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Gabamimetic properties of anxiolytic drugs
Life Sciences, Vol. 32, pp. 613-616 Printed in the U.S.A.
Pergamon Press
GABAMIMETIC PROPERTIES OF ANXIOLYTIC DRUGS 1 Jeffrey M. Goldstein, Linda C. Knobloch and Jeffrey B. Malick Biomedical Research Department Stuart Pharmaceuticals, Division of ICI Americas Inc. Wilmington, Delaware 19897 (Received in final form October 18, 1982)
Summary Diazepam (5 mg/kg, ip) and tracazolate (40 mg/kg, ip), a nonbenzodiazepine anxiolytic, blocked electrically-induced headturning without producing sedation. Bicuculline and picrotoxin, GABA antagonists, at doses not affecting head-turning (2 mg/kg, ip) antagonized the effects of diazepam and tracazolate on headturning. However, at the same dose, bicuculline was more effective as an antagonist of diazepam whereas picrotoxin was more effective as an antagonist of tracazolate. These results suggest that benzodiazepine as well as nonbenzodiazepine anxiolytics possess GABAmimetic activity. The difference in potency between bicuculline and picrotoxin as antagonists of diazepam and tracazolate may be related to their reported differences as GABA antagonists (e.g., site of receptor interaction). There is substantial evidence that many of the pharmacological effects of the benzodiazepines are mediated by gamma-amino butyric acid (GABA). This GABAmimetic activity of the benzodiazepines can be demonstrated in vivo using the rat head-turn model (I) in which the time course for evoking a contralateral head-turn is altered by GABA agonist and antagonist drugs (2-6). Although benzodiazepines reliably and consistently inhibit head-turning (like GABA agonists), and potentiate the effects of muscimol on head-turning (4), it has not been shown that the effects of the benzodiazepines in this model are a specific consequence of their interaction with the GABA receptor and can therefore be blocked with GABA antagonist drugs (e.g., bicuculline, picrotoxin). In this study we investigated the interaction of diazepam with bicuculline and picrotoxin on head-turning. We also investigated whether the ability to block head-turning by benzodiazepines is an effect that is common to other anxiolytic drugs. Tracazolate (ICl 136,753 [4-butylamino-l-ethyl-6-methyl-IH-pyrazolo-(3,4-b)pyridine5-carboxylic acid ethyl ester]) is a new and novel anxiolytic agent which is not chemically related to the benzodiazepines (7). In the present study, the ability of tracazolate to block head-turning was evaluated. Furthermore, studies were also performed with bicuculline and picrotoxin to determine whether the response was due to GABAmimetic activity.
Ipaper presented in part at the FASEB meetings, New Orleans, LA, April 1982 ( A b s t r a c t : Fedn. P r o c . 41:1067, 1982) 0024-3205/83/060613-04503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
614
GABAmimetic Properties of Anxiolytic Drugs
Vol. 32, No. 6, 1983
Methods Male Sprague-Dawley rats, 200-250 g, were stereotaxically implanted with a bipolar nichrome stimulating electrode (Plastic Products Company, Roanoke, VA) in the left caudate nucleus (coordinates from Pellegrino and Cushman [8]: posterior 0.4 mm, lateral 3.5 mm, and horizontal 5.5 mm below the skull). Following a one week recovery period, rats were tested for headturning. Electrical stimulation in the form of balanced biphasic pulse pairs (0.2 msec duration, I00 Hz) was delivered at varying current intensities (20200 ~A) until a head-turn could be elicited within 5 sec. Two sets of five trials each served as the control. Drugs were administered immediately after completion of the second set of trials and rats were tested 15 min later. The actual variable measured was the difference in the mean time it took for the rat to turn its head in the predrug vs postdrug state. A 50% or greater increase in response time over control was used as a significant drug effect
(1). In the drug antagonism studies, bicuculline or picrotoxin was administered immediately after the second set of trials and rats were retested 5 min later. Only those rats that did not show a significant change in response time to the GABA antagonists were subsequently injected with either diazepam or tracazolate and tested 15 min later. At the completion of the studies, rats were sacrificed with an overdose of sodium pentobarbital, perfused with 0.9% saline, followed by 10% formalin and their brains removed. Conventional histological methods were then follo~ed for verification of electrode placement. Results In rats in which head-turning was readily elicited, electrode placement was verified to be within the caudate nucleus. There was some variability as to the exact location of the tips of the stimulating electrodes within the caudate nucleus. In general, electrodes that were situated deep within the nucleus produced head-turning at lower current intensities than electrodes that were situated dorsally. Similar results were reported by Slater et al. (6). Rats in which head-turning could not be elicited due to confounding motor involvement of the limbs had electrode placements in the internal capsule; these rats were not used for the present studies. The effects of diazepam and tracazolate on head-turning are shown in Table I. Both drugs produced a dose-related increase in the latency for head-turning. At the highest dose tested, neither diazepam (5 mg/kg, ip) nor tracazolate (40 mg/kg, ip) produced any overt signs of sedation. Bicuculline and picrotoxin, at doses that did not affect head-turning, antagonized the effects of both diazepam (Table 2) and tracazolate (Table 3) in a dose-related manner. When similar doses of bicuculline and picrotoxin were tested for their ability to block diazepam and tracazolate, it was noted that whereas bicuculline was more effective as an antagonist of diazepam, picrotoxin was more effective as an antagonist of tracazolate.
Vol. 32, No. 6, 1983
GABAmimetic Properties of Anxiolytic Drugs
615
TABLE 1 Dose-response effects of diazepam and tmcazolate on head-tuming
Dose (mg/kg, i.p.)
Treatment
% Blocked*
1.25
7
2.5
8
50
5.0
11
100
20
7
29
4O
13
85
Diazepam
Tracazolate
N
29
• No. of rats with 50% or greater threshold increase at 15 min. post drug.
TABLE 2
TABLE 3
Effect of picrotoxin and bicuculline on dlazepam antagonism of head-turning
Effect of bicuculline and picrotoxin on tracazolate antagonism of head-turning
Treatment
Dose (mg/kg, i.p.)
N
% Blocked*
Treatment
Tracazolate
Dose (mg/kg, i.p.)
N
% Blocked"
40
t0
100
10
70
Diazepam
5.0
11
100
Diazepam + Picrotoxin
5.0 1 .O
4
75
Tracazolate + Bicuculline
40 2
Diazepam + Picrotoxin
5.0 2.0
7
43
Tracazolate + Bicuculline
40 4**
6
33
Diazepam + Picrotoxin
5.0 4.0"*
3
0
Tracazolate + Picrotoxin
40 1
5
80
Diazepam + Bicuculline
5.0 1.0
7
57
Tracazolate + Picrotoxin
40 2
7
14
Diazepam + Bicuculline
5.0 2,0
7
14
* No. of rats with 50% or greater threshold increase at 15 min. post-drug. ** O n e rat convulsed at this dose of picrotoxin.
• No. of rats with 50% or greater threshold increase at 15 min. post drug. ** One rat convulsed at this dose of b i c u c u l l i n e
616
GABAmimetic Propert±es of Anx±olytic Drugs
Vol. 32, No. 6, 1983
Discussion This study has demonstrated that the effects of diazepam on headturning are mediated by an effect at the GABA receptor since the GABA antagonists bicuculline and picrotoxin blocked diazepam's effect. These results confirm the reported GABAmimetic properties of the benzodiazepines in this model (4-6). In addition, we have shown that tracazolate, a nonbenzodiazepine anxiolytic, also produces effects similar to diazepam and both bicuculline and picrotoxin block tracazolate's effects on head-turning. Thus, the reported GABAmimetic properties of the benzodiazepines may be a common effect associated with the anxiolytics and may be related to their mechanism of action. The present findings do not exclude the possibility that the effects of the anxiolytics on head-turning may be a consequence of their sedative properties. However, this would not explain the ability of tracazolate to affect head-turning, since it has been reported that tracazolate has minimal sedative properties in rats (rotorod EDS0 greater than 400 mg/kg9 po) (7). Furthermore, if the sedative properties of a drug interfered with the head-turn response, then chlorpromazine, a neuroleptic with marked sedative properties, should block the head-turn response. In contrast, it has been reported that chlorpromazine, at sedative doses, facilitates head-turning (6). Thus, sedative activity per se may not be correlated with inhibition of head-turning. Of significant interest in the present study is the finding that bicuculline and picrotoxin are not equipotent in antagonizing the effects of diazepam and tracazolate on head-turning. Bicuculline appears to be a more potent antagonist against diazepam, whereas picrotoxin appears to be a more potent antagonist against tracazolate. These effects may be related to the different recognition sites with which ~icuculline and picrotoxin interact. It has been reported that bicuculline blocks the recognition site for GABA through which the benzodiazepine anxiolytics may exert their effects (9,10). On the other hand, picrotoxin blocks the chloride ionophore (11,12), which is also the proposed recognition site for the nonbenzodiazepine anxiolytic tracazolate (13). Our data provide physiological support for these biochemical observations. References 1.
A. BARNETT and J . M . GOLDSTEIN, J .
Pharraacol.
exp.
Ther.
194:296-302
(1975). 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13.
J.B. MALICR and J.M. GOLDSTEIN, Arch. int. Pharmacodyn. 220:269-274 (1976). A.R. CROSSMAN, L.A. LEE and P. SLATER, Br. J. Pharmac. 61:483P (1977). A.R. CROSSMAN, L.A. LEE, D.A. LONGMAN and P. SLATER, Br. J. Pharmac. 66:493P (1979). P. SLATER and D.A. LONCMAN, Life Sci. 2_55:1963-1967 (1979). P. SLATER, L.A. LEE, D.A. LONGMAN and A.R. CROSSMAN, J. Pharmacol. Meth. 3:39-49 (1980). J . B . PATEL and J . B . MALICK, E u r o p . J . P h a r m a c o l . 7 8 : 3 2 3 - 3 3 3 ( 1 9 8 2 ) . L . J . PELLEGRINO and A . J . CUSHMAN~ A S t e r e o t a x i c A t l a s o f t h e R a t B r a i n , M e r e d i t h P u b l i s h i n g C o . , New York ( 1 9 6 7 ) . A. GUIDOTTI, M. BARALDI and E. COSTA, P h a r m a c o l o g y 1 9 : 2 6 7 - 2 7 7 ( 1 9 7 9 ) . A. GUIDOTTI, M. BARALDI, J . P . SCHWARTZ and E. COSTA, P h a r m a c o l . B i o c h e m . Behav. 10:803-807 (1979). M.K. TICKU, M. BAN and R.W. OLSEN, Mol. P h a r m a c o l . 1 4 : 3 9 1 - 4 0 2 ( 1 9 7 8 ) . F. LEEB-LUNDBERG and R.W. OLSEN, i n P s y c h o p h a r m a c o l o g y and B i o c h e m i s t r y of Neurotransmitter Receptors, Edited by H.I. Yamamura, R.W. Olsen and E. Usdin, pp. 593-606~ Elsevier, New York (1980). B.A. MEINERS and A.I. SALAMA, Europ. J. Pharmacol. 78:315-322 (1982).
Pergamon Press
GABAMIMETIC PROPERTIES OF ANXIOLYTIC DRUGS 1 Jeffrey M. Goldstein, Linda C. Knobloch and Jeffrey B. Malick Biomedical Research Department Stuart Pharmaceuticals, Division of ICI Americas Inc. Wilmington, Delaware 19897 (Received in final form October 18, 1982)
Summary Diazepam (5 mg/kg, ip) and tracazolate (40 mg/kg, ip), a nonbenzodiazepine anxiolytic, blocked electrically-induced headturning without producing sedation. Bicuculline and picrotoxin, GABA antagonists, at doses not affecting head-turning (2 mg/kg, ip) antagonized the effects of diazepam and tracazolate on headturning. However, at the same dose, bicuculline was more effective as an antagonist of diazepam whereas picrotoxin was more effective as an antagonist of tracazolate. These results suggest that benzodiazepine as well as nonbenzodiazepine anxiolytics possess GABAmimetic activity. The difference in potency between bicuculline and picrotoxin as antagonists of diazepam and tracazolate may be related to their reported differences as GABA antagonists (e.g., site of receptor interaction). There is substantial evidence that many of the pharmacological effects of the benzodiazepines are mediated by gamma-amino butyric acid (GABA). This GABAmimetic activity of the benzodiazepines can be demonstrated in vivo using the rat head-turn model (I) in which the time course for evoking a contralateral head-turn is altered by GABA agonist and antagonist drugs (2-6). Although benzodiazepines reliably and consistently inhibit head-turning (like GABA agonists), and potentiate the effects of muscimol on head-turning (4), it has not been shown that the effects of the benzodiazepines in this model are a specific consequence of their interaction with the GABA receptor and can therefore be blocked with GABA antagonist drugs (e.g., bicuculline, picrotoxin). In this study we investigated the interaction of diazepam with bicuculline and picrotoxin on head-turning. We also investigated whether the ability to block head-turning by benzodiazepines is an effect that is common to other anxiolytic drugs. Tracazolate (ICl 136,753 [4-butylamino-l-ethyl-6-methyl-IH-pyrazolo-(3,4-b)pyridine5-carboxylic acid ethyl ester]) is a new and novel anxiolytic agent which is not chemically related to the benzodiazepines (7). In the present study, the ability of tracazolate to block head-turning was evaluated. Furthermore, studies were also performed with bicuculline and picrotoxin to determine whether the response was due to GABAmimetic activity.
Ipaper presented in part at the FASEB meetings, New Orleans, LA, April 1982 ( A b s t r a c t : Fedn. P r o c . 41:1067, 1982) 0024-3205/83/060613-04503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
614
GABAmimetic Properties of Anxiolytic Drugs
Vol. 32, No. 6, 1983
Methods Male Sprague-Dawley rats, 200-250 g, were stereotaxically implanted with a bipolar nichrome stimulating electrode (Plastic Products Company, Roanoke, VA) in the left caudate nucleus (coordinates from Pellegrino and Cushman [8]: posterior 0.4 mm, lateral 3.5 mm, and horizontal 5.5 mm below the skull). Following a one week recovery period, rats were tested for headturning. Electrical stimulation in the form of balanced biphasic pulse pairs (0.2 msec duration, I00 Hz) was delivered at varying current intensities (20200 ~A) until a head-turn could be elicited within 5 sec. Two sets of five trials each served as the control. Drugs were administered immediately after completion of the second set of trials and rats were tested 15 min later. The actual variable measured was the difference in the mean time it took for the rat to turn its head in the predrug vs postdrug state. A 50% or greater increase in response time over control was used as a significant drug effect
(1). In the drug antagonism studies, bicuculline or picrotoxin was administered immediately after the second set of trials and rats were retested 5 min later. Only those rats that did not show a significant change in response time to the GABA antagonists were subsequently injected with either diazepam or tracazolate and tested 15 min later. At the completion of the studies, rats were sacrificed with an overdose of sodium pentobarbital, perfused with 0.9% saline, followed by 10% formalin and their brains removed. Conventional histological methods were then follo~ed for verification of electrode placement. Results In rats in which head-turning was readily elicited, electrode placement was verified to be within the caudate nucleus. There was some variability as to the exact location of the tips of the stimulating electrodes within the caudate nucleus. In general, electrodes that were situated deep within the nucleus produced head-turning at lower current intensities than electrodes that were situated dorsally. Similar results were reported by Slater et al. (6). Rats in which head-turning could not be elicited due to confounding motor involvement of the limbs had electrode placements in the internal capsule; these rats were not used for the present studies. The effects of diazepam and tracazolate on head-turning are shown in Table I. Both drugs produced a dose-related increase in the latency for head-turning. At the highest dose tested, neither diazepam (5 mg/kg, ip) nor tracazolate (40 mg/kg, ip) produced any overt signs of sedation. Bicuculline and picrotoxin, at doses that did not affect head-turning, antagonized the effects of both diazepam (Table 2) and tracazolate (Table 3) in a dose-related manner. When similar doses of bicuculline and picrotoxin were tested for their ability to block diazepam and tracazolate, it was noted that whereas bicuculline was more effective as an antagonist of diazepam, picrotoxin was more effective as an antagonist of tracazolate.
Vol. 32, No. 6, 1983
GABAmimetic Properties of Anxiolytic Drugs
615
TABLE 1 Dose-response effects of diazepam and tmcazolate on head-tuming
Dose (mg/kg, i.p.)
Treatment
% Blocked*
1.25
7
2.5
8
50
5.0
11
100
20
7
29
4O
13
85
Diazepam
Tracazolate
N
29
• No. of rats with 50% or greater threshold increase at 15 min. post drug.
TABLE 2
TABLE 3
Effect of picrotoxin and bicuculline on dlazepam antagonism of head-turning
Effect of bicuculline and picrotoxin on tracazolate antagonism of head-turning
Treatment
Dose (mg/kg, i.p.)
N
% Blocked*
Treatment
Tracazolate
Dose (mg/kg, i.p.)
N
% Blocked"
40
t0
100
10
70
Diazepam
5.0
11
100
Diazepam + Picrotoxin
5.0 1 .O
4
75
Tracazolate + Bicuculline
40 2
Diazepam + Picrotoxin
5.0 2.0
7
43
Tracazolate + Bicuculline
40 4**
6
33
Diazepam + Picrotoxin
5.0 4.0"*
3
0
Tracazolate + Picrotoxin
40 1
5
80
Diazepam + Bicuculline
5.0 1.0
7
57
Tracazolate + Picrotoxin
40 2
7
14
Diazepam + Bicuculline
5.0 2,0
7
14
* No. of rats with 50% or greater threshold increase at 15 min. post-drug. ** O n e rat convulsed at this dose of picrotoxin.
• No. of rats with 50% or greater threshold increase at 15 min. post drug. ** One rat convulsed at this dose of b i c u c u l l i n e
616
GABAmimetic Propert±es of Anx±olytic Drugs
Vol. 32, No. 6, 1983
Discussion This study has demonstrated that the effects of diazepam on headturning are mediated by an effect at the GABA receptor since the GABA antagonists bicuculline and picrotoxin blocked diazepam's effect. These results confirm the reported GABAmimetic properties of the benzodiazepines in this model (4-6). In addition, we have shown that tracazolate, a nonbenzodiazepine anxiolytic, also produces effects similar to diazepam and both bicuculline and picrotoxin block tracazolate's effects on head-turning. Thus, the reported GABAmimetic properties of the benzodiazepines may be a common effect associated with the anxiolytics and may be related to their mechanism of action. The present findings do not exclude the possibility that the effects of the anxiolytics on head-turning may be a consequence of their sedative properties. However, this would not explain the ability of tracazolate to affect head-turning, since it has been reported that tracazolate has minimal sedative properties in rats (rotorod EDS0 greater than 400 mg/kg9 po) (7). Furthermore, if the sedative properties of a drug interfered with the head-turn response, then chlorpromazine, a neuroleptic with marked sedative properties, should block the head-turn response. In contrast, it has been reported that chlorpromazine, at sedative doses, facilitates head-turning (6). Thus, sedative activity per se may not be correlated with inhibition of head-turning. Of significant interest in the present study is the finding that bicuculline and picrotoxin are not equipotent in antagonizing the effects of diazepam and tracazolate on head-turning. Bicuculline appears to be a more potent antagonist against diazepam, whereas picrotoxin appears to be a more potent antagonist against tracazolate. These effects may be related to the different recognition sites with which ~icuculline and picrotoxin interact. It has been reported that bicuculline blocks the recognition site for GABA through which the benzodiazepine anxiolytics may exert their effects (9,10). On the other hand, picrotoxin blocks the chloride ionophore (11,12), which is also the proposed recognition site for the nonbenzodiazepine anxiolytic tracazolate (13). Our data provide physiological support for these biochemical observations. References 1.
A. BARNETT and J . M . GOLDSTEIN, J .
Pharraacol.
exp.
Ther.
194:296-302
(1975). 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13.
J.B. MALICR and J.M. GOLDSTEIN, Arch. int. Pharmacodyn. 220:269-274 (1976). A.R. CROSSMAN, L.A. LEE and P. SLATER, Br. J. Pharmac. 61:483P (1977). A.R. CROSSMAN, L.A. LEE, D.A. LONGMAN and P. SLATER, Br. J. Pharmac. 66:493P (1979). P. SLATER and D.A. LONCMAN, Life Sci. 2_55:1963-1967 (1979). P. SLATER, L.A. LEE, D.A. LONGMAN and A.R. CROSSMAN, J. Pharmacol. Meth. 3:39-49 (1980). J . B . PATEL and J . B . MALICK, E u r o p . J . P h a r m a c o l . 7 8 : 3 2 3 - 3 3 3 ( 1 9 8 2 ) . L . J . PELLEGRINO and A . J . CUSHMAN~ A S t e r e o t a x i c A t l a s o f t h e R a t B r a i n , M e r e d i t h P u b l i s h i n g C o . , New York ( 1 9 6 7 ) . A. GUIDOTTI, M. BARALDI and E. COSTA, P h a r m a c o l o g y 1 9 : 2 6 7 - 2 7 7 ( 1 9 7 9 ) . A. GUIDOTTI, M. BARALDI, J . P . SCHWARTZ and E. COSTA, P h a r m a c o l . B i o c h e m . Behav. 10:803-807 (1979). M.K. TICKU, M. BAN and R.W. OLSEN, Mol. P h a r m a c o l . 1 4 : 3 9 1 - 4 0 2 ( 1 9 7 8 ) . F. LEEB-LUNDBERG and R.W. OLSEN, i n P s y c h o p h a r m a c o l o g y and B i o c h e m i s t r y of Neurotransmitter Receptors, Edited by H.I. Yamamura, R.W. Olsen and E. Usdin, pp. 593-606~ Elsevier, New York (1980). B.A. MEINERS and A.I. SALAMA, Europ. J. Pharmacol. 78:315-322 (1982).