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Antagonism of L-baclofen-induced antinociception by CGP 35348 in the spinal cord of the rat
European Journal of Pharmacology, 234 (1993) 255-262
255
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53018
Antagonism of L-baclofen-induced antinociception by CGP 35348 in the spinal cord of the rat D o n n a L. H a m m o n d a n d J o n a t h a n D. W a s h i n g t o n Department of Anesthesia and Critical Care, University of Chicago, Chicago, IL 60637, USA Received 5 October 1992, revised MS received 19 January 1993, accepted 26 January 1993
This study examined the potency and selectivity of the GABA B receptor antagonist CGP 35348 (3-amino-propyl(diethoxymethyl)phosphinic acid) in the spinal cord of the rat. Intrathecal (i.t.) administration of 3-30 /~g CGP 35348 produced a dose-dependent antagonism of the antinociception produced by i.t. administered L-baclofen. Increasing doses of CGP 35348 produced progressive, rightward parallel shifts in the dose-effect relationship of L-baclofen in both the tail flick and hot plate tests. However, in the tail flick test, the magnitude of the shift was not proportional to the dose of CGP 35348 such that doses greater than 10/zg i.t. produced no further antagonism. Schild analysis using all three doses of CGP 35348 yielded a slope of -0.45. However, if the Schild analysis was confined to the two lowest doses of CGP 35348 at which progressive shifts were obtained, a slope of -0.91 and an apparent pA 2 value of 9.3 were obtained. An apparent pA 2 value of 9.0 was also obtained in the hot plate test using the two lowest doses of CGP 35348. These data suggest that CGP 35348 is a competitive GABA B receptor antagonist at low concentrations. However, the failure to observe greater antagonism at higher doses of CGP 35348 suggests that this drug may exhibit additional properties at higher concentrations that can mask or prevent the occurrence of further antagonism. In contrast, the antinociception produced by i.t. administration of the GABA A receptor agonist isoguvacine was not antagonized by 30/xg i.t. CGP 35348, a dose that shifted the dose-effect relationship of L-baclofen 10-fold to the right. These data suggest that CGP 35348 is a potent and selective antagonist of spinal GABA B receptors in the rat. Baclofen; CGP 35348; Antinociception; Spinal cord; GABA B receptors 1. Introduction Since the identification of phaclofen, several competitive antagonists of the GABAB receptor have been synthesized. Like phaclofen, these compounds are structural analogs of baclofen having p A 2 values ranging between 4.0 and 5.3 in the isolated guinea pig ileum preparation and micromolar affinity for the G A B A B receptor in rat brain homogenates (Bowery, 1989; Drew et al., 1990; Kerr et al., 1990). These compounds are also effective antagonists of the G A B A a receptor agonist baclofen in vivo (Aran and Hammond, 1991; Giuliani et al., 1988; Hong and Henry, 1991; Schmid et al., 1989; Wiillner et al., 1989). However, as a class, these antagonists are not remarkably potent in either in vitro or in vivo preparations. More recently, a structurally distinct G A B A B receptor antagonist was synthesized (Bittiger et al., 1990; Olpe et al., 1990). This compound, 3-amino-propyl(diethoxymethyl)phosphinic acid (CGP 35348), is 10 to 30-fold
Correspondence to: D.L. Hammond, Department of Anesthesia and Critical Care, University of Chicago, 5841 South Maryland Avenue MC 4028, Chicago, IL 60637, USA. Tel. 1.312.702 5952, fax 1.312.702 3535.
more potent than phaclofen in the isolated rat vas deferens preparation (Hills et al., 1991b), in rat cortical synaptosomes (Bonanno and Raiteri, 1992), or when administered iontophoretically to rat cortical neurons (Olpe et al., 1990), and has a 3-fold higher affinity for the G A B A B receptor in rat brain homogenates (Olpe et al., 1990). As part of our continuing study of the role of spinal G A B A B receptors in the production of antinociception (Aran and Hammond, 1991; Hammond and Moy, 1992), we examined the ability of intrathecally (i.t.) administered CGP 35348 to antagonize the antinociception produced by i.t. injection of the G A B A B receptor agonist L-baclofen or the G A B A A receptor agonist isoguvacine in the rat. 2. Materials and methods
2.1. Surgical procedures Male Sprague-Dawley rats (Sasco, Madison, WI; 300-350 g) were anesthetized with halothane and implanted with indwelling i.t. catheters (Hammond, 1988). Animals were group housed with free access to food and water and allowed six to seven days to recover from surgery.
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2.2. Analgesiometric procedures Thermal nociceptive threshold was measured using modifications of the tail flick ( D ' A m o u r and Smith, 1941) and hot plate (Woolfe and MacDonald, 1944) tests. In the tail flick test, a high intensity light b e a m was focused on the dorsal surface of the rat's blackened tail. The time interval between the application of the light b e a m and the reflex response was measured electronically to the nearest 0.1 s. The average of two such measurements was defined as the tail flick latency. Animals were removed from the apparatus in the absence of a response within 14 s and assigned this cut-off latency. After determination of tail flick latency, motor function was assessed using the righting reflex test and the inclined plane test (Rivlin and Tator, 1977). In the righting reflex test, the rat was placed on its back and allowed to right itself. The righting reflex was scored as follows: 0, unable to right itself; 0.5, rights u p p e r torso only; 1.0, rights upper and lower torso, but hindlegs abducted; 1.5, rights upper and lower torso with legs properly positioned under body; 2.0, brisk execution of reflex. In the inclined plane test, the rat was placed crosswise to the long axis of an inclined plane that had a rubber surface with horizontal ridges 0.1 cm in height. The initial angle of the inclined plane was 50 ° . The angle was then adjusted in 5 ° increments to determine the largest angle at which the rat was able to maintain its position on the plane. The hot plate test was conducted after the inclined plane test. Each animal was placed on a 55°C copper surface and the time taken to respond either by licking the hind paw or jumping was defined as the hot plate latency. Animals were removed from the hot plate in the absence of a response by 40 s and assigned this cut-off latency.
2.3. Experimental design The effect of p r e t r e a t m e n t with C G P 35348 on L-baclofen-induced antinociception was assessed in the first experiment. After baseline tail flick latency, hot plate latency and motor function were measured, either saline or C G P 35348 (3-30 /zg) was injected i.t. Five minutes later, tail flick latency, hot plate latency, and motor function were redetermined and L-baclofen (0.03-2.0 ~g) was then injected i.t. Response latencies and motor function were redetermined 15, 30, 45, 60 and 90 min later. The dose-response lines for L-baclofen in the absence or presence of the different doses of C G P 35348 were fit using least squares linear regression of the individual data. The resulting dose-response lines were tested for parallelism as described by Tallarida and Murray (1987). The EDs0, defined as the dose of drug that produced one-half the maximum possible
increase in response latency (i.e. to 9 s in the tail flick test and to 25 s in the hot plate test) was determined from the linear regression analysis; 95% confidence limits (CL) were determined by Fieller's theorem (Finney, 1964). Analysis of covariance was used to determine the significance of differences in EDs0s (Zar, 1974). Where increasing doses of C G P 35348 produced progressive, rightward shifts in the dose-response relationship of L-baclofen, the apparent pA 2 value of CGP 35348 was determined by the Schild method using the dose (in tool) administered (Tallarida and Murray, 1987). The effects of C G P 35348 pretreatment on baclofen-induced decrements in righting reflex and inclined plane angle were compared to that of saline p r e t r e a t m e n t using Fisher's exact test. The ability of C G P 35348 to reverse the effects of L-baclofen, and its time of peak effect were ascertained in another experiment. After measurement of baseline tail flick latency, hot plate latency and motor function, 0.3 /xg L-baclofen was injected i.t. Fifteen minutes later, response latencies and motor function were redetermined after which time (time zero) either saline or 30 /xg C G P 35348 was injected i.t. Response latencies and motor function were then redetermined 5, 15, 30 and 45 min later. A two-way A N O V A for repeated measures, in which one factor was drug treatment and the other factor was time, was used to analyze the data. This approach enabled comparison of post-injection latencies to latencies at time zero within each treatment group, as well as between treatment group comparisons of the effects of CGP 35348 to saline at individual time points. Post-hoc comparisons of mean values at individual time points were performed using Newman-Keuls test (Keppel, 1973). A final experiment was conducted to determine whether C G P 35348 retained its selectivity as a G A B A B receptor antagonist when administered i.t. After measurement of baseline tail flick latency, hot plate latency and motor function, 30 /xg of the G A B A a receptor agonist isoguvacine was injected i.t. Fifteen minutes later, response latencies and motor function were redetermined after which time either saline or 30 ~zg C G P 35348 was injected i.t. Response latencies and motor function were then redetermined 5, 15, 30 and 45 min later. The data were analyzed as described for the corresponding study with L-baclofen.
2.4. Drugs and injections Each animal was used only once. All drugs were dissolved in saline, the p H adjusted to 7, and injected i.t. in a volume of 10 /zl followed by 10 ~1 of saline. The injection was monitored by following the movement of an air bubble through polyethylene tubing over a period of 60 s. At the end of the experiment a laminectomy was performed and the patency and loca-
257
tion of the catheter tip was verified by injection of India ink. Animals in which the catheter leaked or was incorrectly placed were discarded from the study. LBaclofen was provided courtesy of Drs. Szeszak and Scheibli, while CGP 35348 was provided courtesy of Drs. Scholer and Maitre, all of Ciba-Geigy, Basel, Switzerland. Isoguvacine hydrochloride was purchased from Research Biochemicals, Inc. (Natick, MA).
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In the tail flick test, the EDs0 value (and 95% CL) of L-baclofen 15 rain after its injection in saline-pretreated rats was 0.14 (0.10-0.19) /xg i.t. (fig. 1A). Pretreatment with 3, 10 or 30 txg i.t. CGP 35348 produced a parallel, rightward shift in the dose-effect relationship of L-baclofen, increasing its EDs0 (and 95% CL) to 0.58 (0.42-0.82) txg, 1.44 (indeterminate) txg, and 1.32 (0.9-2.0) p~g i.t., respectively. These EDs0 values represented increases of 4.1, 10.3 and 9.4-fold and were significantly different from the EDs0 of Lbaclofen in saline-pretreated rats (P < 0.01 each dose). Thus, while the dose-effect relationship of L-baclofen was shifted further right as the dose of CGP 35348 was increased, the magnitude of the shift was not proportional to the dose of the antagonist. This finding was also apparent when the data were subjected to Schild analysis, which yielded a slope of - 0 . 4 5 . However, if the Schild analysis was restricted to the two lowest doses of CGP 35348 at which progressive shifts were obtained, the slope of the regression line was -0.91. Constraint of this slope to - 1 yielded an apparent pA 2 value of 9.3. In the hot plate test, the EDs0 value (and 95% CL) of L-baclofen 15 rain after its injection in saline-pretreated rats was 0.46 (0.32-0.91) Ixg i.t. (fig. 1B). Pretreatment with 3 or 10 ~ g CGP 35348 produced a parallel, rightward shift in the dose-effect relationship of L-baclofen, increasing its EDs0 value (and 95% CL) by 2- and 5-fold to 0.97 (0.76-1.32) Ixg and 2.5 (1.340.6) txg i.t., respectively. These increases were statistically significant (P < 0.01). A further rightward shift in the dose-effect relationship of L-baclofen occurred in rats pretreated with 30 /xg CGP 35348; however, an EDso value and 95% CL limits could not be determined. Although the effect of 30 /xg CGP 35348 appeared to be noncompetitive and associated with a decrease in maximal effect, limitations in the dose combinations that could feasibly be administered precluded such a conclusion. I.t. injection of 2.0 txg Lbaclofen in saline-treated rats produced profound flaccid paralysis (see below). Although 30 ~g CGP 35348
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3.1. Effects of i.t. pretreatment with CGP 35348 on baclofen-induced antinociception
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Dose L-Baclofen (#g) Fig. 1. Dose-response relationship of L-baclofen determined 15 min after its i.t. injection in rats pretreated with saline ( I ) or with 3 /xg (e), 10/~g ( • ) or 30 txg ( • ) i.t. CGP 35348 in the tail flick (A) or hot plate (B) test. Abscissas: Dose of L-baclofen in ,ag. Ordinates: response latency in s. Each point represents the mean of determinations made in 6 - l l rats. Lines of best fit were calculated using a least-squares linear regression. In the tail flick test, the dose-response relationships determined for L-baclofen in rats pretreated with 3, 10 or 30 # g CGP 35348 were parallel to that determined in saline-pretreated rats. in the hot plate test, only the dose-response relationships determined for L-baclofen in rats pretreated with 3 or 10 # g CGP 35348 were parallel to that determined in saline-pretreated rats.
was able to nearly completely antagonize this effect, it was not clear that it would also be able to completely antagonize the flaccidity produced by even higher doses of L-baclofen. In the absence of such a complete antagonism, any increase in hot plate latency produced by higher doses of L-baclofen in combination with 30 Ixg CGP 35348 could not be confidently interpreted as antinociception. For this reason, doses of L-baclofen that exceeded 2.0 /xg were not tested in combination with 30 # g CGP 35348. It is therefore not possible to state whether the antagonism produced by 30 ~g CGP 35348 was noncompetitive or simply represented the lower aspect of a dose-response relationship that was shifted right by more than 5-fold. Schild analysis using the two lowest doses of CGP 35348 yielded a regression line of slope -1.14. When this slope was con-
258 s t r a i n e d to - 1 , an a p p a r e n t p A 2 v a l u e o f 9.0 w a s obtained. T h e t i m e c o u r s e o f t h e a n t a g o n i s m o f 0.3 Ixg Lb a c l o f e n by p r e t r e a t m e n t w i t h v a r i o u s d o s e s o f C G P 35348 is i l l u s t r a t e d in fig. 2. I n b o t h t h e tail flick a n d h o t p l a t e tests, t h e a n t a g o n i s m by C G P 35348 was m a x i m a l w h e n first e x a m i n e d 15 m i n a f t e r t h e i n j e c t i o n o f L - b a c l o f e n , a t i m e t h a t c o r r e s p o n d e d to 20 m i n a f t e r t h e i n j e c t i o n o f C G P 35348. T h e a n t a g o n i s m p e r s i s t e d for at l e a s t 60 m i n .
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3.2. Effects of i.t. posttreatment with CGP 35348 on baclofen-induced antinocieeption
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Fig. 3. Time course of the antagonism of L-baclofen by CGP 35348 in the tail flick (A) and hot plate (B) tests. Abscissas: time in min. Ordinates: response latency in s. Rats were injected first with 0.3/xg i.t. L-baclofen (left arrowhead) and followed 15 rain later (right arrowhead; time zero) by i.t. injection of either saline (11; n = 11) or 30/zg CGP 35348 (e; n = 9). Each point represents the mean +_S.E.M. Where not visible, the S.E.M. bar is encompassed by the symbol. Daggers indicate values in CGP 35348-treated rats that were significantly different from those in saline-treated rats at the corresponding time point (P < 0.01). Data from 9 of the 11 rats in the saline-treated group were obtained in the course of a previous, related study (Hammond and Moy, 1992), and were included here to minimize the number of animals used.
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Fig. 2. Time course of the effect of i.t. pretreatment with saline ( I ; n = 7) or with 3 /xg (e; n = 8), 10/xg (A; n = 8) or 30 izg (T; n = 7) CGP 35348 on the increase in tail flick (A) and hot plate (B) latency produced by 0.3 txg i.t. L-baclofen. Saline or CGP 35348 was injected 5 min before (indicated by left arrowhead) injection of L-baclofen (indicated by right arrowhead). Abscissas: time in min. Ordinates: response latency in s. Each point represents the mean+S.E.M. Where not visible, the S.E.M. bar is encompassed by the symbol. Values significantly different from those in saline-treated rats at the corresponding time point are indicated by asterisks (P < 0.05) or daggers (P < 0.01).
i s h e d by i.t. i n j e c t i o n o f s a l i n e at any t i m e (P > 0.05). H o w e v e r , tail flick l a t e n c y was s i g n i f i c a n t l y d e c r e a s e d w i t h i n 5 m i n a n d m a x i m a l l y d e c r e a s e d 15 m i n a f t e r t h e i.t. i n j e c t i o n o f 30 /xg C G P 35348 as c o m p a r e d to v a l u e s at t i m e z e r o in this t r e a t m e n t g r o u p . T h e a n t a g o n i s m p e r s i s t e d for at l e a s t a n a d d i t i o n a l 30 m i n . Comparison between treatment groups indicated that C G P 35348 also s i g n i f i c a n t l y r e d u c e d tail flick l a t e n c y as c o m p a r e d to v a l u e s in t h e s a l i n e - t r e a t e d g r o u p (P < 0.01) b e g i n n i n g 5 m i n a f t e r its i n j e c t i o n a n d c o n t i n u i n g for t h e d u r a t i o n o f t h e e x p e r i m e n t . H o t p l a t e l a t e n c y was m a x i m a l l y i n c r e a s e d w i t h i n 15 m i n o f t h e i n j e c t i o n o f 0 . 3 / x g i.t. L - b a c l o f e n (fig. 3B). Within session comparison of post-injection latencies to l a t e n c i e s at t i m e z e r o in t h e s a l i n e - t r e a t e d g r o u p i n d i c a t e d t h a t h o t p l a t e l a t e n c y was n o t d i m i n i s h e d by
259
i.t. injection of saline at any time (P > 0.05). However, hot plate latency was significantly decreased within 5 rain of the injection of 3 0 / z g C G P 35348 as compared to values at time zero in this treatment group. The antagonism persisted for the duration of the session. Comparison between treatment groups indicated that C G P 35348 also significantly reduced hot plate latency as compared to values in the saline-treated group (P < 0.01) beginning 5 min after its injection and continuing for the duration of the experiment.
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Doses of L-baclofen greater than 0.6 /zg i.t. decreased the angle at which rats could maintain their position on the inclined plane. I.t. injection of 2.0 /zg L-baclofen in four saline-pretreated rats decreased the inclined plane angle from a baseline value of 48.7 + 1.3 to 32.5 + 1.4 ° within 15 rain and to 31.3 + 1.3 ° by 30 min (fig. 4A). This reduction in angle was antagonized by the lowest dose of C G P 35348 (fig. 4A). Thus, the mean angle maintained on the inclined plane after injection of 2.0 ~ g i.t. L-baclofen in rats pretreated with 3 tzg i.t. C G P 35348 was 42.8 + 1.7 ° at 30 rain (P = 0.01). Higher doses of C G P 35348 did not produce
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3.3. Effects of i.t. posttreatment with CGP 35348 on isogut'acine-induced antinociception Fifteen minutes after i.t. injection of 30 /zg isoguvacine to one group of four rats, tail flick latency and hot plate latency were increased to 11.0_+ 2.8 and 27.1 + 7.5 s, respectively. These increases were therefore comparable to that produced by 0.3 ~ g L-baclofen. Neither the increase in tail flick latency nor the increase in hot plate latency was antagonized at any time after i.t. injection of 30 /xg C G P 35348 in these rats. For example, 15 min after the injection of C G P 35348, when maximal antagonism of L-baclofen had occurred, tail flick latency and hot plate latency remained elevated at 11.4 + 2.6 and 33.0 _+ 4.2 s, respectively. When injected in another group of four rats, 30 ~ g i.t. isoguvacine again increased tail flick latency and hot plate latency to the same extent as in the first group. These increases in tail flick latency and hot plate latency were similarly unaffected at any time after i.t. injection of saline. Thus, 15 rain after the injection of saline, tail flick latency and hot plate latency were 12.1 + 1.9 and 24.2 _+ 7.9 s, respectively. Comparison between treatment groups indicated that the increase in tail flick latency and hot plate latency produced by isoguvacine in rats treated with C G P 35348 did not differ from that of rats treated with saline (P > 0.4, both tests).
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Fig. 4. Plot of the individual data showing the effects of i.t. pretreatment with saline (indicated by 0) or 3, 10 or 30/zg CGP 35348 on the decrease in inclined plane angle (A) and righting reflex (B) produced 30 rain after i.t. injection of 2.0 tzg L-baclofen. Each circle represents the response of a single animal at that dose combination. 'SAL" represents individual data obtained in rats 30 rain after an i.t. injection of saline (no baclofen administered) and serves as a reference for righting reflex and inclined plane angle in untreated animals.
a more complete antagonism, with mean angles ranging from 44.5 + 0.5 to 46.1 + 1.1 ° in the 30 rain postinjection period. Doses of E-baclofen greater than 0.6 g g i.t. also interfered with the righting reflex. Although all four saline-pretreated rats had baseline righting reflex scores >/1.5, within 30 min of 2.0/zg i.t. L-baclofen three of these animals were unable to right themselves and were assigned a score of zero (fig. 4B). At this time all four rats exhibited flaccid paralysis of the hindquarters. Pretreatment with 3, 10 or 30 /zg i.t. C G P 35348 significantly antagonized the decrement in righting reflex (P < 0.02, all doses), with near complete antagonism observed in rats pretreated with the highest dose of C G P 35348 (fig. 4B). Thus, 30 rain after injection of 2.0 Izg L-baclofen in rats pretreated with 30 ~ g i.t. C G P 35348, four of nine rats had a righting reflex of 1.5 and the remaining five rats had a righting reflex of 1.
260
4. Discussion
1.t. administered CGP 35348 antagonized both the antinociceptive and the muscle relaxant effects of intrathecally administered L-baclofen in the rat. In the tail flick and hot plate tests, 3 and 10 /xg CGP 35348 produced dose-dependent and parallel shifts to the right of the dose-effect relationship of L-baclofen. This antagonism was surmountable. Importantly, CGP 35348 retained its selectivity for the GABAB receptor in the spinal cord. Neither the increase in tail flick latency nor the increase in hot plate latency produced by i.t. injection of the GABA A receptor agonist isoguvacine was antagonized by 30 ~g i.t. CGP 35348, a dose that increased the EDs0 of L-baclofen by 10-fold. This finding is consistent with the very poor potency of CGP 35348 at the G A B A A receptor (15% displacement at 1 raM; Olpe et al., 1990) and the results of electrophysiological investigations in which systemic or bath application of CGP 35348 did not antagonize the effects of the G A B A A receptor agonist 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP) on either cortical neurons or on spinal cord neurons in the rat (Olpe et al., 1990). Taken together, these results suggest that CGP 35348 is a selective antagonist of the GABA B receptor in the spinal cord of the rat. CGP 35348 is also the most potent available antagonist of spinal G A B A B receptors mediating antinociception. As little as 3 ~g (13 nmol) i.t. produced a 5-fold rightward shift in the dose-effect relationship of L-baclofen. By comparison, 30 ~g (120 nmol) i.t. phaclofen was required to increase the EDs0 of i.t. administered D,L-baclofen by 4-fold in these same tests (Aran and Hammond, 1991). The finding that CGP 35348 was 10-fold more potent than phaclofen in the spinal cord is consistent with the results of studies in the isolated rat vas deferens (Hills et al., 1991b), in rat cortical synaptosomes (Bonanno and Raiteri, 1992), and iontophoretic studies of rat cortical neurons (Olpe et al., 1990) in which CGP 35348 was 10 to 30 times more potent than phaclofen. CGP 35348 was also more potent than 2-hydroxy-saclofen or 4-amino-3-(5methoxybenzo(b)furan-2-yl) butanoic acid which, at respective doses of 10 p~g (37 nmol) i.t. and 30 p~g (120 nmol) i.t., produced only a 2- to 3-fold shift in the dose-effect relationship of D,L-baclofen (Aran and Hammond, 1991; Hammond and Moy, 1992). Finally, 20 nmol i.t. was the minimum effective dose of Dbaclofen, which is an antagonist of L-baclofen under certain conditions in the spinal cord (Fromm et al., 1990; Sawynok, 1986). Thus, although several G A B A ~ receptor antagonists have been shown to antagonize the antinociception produced by baclofen in the rat, they are uniformly less potent than CGP 35348. Whether CGP 35348 is a true competitive antagonist of G A B A ~ receptors merits further consideration
(see Hills et al., 1991a,b; Seabrook et al., 1990). Although the dose-effect relationship of L-baclofen was shifted further rightward by increasing doses of CGP 35348 in the tail flick test, the magnitude of these shifts was not proportional to the dose of antagonist. Consequently, the slope of the regression line relating log (dose ratio - 1 ) to the negative log concentration of CGP 35348 in the tail flick test deviated significantly from the - 1 expected of a competitive antagonist. A similar finding was made in two studies of the effect of CGP 35348 on the actions of GABA B receptor agonists in isolated smooth muscle preparations (Hills et al., 1991a,b). In both the rat anococcygeus muscle and the rat vas deferens preparations, CGP 35348 produced parallel, rightward shifts in the dose-effect relationship of G A B A ~ receptor agonists (Hills et al., 1991a,b). However, as in the current study, the slope of the Schild regression line plot deviated significantly from - 1 leading Hills and colleagues to conclude that CGP 35348 was not a true competitive antagonist. There are several possible explanations for the shallow Schild slopes obtained in the present study when all three doses were used. The slope will deviate from unity if the agonist and antagonist have not reached equilibrium in the tissue at the time that measurements are made. This explanation is unlikely in the present study as both CGP 35348 and L-baclofen were examined at their respective times of peak effect. Another possible explanation is the existence of heterogenous receptor populations for which the agonist and antagonist have different relative affinities. At least two, and possibly three, subtypes of the G A B A ~ receptor are postulated to exist (see references in Bonanno and Raiteri, 1992). Both presynaptic and postsynaptic mechanisms have been invoked for the antinociceptive actions of baclofen in the spinal cord (see Aran and Hammond, 1991 and Hwang and Wilcox, 1989 for review). Similarly, CGP 35348 has significant affinity for both presynaptic and postsynaptic GABA~ receptors (Olpe et al., 1990). However, it is not known whether the relative affinities of L-baclofen and CGP 35348 for these G A B A B receptor subtypes are comparable. Finally, and of perhaps greatest likelihood, is the possibility that CGP 35348 has additional, unknown pharmacological properties at higher concentrations. Doses of CGP 35348 greater than 10 ~g produced no further rightward shift in the dose-effect curve of Lbaclofen in the tail flick test, suggesting that an additional action prevented or masked any further antagonism. Indeed, when the Schild analysis for the tail flick and hot plate tests was restricted to the two lowest doses of CGP 35348, the slope of the regression line was -0.91 and -1.14, respectively. These slopes are compatible with competitive antagonis~n. Thus, these data suggest that CGP 35348 is a competitive antagonist, albeit at restricted concentrations. Support for
261
this interpretation is found in the recent study by Seabrook and colleagues (1990). Using a rat brain slice preparation in which the drugs were applied in the bathing medium, these investigators obtained a Schild slope of - 0 . 9 9 and concluded that CGP 35348 was a competitive antagonist of the inhibitory effects of G A B A B receptor agonists on neurons in the substantia nigra or striatum. At present, it is unknown what additional properties are expressed by CGP 35348 at higher concentrations. However, activity as a partial agonist at G A B A ~ receptors is unlikely as i.t. injection of doses as high as 3 0 / z g CGP 35348 by itself did not increase either tail flick latency (baseline: 4.6 + 0.1 s; 15 min: 4.2 + 0.2 s; 30 rain: 4.6 + 0.3 s) or hot plate latency (baseline: 9.3 ___1.1 s; 15 rain: 9.3 _+ 1.6 s; 30 rain: 7.9 + 1.2 s) in the rat (McGowan and Hammond, in press). In summary, these results indicate that CGP 35348 is a potent and selective antagonist of GABAB receptors as determined by its ability to antagonize the antinociceptive and muscle relaxant effects of the G A B A 8 receptor agonist L-baclofen, but not those of the G A B A A receptor agonist isoguvacine. While CGP 35348 appears to be a competitive antagonist, the data suggest that the effective dose range may be limited and that additional, unknown pharmacological activities may become apparent at higher concentrations. Systemically or intracerebroventricularly administered CGP 35348 was previously reported to antagonize the antinociceptive and muscle-relaxant effects of systemically administered D,L-baclofen in the mouse or rat (Malcangio et al., 1991). The present results extend these observations to include the i.t. route of administration and spinal G A B A a receptors in the rat. CGP 35348 should prove very useful in future studies of the role of G A B A B receptors in a wide variety of spinal cord functions, such as nociception, autonomic regulation and motor behavior.
Acknowledgement This work was supported by U.S. Public Health Service Grant DA07004 from the National Institute on Drug Abuse
References Aran, S. and D.L. Hammond, 1991, Antagonism of baclofen-induced antinociception by intrathecal administration of phaclofen or 2-hydroxy-saclofen, but not 6-aminovaleric acid in the rat, J. Pharmacol. Exp. Ther. 257, 360. Bittiger, H., W. Froestl, R. Hall, G. Karlsson, K. Klebs, H.-R. Olpe, M.R. Pozza, M.W. Steinmann and H. Van Riezen, 1990, Biochemistry, electrophysiology and pharmacology of a new GABA B antagonist: CGP 35348, in: GABA B Receptors in Mammalian Function, eds. N.G. Bowery, H. Bittiger and H.-R. Olpe (Wiley and Sons, Chicester) p. 47.
Bonanno, G. and M. Raiteri, 1992, Functional evidence for multiple y-aminobutyric acid B receptor subtypes in the rat cerebral cortex, J. Pharmacol. Exp. Ther. 262, 114. Bowery, N.G., 1989, GABA B receptors and their significance in mammalian pharmacology, Trends Pharmacol. Sci. 10, 401. D'Amour, F.E. and D.L. Smith, 1941, A method for determining loss of pain sensation, J. Pharmacol. Exp. Ther. 72, 74. Drew, D.A., G.A.R. Johnston, D.I.B. Kerr and J. Ong, 1990, Inhibition of baclofen binding to rat cerebellar membranes by phaclofen, saclofen, 3-aminopropylphosphonic acid and related GABAB receptor antagonists, N e u r o s c i : ~ L 113, 107. Finney, D.J., 1964, Statistical Method in Biolog~al Assay (Hafnet Publishing Company, New York). Fromm, G.H., T. Shibuya, M. Nakata and C.F. Terrence, 1990, Effects of D-baclofen and L-baclofen on the trigeminal nucleus, Neuropharmacology 29, 249. Giuliani, S., S. Evangelista, F. Borsini and A. Meli, 1988, lntracerebroventricular phaclofen antagonizes baclofen antinociceptive activity in hot plate test with mice, Eur. J. Pharmacol. 154, 225. Hammond, D.L., 1988, Intrathecal administration: methodological considerations, Prog. Brain Res. 77, 313. Hammond, D.L. and M. Moy, 1992, Actions of 4-amino-3-(5methoxybenzo(b)furan-2-yl) butanoic acid and 4-amino-3benzo(b)furan-2-yl) butanoic acid in the rat spinal cord, Eur. J. Pharmacol. 229, 227. Hills, J.M., A.J. Sellers, J. Mistry, M. Broekman and W. Howson, 1991a, Phosphinic acid analogues of GABA are antagonists at the GABA B receptor in the rat anococcygeus, Br. J. Pharmacol, 102, 5. Hills, J.M., M.M. Larkin and W. Howson, 1991b, A comparison of the relative activities of a number of GABA B antagonists in the isolated vas deferens of the rat, Br. J. Pharmacol. 102, 631. Hong, Y. and J.L. Henry, 1991, Phaclofen-reversible effects of GABA in the spinal cord of the rat, Eur. J. Pharmacol. 201, 171. Hwang, A.S. and G.L. Wilcox, 1989, Baclofen, y-aminobutyric acidB receptors and substance P in the mouse spinal cord, J. Pharmacol. Exp. Ther. 248, 1026. Keppel, G., 1973, Design and Analysis: A Researcher's Handbook (Prentice-Hall, Englewood Cliffs) p. 167. Kerr, D.I.B., J. Ong and R.H. Prager, 1990, GABA B receptor agonists and antagonists, in: GABA B Receptors in Mammalian Function, eds. N.G. Bowery, H. Bittiger and H.-R. Olpe (Wiley and Sons, Chicester) p. 29. Malcangio, M., C. Ghelardini, A. Giotti, P. Malmberg-Aiello and A. Bartolini, 199l, CGP 35348, a new GABA B antagonist, prevents antinociception and muscle-relaxant effect induced by baclofen, Br. J. Pharmacol. 103, 1303. McGowan, M.K. and D.L. Hammond, Intrathecal GABA B antagonists attenuate the antinociception produced by microinjection of L-glutamate into the ventromedial medulla of the rat, Brain Res. (in press). Olpe, H-R., G. Karlsson, M.F. Pozza, F. Brugger, M. Steinmann, H. Van Riezen, G. Fagg, R.G. Hall, W. Froestl and H. Bittiger, 1990, CGP 35348: a centrally active blocker of GABAB receptors, Eur. J. Pharmacol. 187, 27. Rivlin, A.S. and C.H. Tator, 1977, Objective clinical assessment of motor function after experimental spinal cord injury in the rat, J. Neurosurg. 47, 577. Sawynok, J., 1986, Baclofen activates two distinct receptors in the rat spinal cord and guinea pig ileum, Neuropharmacology 25, 795. Schmid, K., G. Bohmer and K. Gebauer, 1989, GABA B receptor mediated effects on central respiratory system and their antagonism by phaclofen, Neurosci. Lett. 99, 305. Seabrook, G.R., W. Howson and M.G. Lacey, 1990, Electrophysiological characterization of potent agonists and antagonists at preand postsynaptic GABA B receptors on neurones in rat brain slices, Br. J. Pharmacol. 101,949.
262 Tallarida, R.J. and R.B. Murray, 1987, Manual of Pharmacologic Calculations (Springer Verlag, New York). Woolfe, G. and A.D. MacDonald, 1944, The evaluation of the analgesic action of pethidine hydrochloride (Demerol), J. Pharmacol. Exp. Ther. 80, 300.
WiJllner, U., T. Klockgether and K.-H. Sontag, 1989, Phaclofen antagonizes the depressant effect of baclofen on spinal reflex transmission in rats, Brain Res. 496, 341. Zar, J.H., 1974, Biostatistical Analysis (Prentice-Hall, Englewood Cliffs, N J).
255
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53018
Antagonism of L-baclofen-induced antinociception by CGP 35348 in the spinal cord of the rat D o n n a L. H a m m o n d a n d J o n a t h a n D. W a s h i n g t o n Department of Anesthesia and Critical Care, University of Chicago, Chicago, IL 60637, USA Received 5 October 1992, revised MS received 19 January 1993, accepted 26 January 1993
This study examined the potency and selectivity of the GABA B receptor antagonist CGP 35348 (3-amino-propyl(diethoxymethyl)phosphinic acid) in the spinal cord of the rat. Intrathecal (i.t.) administration of 3-30 /~g CGP 35348 produced a dose-dependent antagonism of the antinociception produced by i.t. administered L-baclofen. Increasing doses of CGP 35348 produced progressive, rightward parallel shifts in the dose-effect relationship of L-baclofen in both the tail flick and hot plate tests. However, in the tail flick test, the magnitude of the shift was not proportional to the dose of CGP 35348 such that doses greater than 10/zg i.t. produced no further antagonism. Schild analysis using all three doses of CGP 35348 yielded a slope of -0.45. However, if the Schild analysis was confined to the two lowest doses of CGP 35348 at which progressive shifts were obtained, a slope of -0.91 and an apparent pA 2 value of 9.3 were obtained. An apparent pA 2 value of 9.0 was also obtained in the hot plate test using the two lowest doses of CGP 35348. These data suggest that CGP 35348 is a competitive GABA B receptor antagonist at low concentrations. However, the failure to observe greater antagonism at higher doses of CGP 35348 suggests that this drug may exhibit additional properties at higher concentrations that can mask or prevent the occurrence of further antagonism. In contrast, the antinociception produced by i.t. administration of the GABA A receptor agonist isoguvacine was not antagonized by 30/xg i.t. CGP 35348, a dose that shifted the dose-effect relationship of L-baclofen 10-fold to the right. These data suggest that CGP 35348 is a potent and selective antagonist of spinal GABA B receptors in the rat. Baclofen; CGP 35348; Antinociception; Spinal cord; GABA B receptors 1. Introduction Since the identification of phaclofen, several competitive antagonists of the GABAB receptor have been synthesized. Like phaclofen, these compounds are structural analogs of baclofen having p A 2 values ranging between 4.0 and 5.3 in the isolated guinea pig ileum preparation and micromolar affinity for the G A B A B receptor in rat brain homogenates (Bowery, 1989; Drew et al., 1990; Kerr et al., 1990). These compounds are also effective antagonists of the G A B A a receptor agonist baclofen in vivo (Aran and Hammond, 1991; Giuliani et al., 1988; Hong and Henry, 1991; Schmid et al., 1989; Wiillner et al., 1989). However, as a class, these antagonists are not remarkably potent in either in vitro or in vivo preparations. More recently, a structurally distinct G A B A B receptor antagonist was synthesized (Bittiger et al., 1990; Olpe et al., 1990). This compound, 3-amino-propyl(diethoxymethyl)phosphinic acid (CGP 35348), is 10 to 30-fold
Correspondence to: D.L. Hammond, Department of Anesthesia and Critical Care, University of Chicago, 5841 South Maryland Avenue MC 4028, Chicago, IL 60637, USA. Tel. 1.312.702 5952, fax 1.312.702 3535.
more potent than phaclofen in the isolated rat vas deferens preparation (Hills et al., 1991b), in rat cortical synaptosomes (Bonanno and Raiteri, 1992), or when administered iontophoretically to rat cortical neurons (Olpe et al., 1990), and has a 3-fold higher affinity for the G A B A B receptor in rat brain homogenates (Olpe et al., 1990). As part of our continuing study of the role of spinal G A B A B receptors in the production of antinociception (Aran and Hammond, 1991; Hammond and Moy, 1992), we examined the ability of intrathecally (i.t.) administered CGP 35348 to antagonize the antinociception produced by i.t. injection of the G A B A B receptor agonist L-baclofen or the G A B A A receptor agonist isoguvacine in the rat. 2. Materials and methods
2.1. Surgical procedures Male Sprague-Dawley rats (Sasco, Madison, WI; 300-350 g) were anesthetized with halothane and implanted with indwelling i.t. catheters (Hammond, 1988). Animals were group housed with free access to food and water and allowed six to seven days to recover from surgery.
256
2.2. Analgesiometric procedures Thermal nociceptive threshold was measured using modifications of the tail flick ( D ' A m o u r and Smith, 1941) and hot plate (Woolfe and MacDonald, 1944) tests. In the tail flick test, a high intensity light b e a m was focused on the dorsal surface of the rat's blackened tail. The time interval between the application of the light b e a m and the reflex response was measured electronically to the nearest 0.1 s. The average of two such measurements was defined as the tail flick latency. Animals were removed from the apparatus in the absence of a response within 14 s and assigned this cut-off latency. After determination of tail flick latency, motor function was assessed using the righting reflex test and the inclined plane test (Rivlin and Tator, 1977). In the righting reflex test, the rat was placed on its back and allowed to right itself. The righting reflex was scored as follows: 0, unable to right itself; 0.5, rights u p p e r torso only; 1.0, rights upper and lower torso, but hindlegs abducted; 1.5, rights upper and lower torso with legs properly positioned under body; 2.0, brisk execution of reflex. In the inclined plane test, the rat was placed crosswise to the long axis of an inclined plane that had a rubber surface with horizontal ridges 0.1 cm in height. The initial angle of the inclined plane was 50 ° . The angle was then adjusted in 5 ° increments to determine the largest angle at which the rat was able to maintain its position on the plane. The hot plate test was conducted after the inclined plane test. Each animal was placed on a 55°C copper surface and the time taken to respond either by licking the hind paw or jumping was defined as the hot plate latency. Animals were removed from the hot plate in the absence of a response by 40 s and assigned this cut-off latency.
2.3. Experimental design The effect of p r e t r e a t m e n t with C G P 35348 on L-baclofen-induced antinociception was assessed in the first experiment. After baseline tail flick latency, hot plate latency and motor function were measured, either saline or C G P 35348 (3-30 /zg) was injected i.t. Five minutes later, tail flick latency, hot plate latency, and motor function were redetermined and L-baclofen (0.03-2.0 ~g) was then injected i.t. Response latencies and motor function were redetermined 15, 30, 45, 60 and 90 min later. The dose-response lines for L-baclofen in the absence or presence of the different doses of C G P 35348 were fit using least squares linear regression of the individual data. The resulting dose-response lines were tested for parallelism as described by Tallarida and Murray (1987). The EDs0, defined as the dose of drug that produced one-half the maximum possible
increase in response latency (i.e. to 9 s in the tail flick test and to 25 s in the hot plate test) was determined from the linear regression analysis; 95% confidence limits (CL) were determined by Fieller's theorem (Finney, 1964). Analysis of covariance was used to determine the significance of differences in EDs0s (Zar, 1974). Where increasing doses of C G P 35348 produced progressive, rightward shifts in the dose-response relationship of L-baclofen, the apparent pA 2 value of CGP 35348 was determined by the Schild method using the dose (in tool) administered (Tallarida and Murray, 1987). The effects of C G P 35348 pretreatment on baclofen-induced decrements in righting reflex and inclined plane angle were compared to that of saline p r e t r e a t m e n t using Fisher's exact test. The ability of C G P 35348 to reverse the effects of L-baclofen, and its time of peak effect were ascertained in another experiment. After measurement of baseline tail flick latency, hot plate latency and motor function, 0.3 /xg L-baclofen was injected i.t. Fifteen minutes later, response latencies and motor function were redetermined after which time (time zero) either saline or 30 /xg C G P 35348 was injected i.t. Response latencies and motor function were then redetermined 5, 15, 30 and 45 min later. A two-way A N O V A for repeated measures, in which one factor was drug treatment and the other factor was time, was used to analyze the data. This approach enabled comparison of post-injection latencies to latencies at time zero within each treatment group, as well as between treatment group comparisons of the effects of CGP 35348 to saline at individual time points. Post-hoc comparisons of mean values at individual time points were performed using Newman-Keuls test (Keppel, 1973). A final experiment was conducted to determine whether C G P 35348 retained its selectivity as a G A B A B receptor antagonist when administered i.t. After measurement of baseline tail flick latency, hot plate latency and motor function, 30 /xg of the G A B A a receptor agonist isoguvacine was injected i.t. Fifteen minutes later, response latencies and motor function were redetermined after which time either saline or 30 ~zg C G P 35348 was injected i.t. Response latencies and motor function were then redetermined 5, 15, 30 and 45 min later. The data were analyzed as described for the corresponding study with L-baclofen.
2.4. Drugs and injections Each animal was used only once. All drugs were dissolved in saline, the p H adjusted to 7, and injected i.t. in a volume of 10 /zl followed by 10 ~1 of saline. The injection was monitored by following the movement of an air bubble through polyethylene tubing over a period of 60 s. At the end of the experiment a laminectomy was performed and the patency and loca-
257
tion of the catheter tip was verified by injection of India ink. Animals in which the catheter leaked or was incorrectly placed were discarded from the study. LBaclofen was provided courtesy of Drs. Szeszak and Scheibli, while CGP 35348 was provided courtesy of Drs. Scholer and Maitre, all of Ciba-Geigy, Basel, Switzerland. Isoguvacine hydrochloride was purchased from Research Biochemicals, Inc. (Natick, MA).
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In the tail flick test, the EDs0 value (and 95% CL) of L-baclofen 15 rain after its injection in saline-pretreated rats was 0.14 (0.10-0.19) /xg i.t. (fig. 1A). Pretreatment with 3, 10 or 30 txg i.t. CGP 35348 produced a parallel, rightward shift in the dose-effect relationship of L-baclofen, increasing its EDs0 (and 95% CL) to 0.58 (0.42-0.82) txg, 1.44 (indeterminate) txg, and 1.32 (0.9-2.0) p~g i.t., respectively. These EDs0 values represented increases of 4.1, 10.3 and 9.4-fold and were significantly different from the EDs0 of Lbaclofen in saline-pretreated rats (P < 0.01 each dose). Thus, while the dose-effect relationship of L-baclofen was shifted further right as the dose of CGP 35348 was increased, the magnitude of the shift was not proportional to the dose of the antagonist. This finding was also apparent when the data were subjected to Schild analysis, which yielded a slope of - 0 . 4 5 . However, if the Schild analysis was restricted to the two lowest doses of CGP 35348 at which progressive shifts were obtained, the slope of the regression line was -0.91. Constraint of this slope to - 1 yielded an apparent pA 2 value of 9.3. In the hot plate test, the EDs0 value (and 95% CL) of L-baclofen 15 rain after its injection in saline-pretreated rats was 0.46 (0.32-0.91) Ixg i.t. (fig. 1B). Pretreatment with 3 or 10 ~ g CGP 35348 produced a parallel, rightward shift in the dose-effect relationship of L-baclofen, increasing its EDs0 value (and 95% CL) by 2- and 5-fold to 0.97 (0.76-1.32) Ixg and 2.5 (1.340.6) txg i.t., respectively. These increases were statistically significant (P < 0.01). A further rightward shift in the dose-effect relationship of L-baclofen occurred in rats pretreated with 30 /xg CGP 35348; however, an EDso value and 95% CL limits could not be determined. Although the effect of 30 /xg CGP 35348 appeared to be noncompetitive and associated with a decrease in maximal effect, limitations in the dose combinations that could feasibly be administered precluded such a conclusion. I.t. injection of 2.0 txg Lbaclofen in saline-treated rats produced profound flaccid paralysis (see below). Although 30 ~g CGP 35348
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3.1. Effects of i.t. pretreatment with CGP 35348 on baclofen-induced antinociception
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Dose L-Baclofen (#g) Fig. 1. Dose-response relationship of L-baclofen determined 15 min after its i.t. injection in rats pretreated with saline ( I ) or with 3 /xg (e), 10/~g ( • ) or 30 txg ( • ) i.t. CGP 35348 in the tail flick (A) or hot plate (B) test. Abscissas: Dose of L-baclofen in ,ag. Ordinates: response latency in s. Each point represents the mean of determinations made in 6 - l l rats. Lines of best fit were calculated using a least-squares linear regression. In the tail flick test, the dose-response relationships determined for L-baclofen in rats pretreated with 3, 10 or 30 # g CGP 35348 were parallel to that determined in saline-pretreated rats. in the hot plate test, only the dose-response relationships determined for L-baclofen in rats pretreated with 3 or 10 # g CGP 35348 were parallel to that determined in saline-pretreated rats.
was able to nearly completely antagonize this effect, it was not clear that it would also be able to completely antagonize the flaccidity produced by even higher doses of L-baclofen. In the absence of such a complete antagonism, any increase in hot plate latency produced by higher doses of L-baclofen in combination with 30 Ixg CGP 35348 could not be confidently interpreted as antinociception. For this reason, doses of L-baclofen that exceeded 2.0 /xg were not tested in combination with 30 # g CGP 35348. It is therefore not possible to state whether the antagonism produced by 30 ~g CGP 35348 was noncompetitive or simply represented the lower aspect of a dose-response relationship that was shifted right by more than 5-fold. Schild analysis using the two lowest doses of CGP 35348 yielded a regression line of slope -1.14. When this slope was con-
258 s t r a i n e d to - 1 , an a p p a r e n t p A 2 v a l u e o f 9.0 w a s obtained. T h e t i m e c o u r s e o f t h e a n t a g o n i s m o f 0.3 Ixg Lb a c l o f e n by p r e t r e a t m e n t w i t h v a r i o u s d o s e s o f C G P 35348 is i l l u s t r a t e d in fig. 2. I n b o t h t h e tail flick a n d h o t p l a t e tests, t h e a n t a g o n i s m by C G P 35348 was m a x i m a l w h e n first e x a m i n e d 15 m i n a f t e r t h e i n j e c t i o n o f L - b a c l o f e n , a t i m e t h a t c o r r e s p o n d e d to 20 m i n a f t e r t h e i n j e c t i o n o f C G P 35348. T h e a n t a g o n i s m p e r s i s t e d for at l e a s t 60 m i n .
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Fig. 3. Time course of the antagonism of L-baclofen by CGP 35348 in the tail flick (A) and hot plate (B) tests. Abscissas: time in min. Ordinates: response latency in s. Rats were injected first with 0.3/xg i.t. L-baclofen (left arrowhead) and followed 15 rain later (right arrowhead; time zero) by i.t. injection of either saline (11; n = 11) or 30/zg CGP 35348 (e; n = 9). Each point represents the mean +_S.E.M. Where not visible, the S.E.M. bar is encompassed by the symbol. Daggers indicate values in CGP 35348-treated rats that were significantly different from those in saline-treated rats at the corresponding time point (P < 0.01). Data from 9 of the 11 rats in the saline-treated group were obtained in the course of a previous, related study (Hammond and Moy, 1992), and were included here to minimize the number of animals used.
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Fig. 2. Time course of the effect of i.t. pretreatment with saline ( I ; n = 7) or with 3 /xg (e; n = 8), 10/xg (A; n = 8) or 30 izg (T; n = 7) CGP 35348 on the increase in tail flick (A) and hot plate (B) latency produced by 0.3 txg i.t. L-baclofen. Saline or CGP 35348 was injected 5 min before (indicated by left arrowhead) injection of L-baclofen (indicated by right arrowhead). Abscissas: time in min. Ordinates: response latency in s. Each point represents the mean+S.E.M. Where not visible, the S.E.M. bar is encompassed by the symbol. Values significantly different from those in saline-treated rats at the corresponding time point are indicated by asterisks (P < 0.05) or daggers (P < 0.01).
i s h e d by i.t. i n j e c t i o n o f s a l i n e at any t i m e (P > 0.05). H o w e v e r , tail flick l a t e n c y was s i g n i f i c a n t l y d e c r e a s e d w i t h i n 5 m i n a n d m a x i m a l l y d e c r e a s e d 15 m i n a f t e r t h e i.t. i n j e c t i o n o f 30 /xg C G P 35348 as c o m p a r e d to v a l u e s at t i m e z e r o in this t r e a t m e n t g r o u p . T h e a n t a g o n i s m p e r s i s t e d for at l e a s t a n a d d i t i o n a l 30 m i n . Comparison between treatment groups indicated that C G P 35348 also s i g n i f i c a n t l y r e d u c e d tail flick l a t e n c y as c o m p a r e d to v a l u e s in t h e s a l i n e - t r e a t e d g r o u p (P < 0.01) b e g i n n i n g 5 m i n a f t e r its i n j e c t i o n a n d c o n t i n u i n g for t h e d u r a t i o n o f t h e e x p e r i m e n t . H o t p l a t e l a t e n c y was m a x i m a l l y i n c r e a s e d w i t h i n 15 m i n o f t h e i n j e c t i o n o f 0 . 3 / x g i.t. L - b a c l o f e n (fig. 3B). Within session comparison of post-injection latencies to l a t e n c i e s at t i m e z e r o in t h e s a l i n e - t r e a t e d g r o u p i n d i c a t e d t h a t h o t p l a t e l a t e n c y was n o t d i m i n i s h e d by
259
i.t. injection of saline at any time (P > 0.05). However, hot plate latency was significantly decreased within 5 rain of the injection of 3 0 / z g C G P 35348 as compared to values at time zero in this treatment group. The antagonism persisted for the duration of the session. Comparison between treatment groups indicated that C G P 35348 also significantly reduced hot plate latency as compared to values in the saline-treated group (P < 0.01) beginning 5 min after its injection and continuing for the duration of the experiment.
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Doses of L-baclofen greater than 0.6 /zg i.t. decreased the angle at which rats could maintain their position on the inclined plane. I.t. injection of 2.0 /zg L-baclofen in four saline-pretreated rats decreased the inclined plane angle from a baseline value of 48.7 + 1.3 to 32.5 + 1.4 ° within 15 rain and to 31.3 + 1.3 ° by 30 min (fig. 4A). This reduction in angle was antagonized by the lowest dose of C G P 35348 (fig. 4A). Thus, the mean angle maintained on the inclined plane after injection of 2.0 ~ g i.t. L-baclofen in rats pretreated with 3 tzg i.t. C G P 35348 was 42.8 + 1.7 ° at 30 rain (P = 0.01). Higher doses of C G P 35348 did not produce
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3.4. Effects of i.t. pretreatment with CGP 35348 on baclofen-induced decrements in inclined plane angle and righting reflex
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3.3. Effects of i.t. posttreatment with CGP 35348 on isogut'acine-induced antinociception Fifteen minutes after i.t. injection of 30 /zg isoguvacine to one group of four rats, tail flick latency and hot plate latency were increased to 11.0_+ 2.8 and 27.1 + 7.5 s, respectively. These increases were therefore comparable to that produced by 0.3 ~ g L-baclofen. Neither the increase in tail flick latency nor the increase in hot plate latency was antagonized at any time after i.t. injection of 30 /xg C G P 35348 in these rats. For example, 15 min after the injection of C G P 35348, when maximal antagonism of L-baclofen had occurred, tail flick latency and hot plate latency remained elevated at 11.4 + 2.6 and 33.0 _+ 4.2 s, respectively. When injected in another group of four rats, 30 ~ g i.t. isoguvacine again increased tail flick latency and hot plate latency to the same extent as in the first group. These increases in tail flick latency and hot plate latency were similarly unaffected at any time after i.t. injection of saline. Thus, 15 rain after the injection of saline, tail flick latency and hot plate latency were 12.1 + 1.9 and 24.2 _+ 7.9 s, respectively. Comparison between treatment groups indicated that the increase in tail flick latency and hot plate latency produced by isoguvacine in rats treated with C G P 35348 did not differ from that of rats treated with saline (P > 0.4, both tests).
00
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Fig. 4. Plot of the individual data showing the effects of i.t. pretreatment with saline (indicated by 0) or 3, 10 or 30/zg CGP 35348 on the decrease in inclined plane angle (A) and righting reflex (B) produced 30 rain after i.t. injection of 2.0 tzg L-baclofen. Each circle represents the response of a single animal at that dose combination. 'SAL" represents individual data obtained in rats 30 rain after an i.t. injection of saline (no baclofen administered) and serves as a reference for righting reflex and inclined plane angle in untreated animals.
a more complete antagonism, with mean angles ranging from 44.5 + 0.5 to 46.1 + 1.1 ° in the 30 rain postinjection period. Doses of E-baclofen greater than 0.6 g g i.t. also interfered with the righting reflex. Although all four saline-pretreated rats had baseline righting reflex scores >/1.5, within 30 min of 2.0/zg i.t. L-baclofen three of these animals were unable to right themselves and were assigned a score of zero (fig. 4B). At this time all four rats exhibited flaccid paralysis of the hindquarters. Pretreatment with 3, 10 or 30 /zg i.t. C G P 35348 significantly antagonized the decrement in righting reflex (P < 0.02, all doses), with near complete antagonism observed in rats pretreated with the highest dose of C G P 35348 (fig. 4B). Thus, 30 rain after injection of 2.0 Izg L-baclofen in rats pretreated with 30 ~ g i.t. C G P 35348, four of nine rats had a righting reflex of 1.5 and the remaining five rats had a righting reflex of 1.
260
4. Discussion
1.t. administered CGP 35348 antagonized both the antinociceptive and the muscle relaxant effects of intrathecally administered L-baclofen in the rat. In the tail flick and hot plate tests, 3 and 10 /xg CGP 35348 produced dose-dependent and parallel shifts to the right of the dose-effect relationship of L-baclofen. This antagonism was surmountable. Importantly, CGP 35348 retained its selectivity for the GABAB receptor in the spinal cord. Neither the increase in tail flick latency nor the increase in hot plate latency produced by i.t. injection of the GABA A receptor agonist isoguvacine was antagonized by 30 ~g i.t. CGP 35348, a dose that increased the EDs0 of L-baclofen by 10-fold. This finding is consistent with the very poor potency of CGP 35348 at the G A B A A receptor (15% displacement at 1 raM; Olpe et al., 1990) and the results of electrophysiological investigations in which systemic or bath application of CGP 35348 did not antagonize the effects of the G A B A A receptor agonist 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP) on either cortical neurons or on spinal cord neurons in the rat (Olpe et al., 1990). Taken together, these results suggest that CGP 35348 is a selective antagonist of the GABA B receptor in the spinal cord of the rat. CGP 35348 is also the most potent available antagonist of spinal G A B A B receptors mediating antinociception. As little as 3 ~g (13 nmol) i.t. produced a 5-fold rightward shift in the dose-effect relationship of L-baclofen. By comparison, 30 ~g (120 nmol) i.t. phaclofen was required to increase the EDs0 of i.t. administered D,L-baclofen by 4-fold in these same tests (Aran and Hammond, 1991). The finding that CGP 35348 was 10-fold more potent than phaclofen in the spinal cord is consistent with the results of studies in the isolated rat vas deferens (Hills et al., 1991b), in rat cortical synaptosomes (Bonanno and Raiteri, 1992), and iontophoretic studies of rat cortical neurons (Olpe et al., 1990) in which CGP 35348 was 10 to 30 times more potent than phaclofen. CGP 35348 was also more potent than 2-hydroxy-saclofen or 4-amino-3-(5methoxybenzo(b)furan-2-yl) butanoic acid which, at respective doses of 10 p~g (37 nmol) i.t. and 30 p~g (120 nmol) i.t., produced only a 2- to 3-fold shift in the dose-effect relationship of D,L-baclofen (Aran and Hammond, 1991; Hammond and Moy, 1992). Finally, 20 nmol i.t. was the minimum effective dose of Dbaclofen, which is an antagonist of L-baclofen under certain conditions in the spinal cord (Fromm et al., 1990; Sawynok, 1986). Thus, although several G A B A ~ receptor antagonists have been shown to antagonize the antinociception produced by baclofen in the rat, they are uniformly less potent than CGP 35348. Whether CGP 35348 is a true competitive antagonist of G A B A ~ receptors merits further consideration
(see Hills et al., 1991a,b; Seabrook et al., 1990). Although the dose-effect relationship of L-baclofen was shifted further rightward by increasing doses of CGP 35348 in the tail flick test, the magnitude of these shifts was not proportional to the dose of antagonist. Consequently, the slope of the regression line relating log (dose ratio - 1 ) to the negative log concentration of CGP 35348 in the tail flick test deviated significantly from the - 1 expected of a competitive antagonist. A similar finding was made in two studies of the effect of CGP 35348 on the actions of GABA B receptor agonists in isolated smooth muscle preparations (Hills et al., 1991a,b). In both the rat anococcygeus muscle and the rat vas deferens preparations, CGP 35348 produced parallel, rightward shifts in the dose-effect relationship of G A B A ~ receptor agonists (Hills et al., 1991a,b). However, as in the current study, the slope of the Schild regression line plot deviated significantly from - 1 leading Hills and colleagues to conclude that CGP 35348 was not a true competitive antagonist. There are several possible explanations for the shallow Schild slopes obtained in the present study when all three doses were used. The slope will deviate from unity if the agonist and antagonist have not reached equilibrium in the tissue at the time that measurements are made. This explanation is unlikely in the present study as both CGP 35348 and L-baclofen were examined at their respective times of peak effect. Another possible explanation is the existence of heterogenous receptor populations for which the agonist and antagonist have different relative affinities. At least two, and possibly three, subtypes of the G A B A ~ receptor are postulated to exist (see references in Bonanno and Raiteri, 1992). Both presynaptic and postsynaptic mechanisms have been invoked for the antinociceptive actions of baclofen in the spinal cord (see Aran and Hammond, 1991 and Hwang and Wilcox, 1989 for review). Similarly, CGP 35348 has significant affinity for both presynaptic and postsynaptic GABA~ receptors (Olpe et al., 1990). However, it is not known whether the relative affinities of L-baclofen and CGP 35348 for these G A B A B receptor subtypes are comparable. Finally, and of perhaps greatest likelihood, is the possibility that CGP 35348 has additional, unknown pharmacological properties at higher concentrations. Doses of CGP 35348 greater than 10 ~g produced no further rightward shift in the dose-effect curve of Lbaclofen in the tail flick test, suggesting that an additional action prevented or masked any further antagonism. Indeed, when the Schild analysis for the tail flick and hot plate tests was restricted to the two lowest doses of CGP 35348, the slope of the regression line was -0.91 and -1.14, respectively. These slopes are compatible with competitive antagonis~n. Thus, these data suggest that CGP 35348 is a competitive antagonist, albeit at restricted concentrations. Support for
261
this interpretation is found in the recent study by Seabrook and colleagues (1990). Using a rat brain slice preparation in which the drugs were applied in the bathing medium, these investigators obtained a Schild slope of - 0 . 9 9 and concluded that CGP 35348 was a competitive antagonist of the inhibitory effects of G A B A B receptor agonists on neurons in the substantia nigra or striatum. At present, it is unknown what additional properties are expressed by CGP 35348 at higher concentrations. However, activity as a partial agonist at G A B A ~ receptors is unlikely as i.t. injection of doses as high as 3 0 / z g CGP 35348 by itself did not increase either tail flick latency (baseline: 4.6 + 0.1 s; 15 min: 4.2 + 0.2 s; 30 rain: 4.6 + 0.3 s) or hot plate latency (baseline: 9.3 ___1.1 s; 15 rain: 9.3 _+ 1.6 s; 30 rain: 7.9 + 1.2 s) in the rat (McGowan and Hammond, in press). In summary, these results indicate that CGP 35348 is a potent and selective antagonist of GABAB receptors as determined by its ability to antagonize the antinociceptive and muscle relaxant effects of the G A B A 8 receptor agonist L-baclofen, but not those of the G A B A A receptor agonist isoguvacine. While CGP 35348 appears to be a competitive antagonist, the data suggest that the effective dose range may be limited and that additional, unknown pharmacological activities may become apparent at higher concentrations. Systemically or intracerebroventricularly administered CGP 35348 was previously reported to antagonize the antinociceptive and muscle-relaxant effects of systemically administered D,L-baclofen in the mouse or rat (Malcangio et al., 1991). The present results extend these observations to include the i.t. route of administration and spinal G A B A a receptors in the rat. CGP 35348 should prove very useful in future studies of the role of G A B A B receptors in a wide variety of spinal cord functions, such as nociception, autonomic regulation and motor behavior.
Acknowledgement This work was supported by U.S. Public Health Service Grant DA07004 from the National Institute on Drug Abuse
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