Therap. 96, 99. Long, J.P. and N.Reltzel, 1959, The neuromuscular blocking properties of ot,Ot'-dtmethylethanolamino 4,4'-biacetophenone (hemicholinmm), J. Pharmacol. Exptl. Therap. 122, 44A. Macintosh, F.C., R.I.Btrks and P.B.Sastry, 1956, Pharmacological mhlbltion of acetylcholine synthesis, Nature 178, 118. Macintosh, F.C., R.I.Birks and P.B.Sastry, 1959, Mode of action of an mhlbitor of acetylchohne synthesis, Neurology 8, Suppl 1, 90. Macintosh, F.C., 1961, Effect of HC-3 on acetylcholine turnover, Federation Proc 20, 562. McEwen, L.M., 1956, The effect on the isolated rabbit heart of vagal stimulation and its modification by cocaine, hexamethonium and ouabam, J. Physiol. (London) 131, 678. Mitchell, J.F., 1963, The spontaneous and evoked release of acetylchohne from the cerebral cortex, J. Physiol. (London) 165, 98. Reitzel, N.L. and J.P.Long, 1959a, The neuromuscular blockmg properties of a,tt'-dtmethylethanolamino 4,4'-biacetophenone (hemlchohnium), Arch. Intern. Pharmacodyn. 119, 20. Reitzel, N.L. and J.P.Long, 1959b, Hemichohmum antagonism by chohne analogues, J. Pharmacol. Exptl. Therap. 127, 15 Slater, P., 1968, The effects of triethylcholine and hemlchohmum-3 on the acetylcholine content of rat brain, Intern. J. Neuropharmacol. 7,421. Smith, J.C., C.J.Cavailito and F.F.Foldes, 1967, Choline acetyltransferase mhibltors: a group of styrylpyndine analogs, Biochem. Pharmacol. 16, 2438.