Biochemical characterization of the interaction of three pyridazinyl-GABA derivatives with the GABAA receptor site

Brain Research. 384 (1986) 224-231

224

Elsevier BRE 12045

Biochemical Characterization of the Interaction of Three Pyridazinyl-GABA Derivatives with the GABA A Receptor Site MICHEL HEAULME l. JEAN-PIERRE CHAMBON I, ROGER LEYRIS I, JEAN-CHARLES MOLIMARD 1. CAMILLE G. WERMUTH z and KATHLEEN BIZIERE I

ISanofi Recherche, Department of Neurobiology, Montpellier and 2Laboratoire de Chimie Organique, Facult( de Pharmacie (UA 501, CNRS), Universit~ Louis Pasteur, Strasbourg, [llkirch-Graffenstaden (France) (Accepted 4 March 1986)

Key words: 7-Aminobutyric acid - - Bicucullinc - - SR 95103 - - SR 95531 - - SR 42641 - - GABA A receptor

An arylaminopyridazine derivative of 7-aminobutyric acid (GABA), SR 95103, has been shown to be a selective antagonist of GABA at the G A B A A receptor site. Subsequent structure-activity studies showed that suppressing the methyl in the 4-position of the pyridazine ring, and substituting the phenyl ring at the para position with a chlorine (SR 42641) or a methoxy group (SR 95531) ted to compounds which exhibited the highest affinities for the GABA receptor site in this series, In the present study we examined the biochemical interaction of these compounds with the GABA receptor as well as their biochemical selectivity for this receptor. SR 95531 and SR 42641 displaced [3H]GABA from rat brain membranes with apparent K i values of 0.15/~M and 0.28,uM respectively and Hit! numbers near 1.0. The two compounds antagonized the GABA-elicited enhancement of [3H]diazepam-binding in a concentration-dependent manner without affecting [3H]diazepam-binding per se. Scatchard and Lineweaver-Burk analysis of the interaction of the two compounds with the GABA A receptor sites, revealed that the compounds were competitive at the high affinity site, but non-competitive at the low affinity site. Neither compound interacted with other GABAergic processes or with a variety of central receptor sites. When administered intravenously, SR 95531 and SR 42641 elicited tonic-clonic seizures in mice. Based on these results, it is postulated that SR 95531 and SR 42641 are specific, potent and competitive GABA A antagonists.

ga suffructicosa 3 and S R 95103 is a synthetic G A B A

INTRODUCTION

d e r i v a t i v e s . B o t h c o m p o u n d s a p p e a r to be relatively R e c e p t o r a n t a g o n i s t s are essential for d e f i n i n g the pharmacological,

functional

and

structural

prop-

specific for the G A B A a r e c e p t o r : h o w e v e r securinine is s o m e w h a t w e a k e r than bicuculline in displac-

erties of n e u r o t r a n s m i t t e r r e c e p t o r s . A l t h o u g h a va-

ing tritiated G A B A

riety of d i f f e r e n t c h e m i c a l structures h a v e b e e n de-

w h e r e a s S R 95103 exhibits an a p p r o x i m a t e l y 20-fold

agonists 1"2,

g r e a t e r affinity t h a n bicuculline for the G A B A A re-

only a limited n u m b e r o f c o m p o u n d s h a v e b e e n rep o r t e d to be s e l e c t i v e G A B A A antagonists. A m o n g

ceptor ~ T h e c h e m i c a l s t r u c t u r e of S R 95103 (Fig. 1) is

these, bicuculline 1'1° is n o w g e n e r a l l y a c c e p t e d as a

c h a r a c t e r i z e d by a G A B A m o l e c u l e i n c o r p o r a t e d by

selective G A B A A a n t a g o n i s t , a l t h o u g h it has b e e n

its N - t e r m i n a l g r o u p to an a m i n o - p h e n y l - p y r i d a z i n e

criticized for not b e i n g p o t e n t e n o u g h 35 and has b e e n

scribed as y - a m i n o b u t y r i c acid ( G A B A )

f r o m its r e c e p t o r site m vitro 3.

r e p o r t e d to a n t a g o n i z e i n h i b i t i o n elicited by c o m -

h e t e r o c y c l e . P r e l i m i n a r y s t r u c t u r e - a c t i v i t y relationship studies h a v e s h o w n that for G A B A r e c e p t o r rec-

p o u n d s o t h e r t h a n G A B A ~'15. R e c e n t l y , on the basis

o g n i t i o n the G A B A

of n e u r o c h e m i c a l and e l e c t r o p h y s i o l o g i c a l results,

surprisingly the p h e n y l g r o u p in t h e 6-position o f the

side-chain ts essential and that

two n e w and c h e m i c a l l y d i f f e r e n t selective G A B A A

pyridazine ring also appears to play an i m p o r t a n t role.

receptor antagonists have been described.

Subsequent

Securi-

nine is an alkaloid e x t r a c t e d f r o m the s h r u b Securine-

structure-activity

relationship

studies

w e r e to s h o w that s u p p r e s s i n g the methyl in t h e 4-

Correspondence: K. Biziere. Sanofi Recherche, Department of Neurobiology, rue du Professeur J. Blayac 34082 Montpellier Cedex, France. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical I)ivision)

225

N

~

~

were kindly provided by Dr. P. Krogsgaard-Larsen (Royal Danish School of Pharmacy, Copenhagen, Denmark) and R 5135 by Dr. P. Hunt (RousselUCLAF, France).

OH

NH 2

SR 95103

H3CO~.~.....

Membrane preparation for GABA binding

oc,o o

o

NH 2

SR 4 2 6 4 1

NH2

SR

95531

Fig. 1. Chemical structure of the pyridazinyl-GABA derivatives examined in this study.

position of the pyridazine ring and parasubstituting the phenyl in the 6-position by a chlorine or a methoxy group (Fig. 1) led to compounds which exhibited the highest affinities for the G A B A receptor site in this series and which were respectively and approximately 250 and 150 times more potent than bicuculline in displacing tritiated G A B A from its receptor site~L However, suppressing the methyl on the pyridazine ring and introducing a substituent on the phenyl ring could have led to modifications of the pharmacological properties of the lead structure, SR 95103. We therefore examined the interaction of SR 95531 (2-[carboxy-Y-propyl]-3-amino-6-paramethoxy-phenyl-pyridazinium bromide) and of SR 42641 (2-[carboxy - 3' - propyl] - 3 - amino - 6 - parachloro - phenylpyridazinium chloride) with the G A B A receptor site and with a variety of other neurotransmitter and drug binding sites, in order to determine whether these compounds were, like the lead structure, selective competitive G A B A A antagonists. MATERIALS AND METHODS

Animals Male Sprague-Dawley rats (Charles River Breeding Laboratories, France) weighing 200-220 g were used.

Drugs Radiochemicals were purchased from New England Nuclear. All chemicals were purchased in the highest grade available from commercial sources. SR 95103, SR 95531 and SR 42641 were prepared according to Chambon et al. 8. Iso-THAZ and THIP

Rats were stunned, decapitated and their brains were rapidly removed. A crude synaptic membrane fraction was prepared as described by Enna and SnydeP 1. In brief, whole brains were homogenized in 20 ml of ice-cold 0.32 sucrose using a PT 10 Polytron homogenizer. The homogenate was centrifuged (1000 g, 4 °C, 10 rain), the supernatant was collected and recentrifuged (20000 g, 4 °C, 20 rain). The resulting pellet was resuspended in 20 ml of ice-cold distilled water and homogenized. The homogenate was centrifuged (8000 g, 4 °C, 20 min), the supernatant was collected, recentrifuged (48,000 g, 4 °C, 20 rain) and the final crude synaptic membrane pellet was frozen for at least 18 h. The pellet was then resuspended in 30 ml Tris-citrate 0.05 M buffer, pH 7.1, containing a 0.05% solution of Triton X-100 and incubated at 37 °C for 30 rain. After centrifugation (48,000 g, 4 °C, 10 rain), the pellet was resuspended in 4 ml of Tris-citrate 0.05 M buffer, pH 7.1.

Standard [~H]GA BA-binding assay [3H]GABA-binding was performed as previously described by Enna and Snyder 11. In brief, 200ul aliquots of synaptic membrane suspensions (1 mg protein) were transferred to 5 ml plastic tubes containing 1.6 ml of Tris-citrate buffer (0.05 M, pH 7.1) and [3H]GABA (2.9 nM final concentration; 83 Ci/mmol spec. act.). Varying concentrations of test compounds dissolved in distilled water were then added to each tube in a volume of 200 ul. The final mixture was incubated for 5 min at 4 °C and the assay terminated by filtration through Whatman GF/C filters. After washing with 10 ml of ice-cold distilled water, filters were placed in scintillation vials and allowed to dry overnight; 10 ml of scintillation fluid (Biofluor, New England Nuclear) were then added and radioactivity was determined in a Kontron Betamatic liquid scintillation counter. Specific binding of [3H]GABA was defined as the difference between total binding in the presence of radioligand alone and non-specific binding in the presence of 10uM muscimol.

226 The effects of ammonium thiocyanate on the ability of the pyridazinyl-GABA derivatives to displace [3H]GABA from its receptor sites were examined as described by Enna and Snyder 12. The saturation isotherm of [3H]GABA binding to this membrane preparation was determined for concentrations of [3H]GABA ranging from 0.5 to t50 nM. Data were analyzed by a non-linear regression computer program. Dose-inhibition curves were generated with 4-7 concentrations of drug in triplicate incubations.

Antagonism of the GABA-mediated enhancement of [3H]diazepam-binding The effects of the pyridazinyl-GABA derivatives and of bicuculline on the GABA-elicited enhancement of [3H]diazepam-binding in rat brain membranes was evaluated according to Fujimoto and Okabayashi 13. In brief, rats were stunned and decapitated; whole brains (minus cerebellum) were removed and homogenized in 20 ml ice-cold 0.32 M sucrose, using a PT 10 Polytron homogenizer. The homogenate was centrifuged (1000 g, 4 °C, 10 min), the supernatant was collected and recentrifuged (30,000 g, 4 °C, 20 min). The resulting pellet was resuspended in ice-cold distilled water, centrifuged (8000 g, 4 °C, 20 min), the supernatant was collected and recentrifuged (48,000 g, 4 °C, 15 min). The final crude synaptic membrane pellet was resuspended in 10 vols. of 50 mM Tris-citrate buffer (pH 7.4) and repelleted twice, resuspended in 5 ml of Tris-citrate buffer and frozen for at least 18 h. Before use, the suspension was thawed, diluted in 25 ml of 50mM Tris-citrate buffer (pH 7.4) and centrifuged (30,000 g, 4 °C, 20 min). The final pellet was resuspended in 25 ml of 50 mM Tris-citrate buffer (pH 7.4). For the binding assays 100~! aliquots of membrane suspension (0.4 mg protein) were transferred to 5 ml plastic tubes containing 400 ~1 of Tris-citrate buffer (50 mM, pH 7.4), [3H]diazepam (1.9 nM final concentration, 72 Ci/mmol spec. act.) and unlabelled G A B A (10/~M final concentration). Various concentrations of test compounds were then added to each tube in a Vol. of 100 bd. The final mixture was incubated for 50 min at 4 °C. The assay was terminated by the addition of 3 ml of ice-cold Tris-citrate buffer and filtration through Whatman GF/C glass fiber fil-

ters. The filters were washed with 6 ml of ice-cold buffer. After washing, bound radioactivity was measured in a liquid scintillation counter (Kontron Betamatic). Non-stimulated binding w,~s determined in the absence of GABA. Non-specific binding was determined in the presence of 100uM unlabelled diazepam.

Other ligand-receptor interactions The possible effects of the pyridazinyl-GABA derivatives on the specific binding of [3H]baclofen (30 nM, 37 Ci/mmol) to GABAB rat whole brain receptors v, [3H]strychnine (6 riM, 15 Ci/mmol) to glycine rat pons medulla receptors 36, L-13H]glutamate (3 nM, 53 Ci/mmol) to rat glutamate frontal cortex receptors 5, [3H]diazepam (1,9 nM, 72 Cifmmol) to rat brain benzodiazepine receptors 29, [3H]quinuclidinyl benzilate (QNB, 0.2 nM, 42 Ci/mmol) to rat cortical muscarinic receptors z°, [3H]WB 4101 (0.2 nM, 26 Ci/mmol) to rat brain cq-adrenergic receptors 32, [3H]serotonin (2 nM, 30 Ci/mmol) to rat hippocampal serotonin receptors 24, [3H]spiroperidol (0.4 nM, 24 Ci/mmol) to rat striatal dopamine 2 (D2) receptors and cortical serotonin 2 (5-HT 2) receptors 9, [35S]tbutylbicyclophosphorothionate (TBPS, 2 nM, 30 Ci/mmol) to rat forebrain TBPS binding sites27 were also examined. Interaction with other GA BA recognition sites Rat brain L-glutamic acid decarboxylase (EC 4.1. I. 15) activity was measured according to Tappaz et al. 31. GABA-a-ketoglutarate transaminase (EC 2.6.1.19) and succinyl-semialdehyde dehydrogenase (EC 1.2.1.24) activities were measured according to Jung et al. TM. In vitro Na+-dependent synaptosomal GABA uptake was measured as described by Ramsay et al. 2s. Con vulsant effects The convulsant effects of SR 95103, SR 95531 and SR 42641 were compared to those of (_+_)bicuculline in female Swiss albino CD1 mice (Charles River, France). Compounds were administered i.v. to groups of 10 mice and animals were observed up to 3 h following drug administration. The CDs0 (95% confidence limit), the dose at which~50% of the animals exhibited seizures, was calculated by probit analysis.

227 RESULTS

TABLE I

Inhibition of specific [3H]GABA binding

GA BA receptoraffinities of pyridazinyl-GABA derivativesand referencecompounds

As previously r e p o r t e d , SR 95103 exhibited an affinity in the micromolar range for the G A B A A receptor with an a p p a r e n t Kg value of 2.2BM (Fig. 2). Both the para-methoxy and the para-chloro substituted derivatives of SR 95103 displaced [ 3 H ] G A B A from rat brain m e m b r a n e s with a mass-action type of kinetics (Fig. 2) and exhibited an approximately 10fold greater affinity for the G A B A A receptor than SR 95103 (Table 1). Bicuculline and i s o - T H A Z were weaker inhibitors of [ 3 H ] G A B A binding than all 3 p y r i d a z i n y l - G A B A derivatives tested in this study (Fig. 2, Table I), whereas R 5135 revealed an approximately 5-fold greater affinity for the G A B A receptor than SR 42641 and SR 95531. Unlike what has previously been described for other G A B A A antagonists 12'21, 50 m M a m m o n i u m thiocyanate did not modify the affinity of the 3 pyridazin y I - G A B A derivatives examined in this study for the G A B A A receptor (results not shown).

Antagonism of" the GABA-mediated enhancement of F H]diazepam-binding It has previously been r e p o r t e d that G A B A (10 u M ) enhances [3H]diazepam-binding in rat synaptosomal m e m b r a n e s in vitro and that this effect can be antagonized bv G A B A antagonists 19'3°, In our expe-

Dose-inhibition curves were generated with 4-7 concentrations of drug in triplicate incubations. ICs0-values were calculated by log-probit analysis, Ki values were calculated using the equation Ki = IC~o/[l+(C/Kd) j, in which C - concentration of [~H]GABA (2.9 nM) and K,t = dissociation constant for dissociation of GABA from the high-affinity receptor site (3(}nM).

Compound

KiJbr inhibition of [3H]GA BA binding (IzM)

GABA Bicuculline ISO-THAZ R 5135 SR 95103 SR 42641 SR 95531

O.O27 38 5O 0.047 22 0.28 0.15

70% increase of [3H]diazepam-binding, whereas none of the p y r i d a z i n y l - G A B A derivatives (100/,M) affected [3H]diazepam-binding. Like bicuculline, all 3 p y r i d a z i n y l - G A B A derivatives antagonized the G A B A - e l i c i t e d enhancement of [3H]diazepam-binding in a c o n c e n t r a t i o n - d e p e n d e n t fashion (Fig. 3). The p a r a - m e t h o x y and the para-chloro substituted derivatives of SR 95103 were a p p r o x i m a t e l y and respectively 25 and 45 times more potent than SR 95103 itself. (Fig. 3).

rimental conditions G A B A (10 uM) induced a mean 7~

=

100

, :lb,4p(~l "q[~

E 100

I

-

It}',

h J~ll I '

[


\

1'(4

: 1

•

o ':

802Z ~

<~

)')

E-

Z'5

£ & 0

8

7

!

::

log I d r u g ¢P,,~)]

0

.

.

.

.

-9

.

.

.

8

.

.

,

7

6 --

. . . .

--4 --3

log ldrug (M)]

Fig. 2. Displacement curves for bicuculline and for the pyridazinyl-GABA derivatives versus [3H]GABA-binding (at, SR 95531; I , SR 42641; [Z], SR 95103; O, bicuculline). All incubations were performed in triplicate.

Fig. 3. Antagonism by bicuculline and by the pyridazinylGABA derivatives of the GABA-mediated enhancement of [3H]diazepam-binding in vitro. Enhancement by GABA 10~uM (control) was normalized to 100%. GABA antagonists were added at varying concentrations at the time of the incubation. Results are the means of 3 experiments carried out in triplicate. IC50 values were defined as the concentration of compound which inhibits 50% of the increase of [3H]diazepam elicited by GABA 10~M.

228 those previously reported in the literature :~'-'~34. Fig. t-

x

500

1o

~ 250

5o

4 represents two Scatchard plots obtained, one in the

0.01

,?

E

absence and the other in the presence of SR 95531 (0.1 ~tM). The apparent K,~ for the high affinity site

x 1

2

3

02

ol

significantly increased in the presence of SR 95531.

03

whereas Bmax was not affected, in contrast, the apparent K d of the low affinity site was not modified in

o.oo

the presence of SR 95531, whereas Bma~ was significantly decreased. Similar results were obtained with

c0

SR 95103 and SR 42641 and are given in Table II. 0.1

0.2

0.3

0.4

0.5

0.6

Thus the antagonism of G A B A binding appeared to be competitive at the high affinity site, but non-com-

Bound (pmol./mg prot.)

petitive at the low affinity site.

Fig. 4. Scatchard plot analysis of [3H]GABA saturation isotherm. Membranes were incubated with [3H]GABA at concentrations varying from 0.5 to 150 nM. Specific binding was measured in the absence ( ~ ) or in the presence (A) of SR 95531 0.1 #M. Plots represent the results obtained from one experiment, individual data points were determined in triplicate. Identical results were obtained from two different experiments. Inset: Lineweaver-Burk analysis of the same data (A: high affinity bindingsites of [3H]GABA; B: low affinity binding sites of [3H]GABA).

A double-reciprocal plot analysis of the saturation isotherm of [3H]GABA binding in the presence of SR 95531 (Fig. 4) confirmed a competitive type interaction of SR 95531 with the high affinity G A B A receptor site and a non-competitive type interaction with , the low affinity binding site. Similar results were observed with SR 95103. SR 42641 and bicuculline (results not shown). Hill plots of the saturation isotherms of [~H]G A B A - b i n d i n g in the presence of SR 95531 re-

Kinetic analysis o f the interaction at the G A B A A receptor site W h e n the saturation isotherms for [3H]GABA

vealed slopes not significantly different from 1.0. Fig. 5 represents a Hill plot for the data from Fig. 4; the

were subjected to Scatchard analysis (Fig. 4), two separate binding sites were revealed: a high affinity

slopes of 1.114 and 1.0, for the high and low affinity G A B A - b i n d i n g sites respectively, indicate the absence of co-operativity. Similar results were obtained

site (Kdl: 3.4 nM; Bmaxl: 0.03 pmol/mg protein) and a low affinity site (Kd2:106.4 nM, Bmax2:0.65 pmol/mg protein). These results are in good agreement with

with SR 95103 and SR 42641 (Table II).

TABLE II Kinetic parameters of [SH]GABA binding in the absence or in the presence of pyridazinyl-GABA derivatives K d and Bmaxvalues were determined by Lineweaver-Burk analysis of saturation isotherms using 7 concentrations of [!H]GABA (ranging from 0.5 to 150 nM). The same data were used to obtain Hill coefficients (nil). Results are the mean _+S.E.MI Of 3 experiments. Compound

High affinity site

Low affini O,site

Kal (nM)

B,,,~ z (pmol/ mg protein)

ntgl

Control SR 95103 101~M

3.2 ± 0.3 7.1 ± 0.6**

0.01 ± 0.002 0.01 + 0.003

1.00 ± 0.31) 1.04 + 0.12

Control SR 426411/~M

4.2 _+0.7 6.2_+0.8*

0.04 _+0.003 0.03_+0.003

Control SR 95531 0.1 #M

2.5 ± 0.7 4.5_+0.4**

Control Bicuculline 50pM

5.5 +__0.7 7.2 _+0.5*

B,na~: (pmoll mg protein)

nil2

100 -+ 10 105 -+ 17

//.25 -+ 0.034 0.10 _+I).022"*

1.00 ± 0.12 1.00 _+0.13

1.03 _+0.11 1.00-+0.07

100 +__6 100 ± 4

0.66 __+0.05t) 0.40 __+0.037*

1.03 +- 0.117 1.03 ± 11.17

0.03 ± 0.002 0.03_+0.003

1.00 _+0.09 1.00 ± 0.0l

100 +_ l 100 _+4

1/.60 -+ 0.04 0.40 _+0.03**

1.00 +_0.03 t.00 _+0.15

0.04 + 0.004 0.05 + 0.004

0.96 + 0.07 1.00 + 0.17

83.3 -+ 6 83.3 ± 6

0.58 + 0.04 0.50 ± 0.035*

1.00 + 0.15 1.00 + (MI9

*P < 0.05 and **P < 0.01, F-test (analysis of variance and covariance).

Kd2 (nM)

229 Log C

Log C

DISCUSSION

m HIGH AFFINITY BINDING SITES

served with bicuculline. As shown in Table 1II, SR 42641 and SR 95531 were 10 times more potent than SR 95103 in inducing seizures, but were 20 times less potent than bicuculline.

~m

Fig. 5. Hill plots of specific binding of [~H]GABA in the absence (A) or in the presence (at) of SR 95531 0.1 ruM. The data for the saturation isotherm of [3H]GABA in Fig. 4 were subjected to Hill plot analysis. B represents the concentration of [3H]GABA specifically bound (pmol/mg protein), Bm~x is the maximal number of specific binding sites for [3H]GABA, C is the concentration (nM) of the displacing agent.

Specificity of action As the pyridazinyl-GABA derivatives tested in this study all carry a G A B A side chain, we suspected they could interact with other G A B A recognition sites. However, the 3 pyridazinyl-GABA derivatives (100 /~M) did not inhibit Na+-dependent synaptosomal uptake and did not affect GABA-T, G A D and SSADH activities. Similarly, these compounds did not interact with other sites of the macromolecular G A B A receptor complex, such as the benzodiazepine and the TBPS sites, and did not affect [3H]baclofen binding to the G A B A B receptor site. Finally, the 3 pyridazinyl-GABA derivatives examined in this study did not interact with a variety of central receptors, notably the strychnine, the glutamate, the dopaminergic D2, the serotoninergic 5-HT1 and 5-HT> the aj-noradrenergic and the cholinergic muscarinic binding sites.

Convulsant effects In mice, all 3 pyridazinyl-GABA derivatives elicited seizures which were comparable to those obTABLE III Con vulsant effects of pyridazinyl- GA BA derivatives in mice Compound

Clonic seizures CDso (mg/kg i. v. )

SR 95103 SR 42641 SR95531 Bicuculline

139 15 10 0.6

(101-192) (9-23) (8-12) (0.5-0.7)

The results of the present study show that both the para-methoxy-phenyl and the para-chloro-phenyl derivatives of SR 95103 appear to be potent, selective and competitive antagonists for G A B A A recognition sites on mammalian central neurons. Both compounds are ca. 200 times more potent than bicuculline and 10 times more potent than SR 95103 in displacing tritiated G A B A from its specific binding sites in rat brain membranes. Both compounds antagonize GABA-mediated enhancement of diazepam binding, a biochemical measure of G A B A receptor activity. In vivo, all 3 pyridazinyl-GABA derivatives induce seizures in mice, with a rank order of potency which is similar to their affinity for the G A B A receptor in vitro. Bicuculline is considerably more potent than all 3 pyridazinyl-GABA derivatives in eliciting seizures in mice, although it exhibits a weaker affinity for the G A B A receptor. In contrast, in vivo microiontophoretic studies in the rat cuneate nucleus have shown that SR 95531 and SR 42641 antagonize GABA-elicited responses with a potency comparable to that of bicuculline 22. Taken together these results suggest that the pyridazinyl-GABA derivatives examined in this study do not readily cross the bloodbrain barrier; this could be due to the highly ionized nature of the exo-endo amidinic function 33. Scatchard and Lineweaver-Burk analysis of the molecular interaction of the pyridazinyl-GABA derivatives with the G A B A a receptor sites, on the basis of the two sites model classically described 23,2<34, revealed that all 3 compounds were competitive at the high affinity site, but non-competitive at the low affinity site. This type of interaction is identical to that classically described for bicuculline 1:. These results are in good agreement with the electrophysiological results obtained with SR 95103, showing that this compound competitively antagonizes GABA-induced responses in the rat spinal root ganglia s. Finally Hill analysis of dose-response curves yielded Hill coefficients of 1, indicating the absence of positive or negative co-operative interaction.

230 Unlike what has previously been described for G A B A A antagonists 3'~2'21, the affinity for the G A B A A receptor of the 3 pyridazinyl-GABA derivatives was not increased in the presence of ammonium thiocyanate. This could be due to the fact that, unlike all other G A B A A antagonists, these compounds contain in their structure a G A B A sequence terminated by an acidic function. Both the para-methoxy-phenyl and the parachloro-phenyl derivatives of SR 95103 appear to be selective for G A B A A receptors, since neither compound had any significant effect on a variety of brain neurotransmitter or drug-binding sites and notably did not interact with the G A B A B, the benzodiazepine, the glutamate or the strychnine-binding sites. The selectivity of these compounds for the G A B A A receptor has been further confirmed by microiontophoretic studies in the rat cuneate nucleus, showing that these compounds do not affect glycine-induced responses 22. Finally, although these compounds carry a G A B A side chain, they did not affect high affinity G A B A uptake and they did not interact with the enzymes involved in G A B A synthesis or degradation. Thus, suppressing the methyl in the 4-position of the pyridazine ring of SR 95103 and para-substituting the phenyl ring with a methoxy group or a chlor-

ine did not seem to lead to a loss of selectivity for the G A B A A receptor site. Other G A B A A antagonists have recently been described. Securinine appears to be selective for G A B A A receptors, but is somewhatweaker than bicuculline 3. I s o - T H A Z is a weaker inhibitor of G A B A binding than bicuculline and also blocks neuronal inhibition elicited by glycine 2. R 5135 and pitrazepine exhibit a strong affinity for the G A B A A receptor, but lack specificity since R 5135 strongly interacts with benzodiazepine and glycine receptors 16 and pitrazepine interacts with benzodiazepine receptors I~. Thus the pyridazinyl-GABA derivatives described in this study present the advantage of being both potent and apparently selective antagonists for G A B A A receptor sites. A frequent limitation to the use of bicucul[ine as a G A B A receptor antagonist is that it is relatively insoluble in water and unstable at physiological p H 2~, although both shortcomings can be circumvented by the use of more soluble and more stable quaternary salts 35. In contrast the pyridazinyl-GABA derivatives described in the present study are water soluble and stable in a wide range of pH: Thus these compounds should be useful substances to further characterize G A B A receptor sites.

REFERENCES

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