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Central and peripheral actions of anticholinergic drugs when administered with triflupromazine
A;rrrrophan~tcrto/~~y, 1971, 10,93-101
Pergamon Press.
Printed in Gt. Britain.
CENTRAL AND PERIPHERAL ACTIONS OF ANTICHOLINERGIC DRUGS WHEN ADMINISTERED TRIFLUPROMAZINE
WITH
R. W. BRIMBLECOMBE, D. M. GREEN, F. A. B. ALDOUS and PAMELA B. J. THOMPSON Chemical Defence Establishment, (Accepted
Porton Down, Salkbury, Wilts
8 April
1970)
Summary-Triflupromazine was found to potentiate the actions of six anticholinergic drugs in producing elevation of EEG arousal thresholds, and dissociation between EEG and behavioural arousal thresholds, in cat enc~phale isol& preparations. The effects of triflupromazine on the anticholinergic activity of the six drugs were studied using as tests antagonism of oxotremorine-induced salivation and tremors and production of mydriasis in mice. It is concluded that the potentiating effect of triflupromazine on the EEG cannot be explained on the basis of an addition of anticholinergic activities and that other, as yet not understood, synergistic mechanisms are involved.
is well known that atropine and other anticholinergic drugs produce a pattern of slow wave activity in the electroencephalogram independent of the behavioural state of the animal. BRADLEY and HANCE (1957) reported that the phenothiazine derivatives chlorpromazine and methopromazine acted synergistically with atropine in producing this slow wave activity in the EEG, and they suggested that this action of the phenothiazines might be related to their weak anticholinergic activity. COLEMAN et al. (1962) described another effect of administering anticholinergic drugs with the phenot,hiazine derivative, triflupromazine. This compound rendered anticholinergic drugs more effective in protecting mice from the lethal effects of the anticholinesterase agent, sarin. This effect also could be due to the drugs acting synergistically. It seemed important, therefore, to study quantitatively the effects of triflupromazine (TFP) on the central and peripheral anticholinergic activities of various drugs, in order to establish whether any synergism could be related to a summation of anticholinergic activities, or to another effect of TFP independent of any anticholinergic activity it may possess. A preliminary communication on this work was published recently (BRIMBLECOMBE and GREEN, 1969). IT
METHODS
Central anticholinergic activity (a) E&cts on brain electrocortical
activity (EncPphale isok preparations). Cats (2-2.6 kg) were used. The animals were prepared under halothane anaesthesia using the operative procedure described by BRADLEY and KEY (1958). Ten stainless steel screw electrodes were inserted over the lateral and suprasylvian gyri of the cerebral hemispheres, and a concentric bipolar stainless steel stimulating electrode was orientated in the brain stem reticular formation using Horsley Clarke stereotactic coordinates A 5.0, L 1.5, H +0*5-2.0. 93
94
R. W. BRIMBLECOMBE, D. M. GREEN, F. A. B. ALDOUS and PAMELA B. J. THOMPSON
When all operative procedures had been completed, the wound edges and pressure points were infiltrated with 2% Procaine HCl solution and the administration of halothane was then terminated. The preparation was then allowed to recover for at least 1 hr. When the electrocortical activity of the animal showed a sleep or synchronized pattern (low frequency, high amplitude waves) the arousal threshold was determined. A stimulus from a Grass S4 stimulator (frequency 300 c/s, pulse width O-5 msec, duration 6 set) was applied to the reticular formation electrode; the voltage was gradually increased in 0.1 V steps from 0.1 V until both behavioural arousal (opening of the eyes and movements of the vibrissae) and electroencephalographic (EEG) arousal (change in electrocortical activity to an alert, desynchronized pattern with high frequency, low amplitude waves) were obtained. This method was described by BRADLEY and KEY(1958,1959). Three cat preparations were used for each drug given alone, and for each drug combined with an equimolecular quantity of TFP. The drugs were administered intravenously in normal saline in incremental doses given every 20 min. The EEGs were recorded on an S-channel Elema Mingograph electroencephalograph. Arousal thresholds were determined 20 min after each drug administration and curves were plotted relating dose of drug to the thresholds for behavioural and for EEG arousal. From these curves it was possible to read off the dose required to raise the EEG arousal threshold by 100%. (b) Antagonism of oxotremorine-induced tremors in mice. Female albino mice (18-25 g) were used. A solution of the anticholinergic drug in saline was injected intraperitoneally 15 min before the intravenous injection of 100 pg/kg oxotremorine. Animals were examined at 5, 10 and 15 min after the oxotremorine injection for the presence of tremors. No attempt was made to grade the severity of either response; it was noted as being either present or absent. A preliminary experiment was carried out using two mice per dose to obtain the approximate level of activity of the anticholinergic drug. Then an assay was carried out using 4 groups of 5 mice each with logarithmically spaced doses of the drug. An ED,, for block of tremors was calculated by probit analysis. The ED,,+ of the drug alone and for the same drug combined with an equal quantity of TFP were compared for any significant difference at the P=O*O5 level. Peripheral anticholinergic activity (a) Antagonism of oxotremorine-induced salivation. The mice used to detect the blockade of oxotremorine-induced tremors (see above) were also examined for the presence of salivation. The ED~,, for blockade of salivation was taken to represent a peripheral measure of anticholinergic potency. (b) Production of mydriasis in mice. Drugs were injected intravenously into the tail vein of male albino mice (20-30 g). After preliminary screening to find suitable dose levels, using single mice per dose, 3 groups of 10 mice were used and the pupil diameters measured at fixed intervals after injection. Readings were normally continued so that the total period of drug action was covered. Mice were kept in the dark until immediately before pupil diameters were measured, when readings were measured using an eye piece graticule incorporated into an X20 microscope. The mice were placed so that their eyes were 20 cm from a Watson Barnet lamp, and measurements were made without delay. The maximal pupil diameter at each of the 3 dose levels was read from the graphs, and the relative potency of the drug with and without an equimolecular quantity of triflupromazine was calculated on the basis of a 6-point assay.
Central and peripheral actions of antichoiinergic drugs when administered with triflupromazine
95
Drugs The drugs used are listed in Table 1. Atropine sulphate was purchased from B.D.H. Ltd., and hyoscine hydrobromide from McFarlan Smith Ltd. Trifiupromazine was kindly donated by E. R. Squibb & Sons Ltd., and 03063 was obtained from Dr. I. W. Coleman of the Defence Chemical, Biological and Radiation Laboratories, Ottawa, Canada. The remaining compounds were synthesized in these laboratories.
Formula
Drug
/
N-CHTCHFO-$ 0
CZH5
Trifl~pfom~ine (TFP)
(HCL)
Oxotremarina
Sarin
TABLE
1, LISTOF
DRLKB
USED
96
R. W.
BRIMBLECOMEZE,
D. M.
GREEN,
PAMELA
F. A. B. A~oous and
B. J. THOMPSON
RESULTS
The effect of atropine, hyoscine, PMCG, N-methyl piperidyl benzilate, G3063, caramiphen and TFP were studied in cat enckphale is_&?preparations and the threshold dose/response curves for EEG and behavioural arousal were plotted. Examples of the curves obtained with 4 of the drugs studied are shown in Fig. 1. All the drugs studied, with the exception of TFP, produced a marked divergence or dissociation between EEG and behavioural arousal thresholds. The curves show that with all the anticholinergic drugs there was a tendency for EEG arousal thresholds to flatten at levels of 150-200% above the control levels. When equimolecular quantities of TFP were given with the anticholinergic drugs, the EEG arousal thresholds were potentiated and elevated much further. Each of the 6 anticholinergic drugs, in combination with TFP, elevated the threshold more than 350% above control levels. Above this it was not possible to assess R4.C.G.
I
IO
p I
I
I
Behavioural
I
4 p moles/kg
E.E.G
Tritlupromazine
G 3063
I 2
0
amusol
x-x
arousal
0-9
I.5
f~ moles/kg
moles I kq ix.
6
j’
I
I2
I
ix
(TEE)
I
25
5-O p moles/kg
i.v.
,:*“-..
Drug alone
._--_-_..*
_x
1
I 2,o
I
I
I
o-5
1
I
IO ix.
D:“Q
equimoleculor quontity of TW
FIG. 1. Effects of drugs on thresholds for EEG and behavioural arousal produced by stimulation of the brain stem reticular formation. Mean values for the % change in thresholds, three experiments for each drug and each drug combination, are plotted against dose.
Central and peripheral actions of anticholinergic
drugs when administered
with triflupromazine
97
arousal thresholds, since the stimulation voltage produced motor effects and stimulation artefacts appeared in the records. A record showing the effect of atropine given alone and in combination with TFP is shown in Fig. 2. The potency of the anticholinergic drugs and the TFP-anticholiner~c drug combinations in elevating the EEG arousal thresholds by 100% is shown in Table 2. Combination of TFP with the anticholinergic drugs also raised behavioural arousal thresholds but to a lesser degree than EEG arousal thresholds. Behavioural arousal
FtG. 2. Effect of atropine
combined with triflupromazine on EEC arousal produced by stimulation of the reticular formation. CAT 1 A, control arousal at 0.8 V; B, after 6 pmol/kg i.v. atropine, stimulation at 2.0 Y. CAT 2 A, control arousal at 1-l V;
B, after 3 wmol/kg i.v. atropine-t stimulation at 4.6 V.
3 pmoI/kg i.v. TFP,
R. W. BRIMBLE~MB~,D. M. GREEN,F. A. B. ALDOUS and PAMELA B. .I. THOMPSON
98
TABLET. EFFECISOFTRIFLUPROMAZINEON CENTRALANTICHOLINERGIC ACI-IVIXY
Compound
Atropine Hyoscine N-CH, Pip. Benzilate PMCG G3063 Caramiphen TFP
Dose for elevation of EEG arousal by 100% in cat encdphafe isok! pmoies/kg
Antagonism of oxotremorine-induced tremors in mice ED$~pmoles/kg with 95 y0 limits Potentiation Alone +TFP factor
Potentiation factor
Alone
+TFP
3.4 0.28
1.6 0.14
2.1 2*0
16.1 ( 9.3 -30.3) 1.1 ( 0*59- 2.5)
0.62 0.70 3.00 48.6 42.00
0.20 0.15 0.80 11.00 -
3-l 4.7 3.8 4.4 -
1.8 ( 0.96 3.2) 3.2 ( 2.2 - 4.7) 4.7 ( 1.9 -11.3) 40.3 (24.0 -675) >lOO
7.1 (4-l-12.1) 0.5 (O-2- 0.9) 3-9 3.4 3.1 15.3
(2*1- 7.1) (l-3- 8.4) (1% 53) (8.3-30.0) -
2.3* 2.2* 0.46 o-94 1-5 26*
*Significant at PcO.05.
thresholds were never elevated more than 50% above control levels; the elevations seemed mainly to be due to TFP alone. Four enckphale isol6 preparations were used to assess the ability of the organophosphorus anticholinesterase compound, sarin, to antagonize the elevation of EEG arousal thresholds produced by G3063 and atropine, and combinations of these drugs with TFP. Arousal thresholds were determined 15 min after administration of sarin, and the results are shown graphically in Fig. 3. Comparison of the effects of G3063 and atropine with or without TFP shows that little or no more sarin was required to antagonize the increased elevation of EEG arousal threshold when TFP was added. Oxotremorine experiments
The central and peripheral activities of all the drugs studied and of combinations of the drugs with equimolecular quantities of TFP were assessed by blockade of oxotremorine-induced tremors and salivation in mice respectively. Estimates of central anticholinergic activity are shown in Table 2, where it can be seen that TFP significantly potentiated the anticholinergic activity of only 3 of the 6 drugs studied. TFP itself showed no central anticholinergic activity at a dose level of 100 pmolfkg. Estimates of peripheral anticholinergic activity are given in Table 3, where it can be seen that in 5 cases TABLET. EFFECTSOFTRIFLUIXOMAZINEONPERIPHERAL ANTICHOLINERGIC ACTWITY Antagonism of oxotremorine-induced salivation in mice EDDYpmol/kg with 95 y0 limits
Compound
+TFP
Alone Atropine Hyoscine N-CHI Pip. Benzilate PMCG G3063 Caramiphen TFP *Significant at P CO.05
044 0.05 1.0 2.0 5.4 23.5 >lOO
( 0.25- 0.75) ( 0.02- 0.08) ( 0*52- 190) ( 1.5 - 2.7) ( 3.4 - 8.6) (13.2 4ie1)
044 0.12 12.3 4.0 IO.7 >lOO
(O-25- 0.75) (0.07- 0.20) (7.9 -19.1) (2.2 - 7.6) (7.6 -15.0)
Potentiation factor I.0 0.4* 0.08* 0.5 0.5*
Mouse mydriasis assay Potentiation factor 1.03 0.90 0.74+ 1.01 1.03 I.20
Central and peripheral actions of anticholinergic
/ 25 /L moles/kg i.v. 63063
pg/kg
i.v. sorin
drugs when administered with triflupromazine
25 p moles/kg i.v. 63063 + TEP
w/kg
99
ix sorin
x 100
/ moles/kg i.v. otropine
25 p
FIG.
3.
pg/kg
ix sorin 25 p moles/kg i.v. otropine +lTf?
Effect of sarin
ED,,
as higher dose levels of this combination produced obvious toxic signs.
Mouse mydriasis experiments The results showing relative potencies (expressed as potentiation factors) of the drug given alone and in combination with TFP are given in Table 3. In all 6 cases TFP did not produce any significant potentiation in the degree of mydriasis and no marked effect upon the duration of action. Combination of N-methyl piperidyl benzilate with TFP produced a significant decrease in activity. Examples of the curves obtained with 3 of the drugs studied are shown in Fig. 4. This method of assessing peripheral anticholinergic activity is advantageous in that both time taken to peak effect and duration of actions of the drugs can be studied.
100
R. W.
~RI~BLE~OM~E,
D. M.
GREES,
F. A. B. ALDOUSand PAMELAB. J. THOMPSON
Atropine
30-
x 4
I 10
0
I 20
80
20
80
i
Time.
D .z I IO
0
Time, N-
piperidyl benrilote
I I
I 2
8
Time,
-0 FIG.
Effect of
min
Drug alone Drug equim~e~uior triflu~omozine
drugs and
I6
quantity
of drugs in mice.
triflupromazine
producing
The results show that TFP potentiated the actions of all anticholinergic compounds in elevating EEG arousal thresholds in cat e~~c~~~~~eisoIP preparations. It was found that the EEG arousal threshold dose/response curves obtained with the anticholinergic drugs given alone flattened out at levels 150-200% above control threshold levels; this
Central and peripheral actions of anticholinergic drugs when administered with triflupromazine
101
phenomenon had previously been reported with atropine by BRADLEY and KEY (1958). However, when TFP was used in conjunction with an anticholinergic drug, the curves rose steeply and no plateau level could be reached. This qualitative difference in the patterns of elevation of EEG arousal thresholds suggests that mere addition of anticholinergic actions may not be responsible for the potentiating effect of TFP. Further evidence for this hypothesis lies in the fact that similar doses of the anticholinesterase drug sarin antagonized both the ‘normal’ elevation of EEG arousal and the potentiated elevation produced by the addition of TFP. TFP given alone did not display a typical anticholinergic effect in that it did not produce a divergence, or dissociation, between EEG and behavioural arousal threshold dose/response curves. We have previously shown (BRIMBLECOMBEand GREEN, 1968a) that there is a significant correlation between the central anticholinergic activities of drugs, as measured by blockade of oxotremorine-induced tremors in mice, and their potency in elevating EEG arousal thresholds. Oxotremorine-induced tremors appear to result from the stimulation of central muscarinic receptors (BEBBINGTON and BRIMBLECOMBE, 1965), and efficiency in blocking these tremors can be regarded as a good measure of central anticholinergic potency. The relative merits of the methods described in this paper for determining central and peripheral anticholinergic activities have recently been described by us (BRIMBLECOMBEand GREEN, 1968b). If the increased elevation of EEG arousal threshold produced by TFP is not due to an anticholinergic effect, then TFP would not be expected to potentiate the effects of anticholinergic drugs in blocking oxotremorine-induced tremors. With 3 drugs (G3063, PMCG and N-methyl piperidyl benzilate) there was no such potentiation. The fact that TFP potentiated the tremor-blocking effect of atropine, hyoscine and caramiphen cannot be easily explained. TFP did not significantly potentiate the peripheral anticholinergic activity of any of the 6 compounds tested, and with 1 drug (N-methyl piperidyl benzilate) there was a significant reduction in peripheral anticholinergic activity as measured both by production of mydriasis and blockade of oxotremorine-induced salivation in the mouse, an effect probably worthy of further investigation. TFP given by itself showed no marked central or peripheral anticholinergic activity when given at relatively high dose levels of 100 pmol/kg. This drug failed to block oxotremorine-induced tremors and salivation and also failed to produce mydriasis. The results of the experiments reported here suggest strongly that the potentiating effect of TFP on the elevation of EEG arousal thresholds by anticholinergic drugs is not due to a combination of anticholinergic activities. REFERENCES BEBBINGTON, A. and BRIMBLECOMBE, R. W. (1965). Muscarinic receptors in the peripheral and central nervous system. In: Advances in Drug Research, 2, pp. 143-172. Academic Press, London. BRADLEY,P. B. and HANCE, A. S. (1957). The effect of chlorpromazine and methopromazine on thz electrical activity of the brain in the cat. Electroenceph. cfin. NeurophysioI. 9: 191-215. BRADLEY,P. B. and KEY, B. J. (1958). The effects of drugs on arousal responses produced by electrical stimulation of the reticular formation of the brain. Electroenceph. clin. Neurophysiol. 10: 97-110. BRADLEY,P. B. and KEY, B. J. (1959). A comparative study of the effects of drugs on the arousal system of the brain. Br. J. Pharm. Chemother. 14: 340-349. BRIMBLECOMBE, R. W. and GREEN,D. M. (1968a). Further evidence for cholinergic synapses in the mesodiencephalic activating system. J. Physiok, Land. 194: 16-17P. BRIMBLECOMBE, R. W. and GREEN, D. M. (1968b). The peripheral and central actions of some anticholinergic substances. Int. J. Neuropharmac. 7: 15-21. BRIMBLECOMBE, R. W. and GREEN,D. M. (1969). The effect of triflupromazine on the peripheral and central actions of some anticholinergic drugs. Br. J. Pharmac. 35: 364P. COLEMAN,I. W., LITTLE,P. E. and BANNARD,R. A. B. (1962). Cholinolytics in the treatment of anticholinesterase poisoning. Can. J. Biochem. Physiol. 40: 827-834.
Pergamon Press.
Printed in Gt. Britain.
CENTRAL AND PERIPHERAL ACTIONS OF ANTICHOLINERGIC DRUGS WHEN ADMINISTERED TRIFLUPROMAZINE
WITH
R. W. BRIMBLECOMBE, D. M. GREEN, F. A. B. ALDOUS and PAMELA B. J. THOMPSON Chemical Defence Establishment, (Accepted
Porton Down, Salkbury, Wilts
8 April
1970)
Summary-Triflupromazine was found to potentiate the actions of six anticholinergic drugs in producing elevation of EEG arousal thresholds, and dissociation between EEG and behavioural arousal thresholds, in cat enc~phale isol& preparations. The effects of triflupromazine on the anticholinergic activity of the six drugs were studied using as tests antagonism of oxotremorine-induced salivation and tremors and production of mydriasis in mice. It is concluded that the potentiating effect of triflupromazine on the EEG cannot be explained on the basis of an addition of anticholinergic activities and that other, as yet not understood, synergistic mechanisms are involved.
is well known that atropine and other anticholinergic drugs produce a pattern of slow wave activity in the electroencephalogram independent of the behavioural state of the animal. BRADLEY and HANCE (1957) reported that the phenothiazine derivatives chlorpromazine and methopromazine acted synergistically with atropine in producing this slow wave activity in the EEG, and they suggested that this action of the phenothiazines might be related to their weak anticholinergic activity. COLEMAN et al. (1962) described another effect of administering anticholinergic drugs with the phenot,hiazine derivative, triflupromazine. This compound rendered anticholinergic drugs more effective in protecting mice from the lethal effects of the anticholinesterase agent, sarin. This effect also could be due to the drugs acting synergistically. It seemed important, therefore, to study quantitatively the effects of triflupromazine (TFP) on the central and peripheral anticholinergic activities of various drugs, in order to establish whether any synergism could be related to a summation of anticholinergic activities, or to another effect of TFP independent of any anticholinergic activity it may possess. A preliminary communication on this work was published recently (BRIMBLECOMBE and GREEN, 1969). IT
METHODS
Central anticholinergic activity (a) E&cts on brain electrocortical
activity (EncPphale isok preparations). Cats (2-2.6 kg) were used. The animals were prepared under halothane anaesthesia using the operative procedure described by BRADLEY and KEY (1958). Ten stainless steel screw electrodes were inserted over the lateral and suprasylvian gyri of the cerebral hemispheres, and a concentric bipolar stainless steel stimulating electrode was orientated in the brain stem reticular formation using Horsley Clarke stereotactic coordinates A 5.0, L 1.5, H +0*5-2.0. 93
94
R. W. BRIMBLECOMBE, D. M. GREEN, F. A. B. ALDOUS and PAMELA B. J. THOMPSON
When all operative procedures had been completed, the wound edges and pressure points were infiltrated with 2% Procaine HCl solution and the administration of halothane was then terminated. The preparation was then allowed to recover for at least 1 hr. When the electrocortical activity of the animal showed a sleep or synchronized pattern (low frequency, high amplitude waves) the arousal threshold was determined. A stimulus from a Grass S4 stimulator (frequency 300 c/s, pulse width O-5 msec, duration 6 set) was applied to the reticular formation electrode; the voltage was gradually increased in 0.1 V steps from 0.1 V until both behavioural arousal (opening of the eyes and movements of the vibrissae) and electroencephalographic (EEG) arousal (change in electrocortical activity to an alert, desynchronized pattern with high frequency, low amplitude waves) were obtained. This method was described by BRADLEY and KEY(1958,1959). Three cat preparations were used for each drug given alone, and for each drug combined with an equimolecular quantity of TFP. The drugs were administered intravenously in normal saline in incremental doses given every 20 min. The EEGs were recorded on an S-channel Elema Mingograph electroencephalograph. Arousal thresholds were determined 20 min after each drug administration and curves were plotted relating dose of drug to the thresholds for behavioural and for EEG arousal. From these curves it was possible to read off the dose required to raise the EEG arousal threshold by 100%. (b) Antagonism of oxotremorine-induced tremors in mice. Female albino mice (18-25 g) were used. A solution of the anticholinergic drug in saline was injected intraperitoneally 15 min before the intravenous injection of 100 pg/kg oxotremorine. Animals were examined at 5, 10 and 15 min after the oxotremorine injection for the presence of tremors. No attempt was made to grade the severity of either response; it was noted as being either present or absent. A preliminary experiment was carried out using two mice per dose to obtain the approximate level of activity of the anticholinergic drug. Then an assay was carried out using 4 groups of 5 mice each with logarithmically spaced doses of the drug. An ED,, for block of tremors was calculated by probit analysis. The ED,,+ of the drug alone and for the same drug combined with an equal quantity of TFP were compared for any significant difference at the P=O*O5 level. Peripheral anticholinergic activity (a) Antagonism of oxotremorine-induced salivation. The mice used to detect the blockade of oxotremorine-induced tremors (see above) were also examined for the presence of salivation. The ED~,, for blockade of salivation was taken to represent a peripheral measure of anticholinergic potency. (b) Production of mydriasis in mice. Drugs were injected intravenously into the tail vein of male albino mice (20-30 g). After preliminary screening to find suitable dose levels, using single mice per dose, 3 groups of 10 mice were used and the pupil diameters measured at fixed intervals after injection. Readings were normally continued so that the total period of drug action was covered. Mice were kept in the dark until immediately before pupil diameters were measured, when readings were measured using an eye piece graticule incorporated into an X20 microscope. The mice were placed so that their eyes were 20 cm from a Watson Barnet lamp, and measurements were made without delay. The maximal pupil diameter at each of the 3 dose levels was read from the graphs, and the relative potency of the drug with and without an equimolecular quantity of triflupromazine was calculated on the basis of a 6-point assay.
Central and peripheral actions of antichoiinergic drugs when administered with triflupromazine
95
Drugs The drugs used are listed in Table 1. Atropine sulphate was purchased from B.D.H. Ltd., and hyoscine hydrobromide from McFarlan Smith Ltd. Trifiupromazine was kindly donated by E. R. Squibb & Sons Ltd., and 03063 was obtained from Dr. I. W. Coleman of the Defence Chemical, Biological and Radiation Laboratories, Ottawa, Canada. The remaining compounds were synthesized in these laboratories.
Formula
Drug
/
N-CHTCHFO-$ 0
CZH5
Trifl~pfom~ine (TFP)
(HCL)
Oxotremarina
Sarin
TABLE
1, LISTOF
DRLKB
USED
96
R. W.
BRIMBLECOMEZE,
D. M.
GREEN,
PAMELA
F. A. B. A~oous and
B. J. THOMPSON
RESULTS
The effect of atropine, hyoscine, PMCG, N-methyl piperidyl benzilate, G3063, caramiphen and TFP were studied in cat enckphale is_&?preparations and the threshold dose/response curves for EEG and behavioural arousal were plotted. Examples of the curves obtained with 4 of the drugs studied are shown in Fig. 1. All the drugs studied, with the exception of TFP, produced a marked divergence or dissociation between EEG and behavioural arousal thresholds. The curves show that with all the anticholinergic drugs there was a tendency for EEG arousal thresholds to flatten at levels of 150-200% above the control levels. When equimolecular quantities of TFP were given with the anticholinergic drugs, the EEG arousal thresholds were potentiated and elevated much further. Each of the 6 anticholinergic drugs, in combination with TFP, elevated the threshold more than 350% above control levels. Above this it was not possible to assess R4.C.G.
I
IO
p I
I
I
Behavioural
I
4 p moles/kg
E.E.G
Tritlupromazine
G 3063
I 2
0
amusol
x-x
arousal
0-9
I.5
f~ moles/kg
moles I kq ix.
6
j’
I
I2
I
ix
(TEE)
I
25
5-O p moles/kg
i.v.
,:*“-..
Drug alone
._--_-_..*
_x
1
I 2,o
I
I
I
o-5
1
I
IO ix.
D:“Q
equimoleculor quontity of TW
FIG. 1. Effects of drugs on thresholds for EEG and behavioural arousal produced by stimulation of the brain stem reticular formation. Mean values for the % change in thresholds, three experiments for each drug and each drug combination, are plotted against dose.
Central and peripheral actions of anticholinergic
drugs when administered
with triflupromazine
97
arousal thresholds, since the stimulation voltage produced motor effects and stimulation artefacts appeared in the records. A record showing the effect of atropine given alone and in combination with TFP is shown in Fig. 2. The potency of the anticholinergic drugs and the TFP-anticholiner~c drug combinations in elevating the EEG arousal thresholds by 100% is shown in Table 2. Combination of TFP with the anticholinergic drugs also raised behavioural arousal thresholds but to a lesser degree than EEG arousal thresholds. Behavioural arousal
FtG. 2. Effect of atropine
combined with triflupromazine on EEC arousal produced by stimulation of the reticular formation. CAT 1 A, control arousal at 0.8 V; B, after 6 pmol/kg i.v. atropine, stimulation at 2.0 Y. CAT 2 A, control arousal at 1-l V;
B, after 3 wmol/kg i.v. atropine-t stimulation at 4.6 V.
3 pmoI/kg i.v. TFP,
R. W. BRIMBLE~MB~,D. M. GREEN,F. A. B. ALDOUS and PAMELA B. .I. THOMPSON
98
TABLET. EFFECISOFTRIFLUPROMAZINEON CENTRALANTICHOLINERGIC ACI-IVIXY
Compound
Atropine Hyoscine N-CH, Pip. Benzilate PMCG G3063 Caramiphen TFP
Dose for elevation of EEG arousal by 100% in cat encdphafe isok! pmoies/kg
Antagonism of oxotremorine-induced tremors in mice ED$~pmoles/kg with 95 y0 limits Potentiation Alone +TFP factor
Potentiation factor
Alone
+TFP
3.4 0.28
1.6 0.14
2.1 2*0
16.1 ( 9.3 -30.3) 1.1 ( 0*59- 2.5)
0.62 0.70 3.00 48.6 42.00
0.20 0.15 0.80 11.00 -
3-l 4.7 3.8 4.4 -
1.8 ( 0.96 3.2) 3.2 ( 2.2 - 4.7) 4.7 ( 1.9 -11.3) 40.3 (24.0 -675) >lOO
7.1 (4-l-12.1) 0.5 (O-2- 0.9) 3-9 3.4 3.1 15.3
(2*1- 7.1) (l-3- 8.4) (1% 53) (8.3-30.0) -
2.3* 2.2* 0.46 o-94 1-5 26*
*Significant at PcO.05.
thresholds were never elevated more than 50% above control levels; the elevations seemed mainly to be due to TFP alone. Four enckphale isol6 preparations were used to assess the ability of the organophosphorus anticholinesterase compound, sarin, to antagonize the elevation of EEG arousal thresholds produced by G3063 and atropine, and combinations of these drugs with TFP. Arousal thresholds were determined 15 min after administration of sarin, and the results are shown graphically in Fig. 3. Comparison of the effects of G3063 and atropine with or without TFP shows that little or no more sarin was required to antagonize the increased elevation of EEG arousal threshold when TFP was added. Oxotremorine experiments
The central and peripheral activities of all the drugs studied and of combinations of the drugs with equimolecular quantities of TFP were assessed by blockade of oxotremorine-induced tremors and salivation in mice respectively. Estimates of central anticholinergic activity are shown in Table 2, where it can be seen that TFP significantly potentiated the anticholinergic activity of only 3 of the 6 drugs studied. TFP itself showed no central anticholinergic activity at a dose level of 100 pmolfkg. Estimates of peripheral anticholinergic activity are given in Table 3, where it can be seen that in 5 cases TABLET. EFFECTSOFTRIFLUIXOMAZINEONPERIPHERAL ANTICHOLINERGIC ACTWITY Antagonism of oxotremorine-induced salivation in mice EDDYpmol/kg with 95 y0 limits
Compound
+TFP
Alone Atropine Hyoscine N-CHI Pip. Benzilate PMCG G3063 Caramiphen TFP *Significant at P CO.05
044 0.05 1.0 2.0 5.4 23.5 >lOO
( 0.25- 0.75) ( 0.02- 0.08) ( 0*52- 190) ( 1.5 - 2.7) ( 3.4 - 8.6) (13.2 4ie1)
044 0.12 12.3 4.0 IO.7 >lOO
(O-25- 0.75) (0.07- 0.20) (7.9 -19.1) (2.2 - 7.6) (7.6 -15.0)
Potentiation factor I.0 0.4* 0.08* 0.5 0.5*
Mouse mydriasis assay Potentiation factor 1.03 0.90 0.74+ 1.01 1.03 I.20
Central and peripheral actions of anticholinergic
/ 25 /L moles/kg i.v. 63063
pg/kg
i.v. sorin
drugs when administered with triflupromazine
25 p moles/kg i.v. 63063 + TEP
w/kg
99
ix sorin
x 100
/ moles/kg i.v. otropine
25 p
FIG.
3.
pg/kg
ix sorin 25 p moles/kg i.v. otropine +lTf?
Effect of sarin
ED,,
as higher dose levels of this combination produced obvious toxic signs.
Mouse mydriasis experiments The results showing relative potencies (expressed as potentiation factors) of the drug given alone and in combination with TFP are given in Table 3. In all 6 cases TFP did not produce any significant potentiation in the degree of mydriasis and no marked effect upon the duration of action. Combination of N-methyl piperidyl benzilate with TFP produced a significant decrease in activity. Examples of the curves obtained with 3 of the drugs studied are shown in Fig. 4. This method of assessing peripheral anticholinergic activity is advantageous in that both time taken to peak effect and duration of actions of the drugs can be studied.
100
R. W.
~RI~BLE~OM~E,
D. M.
GREES,
F. A. B. ALDOUSand PAMELAB. J. THOMPSON
Atropine
30-
x 4
I 10
0
I 20
80
20
80
i
Time.
D .z I IO
0
Time, N-
piperidyl benrilote
I I
I 2
8
Time,
-0 FIG.
Effect of
min
Drug alone Drug equim~e~uior triflu~omozine
drugs and
I6
quantity
of drugs in mice.
triflupromazine
producing
The results show that TFP potentiated the actions of all anticholinergic compounds in elevating EEG arousal thresholds in cat e~~c~~~~~eisoIP preparations. It was found that the EEG arousal threshold dose/response curves obtained with the anticholinergic drugs given alone flattened out at levels 150-200% above control threshold levels; this
Central and peripheral actions of anticholinergic drugs when administered with triflupromazine
101
phenomenon had previously been reported with atropine by BRADLEY and KEY (1958). However, when TFP was used in conjunction with an anticholinergic drug, the curves rose steeply and no plateau level could be reached. This qualitative difference in the patterns of elevation of EEG arousal thresholds suggests that mere addition of anticholinergic actions may not be responsible for the potentiating effect of TFP. Further evidence for this hypothesis lies in the fact that similar doses of the anticholinesterase drug sarin antagonized both the ‘normal’ elevation of EEG arousal and the potentiated elevation produced by the addition of TFP. TFP given alone did not display a typical anticholinergic effect in that it did not produce a divergence, or dissociation, between EEG and behavioural arousal threshold dose/response curves. We have previously shown (BRIMBLECOMBEand GREEN, 1968a) that there is a significant correlation between the central anticholinergic activities of drugs, as measured by blockade of oxotremorine-induced tremors in mice, and their potency in elevating EEG arousal thresholds. Oxotremorine-induced tremors appear to result from the stimulation of central muscarinic receptors (BEBBINGTON and BRIMBLECOMBE, 1965), and efficiency in blocking these tremors can be regarded as a good measure of central anticholinergic potency. The relative merits of the methods described in this paper for determining central and peripheral anticholinergic activities have recently been described by us (BRIMBLECOMBEand GREEN, 1968b). If the increased elevation of EEG arousal threshold produced by TFP is not due to an anticholinergic effect, then TFP would not be expected to potentiate the effects of anticholinergic drugs in blocking oxotremorine-induced tremors. With 3 drugs (G3063, PMCG and N-methyl piperidyl benzilate) there was no such potentiation. The fact that TFP potentiated the tremor-blocking effect of atropine, hyoscine and caramiphen cannot be easily explained. TFP did not significantly potentiate the peripheral anticholinergic activity of any of the 6 compounds tested, and with 1 drug (N-methyl piperidyl benzilate) there was a significant reduction in peripheral anticholinergic activity as measured both by production of mydriasis and blockade of oxotremorine-induced salivation in the mouse, an effect probably worthy of further investigation. TFP given by itself showed no marked central or peripheral anticholinergic activity when given at relatively high dose levels of 100 pmol/kg. This drug failed to block oxotremorine-induced tremors and salivation and also failed to produce mydriasis. The results of the experiments reported here suggest strongly that the potentiating effect of TFP on the elevation of EEG arousal thresholds by anticholinergic drugs is not due to a combination of anticholinergic activities. REFERENCES BEBBINGTON, A. and BRIMBLECOMBE, R. W. (1965). Muscarinic receptors in the peripheral and central nervous system. In: Advances in Drug Research, 2, pp. 143-172. Academic Press, London. BRADLEY,P. B. and HANCE, A. S. (1957). The effect of chlorpromazine and methopromazine on thz electrical activity of the brain in the cat. Electroenceph. cfin. NeurophysioI. 9: 191-215. BRADLEY,P. B. and KEY, B. J. (1958). The effects of drugs on arousal responses produced by electrical stimulation of the reticular formation of the brain. Electroenceph. clin. Neurophysiol. 10: 97-110. BRADLEY,P. B. and KEY, B. J. (1959). A comparative study of the effects of drugs on the arousal system of the brain. Br. J. Pharm. Chemother. 14: 340-349. BRIMBLECOMBE, R. W. and GREEN,D. M. (1968a). Further evidence for cholinergic synapses in the mesodiencephalic activating system. J. Physiok, Land. 194: 16-17P. BRIMBLECOMBE, R. W. and GREEN, D. M. (1968b). The peripheral and central actions of some anticholinergic substances. Int. J. Neuropharmac. 7: 15-21. BRIMBLECOMBE, R. W. and GREEN,D. M. (1969). The effect of triflupromazine on the peripheral and central actions of some anticholinergic drugs. Br. J. Pharmac. 35: 364P. COLEMAN,I. W., LITTLE,P. E. and BANNARD,R. A. B. (1962). Cholinolytics in the treatment of anticholinesterase poisoning. Can. J. Biochem. Physiol. 40: 827-834.