Characterisation of [3H]gabapentin binding to a novel site in rat brain: homogenate binding studies

European Journal of Pharmacology - Molecular Pharmacology Section, 244 (1993) 293-301 ~3 1993 Elsevier Science Publishers B.V. All rights reserved 0922-4106/93/$06.00

293

EJPMOL 90408

Characterisation of [3H]gabapentin binding to a novel site in rat brain: homogenate binding studies N i r m a l a S u m a n - C h a u h a n , L o u i s e W e b d a l e , D a v i d R. Hill a n d G e o f f r e y N. W o o d r u f f Parke-Dat:is Neuroscience Researeh Centre, Addenbrookes ttospital Site, Cambridge CB2 2QB, UK Received 25 September 1992, revised MS received 23 October 1992, accepted 3 November 1992

The binding characteristics of [3H]gabapentin, the radiolabelled analogue of the novel anticonvulsant gabapentin (l(aminomethyl)cyclohexaneacetic acid) were studied using purified synaptic plasma membranes prepared from rat cerebral cortex. In 10 mM HEPES buffer [3H]gabapentin bound to a single population of sites with high affinity (K o = 38 _+ 2.8 riM) with a maximum binding capacity of 4.6 + 0.4 pmol/mg protein, reaching equilibrium after 30 rain at 20°C. This novel site was unique to the central nervous system with little or no specific [3H]gabapentin binding being measurable in a range of peripheral tissues. Binding was potently inhibited by a range of gabapentin analogues and 3-alkyl substituted y-aminobutyric acid (GABA) derivatives although GABA itself and the selective GABA B receptor ligand baclofen, were only weakly active. Gabapentin itself (ICs0 = 80 nM) and 3-isobutyl GABA (ICs0 = 8(1 nM) which also has anticonvulsant properties, showed the highest affinity for the binding site. Of a wide range of other pharmacologically active compounds only the polyamines spermine and spermidine influenced [3H]gabapentin binding, with both compounds producing a maximum of 50% inhibition of specific binding. Magnesium ions produced a similar pattern of inhibition but the effect of the polyamines and magnesium ions were not additive. The data provide evidence for the existence in brain of a novel binding site that may mediate the anticonvulsant effects of gabapentin and other potential anticonvulsant compounds. Gabapentin; GABA (y-aminobutyric acid); Baclofen; G A B A A receptors; GABA u receptors; NMDA (N-methyI-D-aspartate); Glycine; Anticonvulsants: Polyamines

1. Introduction Gabapentin (1-(aminomethyl)cyclohexaneacetic acid) is a novel anticonvulsant with an unknown mechanism of action that has recently been shown to be orally effective in a number of animal models of epilepsy, including maximal electroshock in rats and pentylentetrazol or audiogenic-induced seizures in mice (Bartoszyk et al., 1986; Dooley et al., 1986; Chadwick, 1992). Gabapentin is also effective in decreasing the frequency of seizures in medically refractory patients with partial or generalised epilepsy (Crawford et al., 1987; Wieser, 1989; Chadwick, 1992). Although originally synthesized as a lipophilic analogue of the inhibitory amino acid transmitter 7aminobutyric acid (GABA) capable of penetrating the blood-brain barrier, gabapentin does not possess high affinity for either G A B A A or G A B A B receptors

Correspondence to: David R. Hill, Parke-Davis Neuroscience Research Centre, Addenbrookes Hospital Site, Hills Road, Cambridge CB2 2QB, UK. Tel. (223) 210929; Fax (223) 249106.

(Bartoszyk and Reimann, 1985, and this study). Consistent with this is the finding that gabapentin does not affect either the response to iontopheretically applied G A B A in cultured mouse spinal cord neurones (Taylor et al., 1988) or the effects of chloride and potassium channel activation in the hippocampus (Haas and Wieser, 1986). Recent in vitro electrophysiological and in vivo behavioural studies have suggested the involvement of the excitatory amino acid system, and in particular the N-methyl-D-aspartate (NMDA) receptor complex in the actions of gabapentin. Thus, in cultured rat striatal neurons, gabapentin antagonised the glycine-induced potentiation of NMDA-evoked whole-cell currents (K~ = 47/zM), an effect which was fully reversed by the glycine receptor agonist D-serine (Sprosen, 1990). Further, in the absence of exogenous glycine, gabapentin potentiated NMDA-evoked currents in its own right. These results led to the suggestion that gabapentin may act as a partial agonist at the glycine modulatory site of the N M D A receptor complex (Johnson and Ascher, 1987; Sprosen, 1990). Such compounds have been shown to act as anticonvulsants (Singh et al.,

294

1990). Furthermore, other studies have shown that intraperitoneal administration of gabapentin 30 rain prior to seizure induction with pentylenetetrazol or semicarbazide, provided protection against tonic seizures (Oles et al., 1990). This effect of gabapentin could be reversed by i.c.v, administration of D-serine, again supporting the notion that gabapentin may be acting at the glycine modulatory site of the NMDA receptor complex. Despite these apparently clear indications of an interaction at the strychnine-insensitive glycine site, other data obtained in our laboratories indicate that gabapentin does not inhibit strychnine-insensitive [3H]glycine binding to brain membranes, nor does it influence the binding of [3H]MK-801 ([3H](5R, 10S)( + )-5-methyl- 10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine; dizocilpine) or [3H]TCP ([3H]I-[1-(2thienyl)cyclohexyl]piperidine) to the NMDA receptor channel, as would be expected of a glycine partial agonist (Wong et al., 1987, data not shown). Neither does gabapentin influence the development of longterm potentiation in hippocampal slice preparations (Taylor et al., 1988). In order to investigate the site of action of gabapentin, we have developed an in vitro assay to measure [3H]gabapentin binding in brain homogenates. The results of these experiments, which are in contrast to previous reports suggesting no specific binding sites for [3H]gabapentin (Dooley et al., 1986), are described in this report.

2. Materials and methods

2.1. Tissue preparation 2. l. 1. Brain tissue Partially purified synaptic plasma membranes were prepared from rat cortex using sucrose density gradients. The combined cerebral cortex from 10 rats was homogenized in 10 volumes (w/v) of ice-cold 0.32 M sucrose in 5 mM Tris-acetate (pH 7.4) using a glass homogenizer fitted with a motorized Teflon pestle (10-15 strokes at 200 rpm). The homogenate was centrifuged at 1000 x g for 10 min and the supernatant collected and kept on ice. The pellet (P1) was rehomogenized in 20 ml of Tris-sucrose and the homogenate recentrifuged as above. The combined supernatants were centrifuged at 21,500 × g for 20 min. The pellet (P2) was resuspended in 20 volumes (with respect to original wet weight) of 5 mM Tris-acetate, pH 8, stirred on ice for 1 h and the suspension was then centrifuged at 40,000 x g for 30 rain. The resulting pellet was resuspended in 90 ml of 1.2 M Trissucrose and 15 ml of this mixture was added to ultracentrifuge tubes. On top of this, 10 ml of 0.9 M sucrose

were layered followed by a final layer of 5 mM Trisacetate pH 8. The tubes were then centrifuged at 100,000 x g for 90 min. The synaptic plasma membranes located at the 0.9/1.2 M sucrose interface were collected, resuspended in 50 ml 5 mM Tris-acetate, pH 7.4, and centrifuged at 40,000 x g. The final pellet was resuspended in 50 ml of Tris-acetate, pH 7.4, aliquoted and then frozen until use. On the day of use the tissue was thawed, centrifuged at 40,000 x g and then resuspended in 10 mM HEPES for the assay. Membranes that were assayed in parallel with peripheral tissues in the tissue distribution experiments were prepared as above but were not subject to fractionation on sucrose density gradients. 2.1.2. Peripheral tissues Membranes from liver, kidney, heart, lung pancreas and spleen were prepared by Polytron homogenization in 20 ml of 10 mM HEPES buffer, pH 7.4, followed by centrifugation at 40,000 x g for 20 min. The pellet was then washed twice in 10 mM HEPES buffer and stored frozen until used. 2.2. Displacement studies

For the assay, tissue (approx. 0.05-0.1 mg protein) was incubated with 20 nM [3H]gabapentin (Amersham, custom synthesis, specific activity 73 Ci/mmoi) in 10 mM HEPES buffer (pH 7.4 at 22°C, sodium-free) in the presence of varying concentrations of test compound for 30 min at 22°C, before filtering onto Whatman GFB filters under vacuum. Filters were washed 3 times with 5 ml ice-cold 100 mM NaCI and bound radioactivity determined using liquid scintillation counting. Non-specific binding was defined by 100 p.M gabapentin or 10 txM 3-isobutyl GABA. 2.3. Saturation studies

Specific binding was determined following incubation of synaptic plasma membranes with varying concentrations (1 nM-1 mM) of gabapentin for 30 min at room temperature as described above. Concentrations of gabapentin higher than 20 nM were achieved by displacing 20 nM [3H]gabapentin with increasing concentrations of unlabelled gabapentin and correcting for dilution using the equation total gabapentin bound = (specific dpm bound) [non-radioactive gabapentin] x (1 + [radioactive gabapentin] ) 2.4. Kinetic studies

To initiate the association reaction, [3H]gabapentin (20 nM) was added to vessels containing synaptic

295

EFFECT OF pH ON [aH]-GABAPENTINBIN~NGTO RAT CORTICAL SYNAPTICPLASMAMEMBRANES 10000

plasma membranes either in the presence or absence of 100/xM gabapentin. Aliquots were taken from both flasks at various time intervals (0.5-120 min) filtered, washed and specific binding determined. For dissociation studies, association was allowed to occur for 30 min and aliquots taken to determine specific binding at equilibrium (t = 0). An excess of unlabelled gabapentin was then added to both 'total' and 'non-specific' flasks to effect dissociation and aliquots taken at intervals as before to determine specific binding.

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3. Results

3.1. pH dependency, saturation and kinetic studies Under the assay conditions described above, using 20 nM [3H]gabapentin and approximately 0.1 mg protein, total binding observed was 12,000-15,000 dpm, 80-85% of which was specifically displaced by 100/xM non-radioactive gabapentin. Specific binding increased linearly with increasing protein concentration up to at least 120/xg protein (data not shown). All experiments were performed in the linear range. Heating the tissue at 60°C for 10 min abolished all specific binding. Binding of [3H]gabapentin was dependent upon pH with optimal levels of specific binding measured at pH 7.4 (fig. 1). Specific binding declined markedly at acidic pH. Thus, at pH 6.6 the level of specific binding was only 40% of that measured at pH 7.4 (fig. 1). Binding was also reduced above pH 7.4 although the reduction in binding was less dramatic than at acidic pH (fig. 1). Association curves measured at 20°C (fig. 2) were monophasic and showed that equilibrium was reached within 30 min. At 4°C equilibrium was reached only after 180 min (data not shown) although the amount of specific binding was the same as that measured at 20°C. Dissociation studies yielded biphasic curves (fig. 3), with approximately 60% of the total [3H]gabapentin bound at equilibrium dissociating within 20 rain of addition of excess non-radioactive gabapentin. This relatively slow rate of dissociation allowed the use of filtration to separate bound from free radioligand. In saturation studies using isobutyl GABA to define non-specific binding, [3H]gabapentin bound to a single class of non-interacting binding sites (fig. 4). Analysis

8

9

10

pH

2.5. pHstudies Aliquots of tissue were incubated for 30 min with 20 nM [3H]gabapentin in the absence and presence of 10 /xM isobutyl GABA in either 10 mM HEPES buffer (pH range 5.2-7.4; adjusted using KOH) or 10 mM Tris (pH range 7.0-9.9; adjusted with HCI). Following the incubation, the tissue was filtered, washed and the radioactivity counted as described above.

"~'oI ~ . .

Fig. 1. pH dependency of [3H]gabapentin binding to synaptic plasma membranes. Specific binding of 20 nM gabapentin was determined over the pH range 5.2-9.8 using either HEPES (pH 5.2-7.4) or Tris buffer (pH 7.0-9.8). The graph represents the results from a single experiment where each point is the mean of triplicate determinations. The experiment was repeated twice with identical results.

of these data using GRAPHPAD software yielded K D values of 38 + 2.8 nM (fig. 4) which agreed closely with the ICs0 value (80 riM) for displacement of [3H]gabapentin binding by gabapentin itself. The maximum binding capacity (Bmax) was 4.6 _+0.4 pmol/mg protein.

3.2. Pharmacological specificity In order to characterise the nature of the [3H]gabapentin binding site, a range of compounds from different pharmacological classes were examined for their ability to displace [3H]gabapentin binding. The results are summarised in tables 1-4. Fig. 5 shows representative inhibition curves for a number of these compounds.

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30

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90

120

TIME(Mm) Fig. 2. Time course for the association of [3H]gabapentin binding to synaptic plasma membranes. Specific binding of 20 nM gabapentin was determined at various time intervals. The graph represents the combined data from two separate experiments each performed in duplicate. The inset shows the data transformed where B is specific binding at time t and B c is the specific binding measured at equilibrium. The linear nature of the plot indicates pseudo-first order kinetics for the rate of association.

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3.2.1. Gabapentin analogues Twenty-nine analogues of gabapentin were examined for activity in the binding assay. The results obtained for eight key compounds are summarised in table 1. Decreasing the size of the saturated cyclohexyl ring of gabapentin by one carbon atom (compound 1) resulted in only a small change in affinity for the [3H]gabapentin binding site. Similarly, increasing the cyclohexyl ring by one carbon atom failed to change binding affinity. The cyclooctane analogue (compound 3), however, was approximately 23-fold less active than gabapentin (table 1). Substitution of a methyl group on the cyclohexane ring was well tolerated, but sterically larger modifications caused marked loss of activity for this site (data not shown).

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Fig. 3. Time course of the dissociation of [3H]gabapentin binding from synaptic plasma membranes. [3H]Gabapentin (20 nM) was incubated with synaptic plasma membranes for 30 min to achieve equilibrium. Excess non-radioactive gabapentin was added and the amount of specifically bound radioligand was determined at various time intervals. The data are from a single representative experiment. Specific binding expressed as a percentage of the binding at equilibrium is plotted against time. The inset shows the same data transformed as a semilog plot, the curvilinear nature of which suggests dissociation from more than one site.

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2so %0 7so 1000 [3H]-GABAPENTIN (nM)

Fig. 4. Saturation of [3H]gabapentin binding to synaptic plasma membranes. Specific binding defined by 3 p.M isobutyl GABA was determined over a range of concentrations of [3H]gabapentin. The main graph shows the untransformed data from a single experiment in which each point was determined in triplicate. The inset shows a Scatchard plot of the same data, suggesting binding to a single population of binding sites. Data from four separate experiments yielded a mean K D of 38_+2.8 nM and a maximum binding capacity (Bmax) of 4.6 _+0.4 p m o l / m g protein.

Fig. 5. Inhibition of [3H]gabapentin binding to synaptic plasma membranes. The graph shows representative curves for gabapentin, the gabapentin analogue compound 3, the GABA derivative compound 11 and the polyamine spermine. Each point represents the mean of duplicate determinations.The curves of best fit were determined by non-linearleast squares analysis. Isopropylation (compound 4) (or methylation) at the primary amine of gabapentin resulted in only a 2-fold decrease in affinity, but substitution with larger groups at the same position (compound 5) again caused dramatic loss of activity to give ICh0 values of greater than 1 mM. Modification or replacement of the carboxylic acid group also produced marked loss in activity, with compounds 6 and 7 possessing ICh0 values three orders of magnitude greater than that for gabapentin (table 1).

3.2.2. GABA-related compounds [3H]Gabapentin binding was weakly inhibited by GABA itself and the selective GABA B agonist baclofen (lCh0 values 608 izM and 845 p~M respectively; table 2). Other compounds active at GABA receptors, including kojic amine, muscimol, bicuculline, isonipecotic acid, cis-crotonic acid and trans-crotonic acid, did not inhibit [3H]gabapentin binding (IC50 values > 1 mM, data not shown), suggesting that the site(s) labelled by [3H]gabapentin are unlikely to be GABA receptors. Similarly, the GABA uptake inhibitors nipecotic acid, THPO (4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol) and thiomuscimol were also inactive or only weakly active (ICh0 value 100 ~ M for nipecotic acid, > 1 mM for the others). Taken together with the fact that the binding studies were carried out in sodium-free buffer, the probability that [3H]gabapentin was binding to sodium-dependent GABA uptake sites was also very low. Of particular interest was the observation that 3substituted GABA derivatives (Andruszkiewicz and Silverman, 1990; table 2) were potent displacers of [3H]gabapentin binding. Thus, the introduction of a methyl group at the 3-position of GABA (compound 8) resulted in a 40-fold increase in affinity for the gabapentin binding site as compared to GABA itself. Further increases in affinity were observed by increas-

297

ing the size of the alkyl substituent, with optimum activity achieved by 3-isobutyl GABA (compound 13), which possessed an IC50 value (0.08 /zM) comparable to that for gabapentin itself (0.08 ~M). The presence of a hydroxyl group in the molecule at the 3-position (/3-hydroxy-GABA), however, resulted in a marked loss of activity at the gabapentin binding site (IC50 > 1 mM). The 3-phenyl derivative of GABA was only weakly active as an inhibitor of binding (table 2), although with an ICs0 of 34 ttM this compound was approxiTABLE 1 Affinities of gabapentin analogues at the [3H]gabapentin binding site. Data are the geometric mean and range from at least three separate experiments using 20 nM [3H]gabapentin as described in the Materials and methods section. Compound Gabapentin

Structure H oN COOH

IC5o (/zM) 0.08

Range 0.05-0.13

mately 20-fold more potent than baclofen, the 3-chlorophenyl derivative of GABA. Interestingly, 3-phenyl GABA is only weakly active at GABA B receptors (table 2). Substitution of GABA at the 2 and 4 positions did not result in high affinity for the gabapentin binding site (table 3). Thus, the 2-methyl analogue was 6-fold less active, the 4,4-dimethyl analogue 25-fold less active, and the 4-ethyl analogue approximately 100-fold less active than their respective 3-substituted analogues. In general, the 2-substituted analogues displayed higher affinity than the 4-substituted derivatives. Of the 2- and 4-substituted GABA analogues examined, 2-phenyl-GABA possessed the highest affinity (ICs0 value 16 ~M). Activity at the gabapentin binding site of the substituted GABA analogues was lost with substitution at the primary amine (compounds 23, 24 and 25; table 3), and with replacement of the carboxylic acid group with other acidic groups. For example, 3-aminopropylphosphonic acid and 3-amino-l-propanesulfonic acid were both inactive at 10 mM.

3.2.3. NMDA / glycine receptor complex ligands 1

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A range of ligands active at the NMDA/glycine receptor complex, listed below, were evaluated. Glutamate, glycine, D-serine, N-methyl-D-aspartic acid, 7chlorokynurenic acid, (+)-MK-801, strychnine and 3(( + )-2-carboxypiperizine-4-yl)propyl-1-phosphonic acid (CPP), all possessed ICs0 values of 1 mM or greater, with the exception of (+)-MK-801 (ICs0 approximately 200/xM). Considering the potency of MK-801 for the NMDA-gated ionophore, it is unlikely that the interaction of such a high concentration of MK-801 with the gabapentin binding site can account for its activity at the NMDA receptor. Recent studies have shown that polyamines may play a modulatory role in the NMDA receptor complex (see Williams et al., 1991). In our studies, spermine and spermidine inhibited approximately 50% of specifically bound [3H]gabapentin with ICs0 values of 12/zM and 15/.tM, respectively (table 4; fig. 5). Putrescine, the diamine precursor from which spermine and spermidine are synthesized in vivo, and difluoromethyl ornithine (DFMO), an inhibitor of ornithine decarboxylase (the rate-limiting enzyme involved in their biosynthesis), were much less active (ICs0 values greater than 1 mM). Similarly, the putative polyamine antagonists ifenprodil (Reynolds and Miller, 1989), arcaine and agrnatine (Reynolds, 1990), and three acetylated derivatives of spermine and spermidine previously shown to be active at the polyamine binding site (Sprosen, 1990) were also much less active (ICs0 values > 1 mM). Scatchard analysis indicated that the polyamines inhibited binding by reducing the maximum binding

298 ~IABLE 2 Affinities of GABA and 3-substituted GABA analogues for GABAA, GABA B and gabapentin binding sites. Assays were performed using 20 nM [3H]gabapentin and 10 nM [3H]GABA. Each value is the geometric mean (and range) from 2-4 separate experiments. R

H2 N " ~ ' ~ Compound

R

Gabapentin GABA Baclofen 8 9 10 11 12 13 14 15 /3-PhenyI-GABA /3-Hydroxy-GABA

H p-Chlorophenyl CH 3 CH2CH 3 CH(CH 3)2 (CH2)3CH 3 CH(CH3)(CH2CH 3) CHzCH(CH3) 2 (CH2)2CH(CH3) 2 Cyclohexyl Phenyl OH

CO2 H

[3H]Gabapentin IC50 (p,M)

[3H]GABAA IC50 (/xM)

[3H]GABAB IC5o (/zM)

0.08 (0.05-0.13) 610 (440-770) 850 (450-1700) 14 (9-21) 0.92 (0.8-1.2) 0.26 (0.1-0.4) 0.94 (0.6-1.5) 0.17 (0.1-0.3) 0.08 (0.06-0.11) 5.4 (4-9) 1.6 (1.2-2.6) 34 (21-45) > 1,000

> 1,000 0.036 a 24 > 1,000 > 1,000 > 1,000 > 1,000 > 1,000 > 1,000 > 1,000 N.D. b 2.0

> 1,000 0.025 0.017 84 > 1,000 > 1,000 68 > 1,000 > 1,000 190 > 1,000 N.D. 0.7

a Baclofen was included in the assay to prevent binding to GABA A receptors, b N.D. = not determined.

rather than affecting the affinity of gabapentin for the binding site (Control Bmax = 4.6 + 0.4 p m o l / m g protein, K D = 38.0 + 2.8 nM; in the presence of 3 mM spermidine Bma x = 2.7 + 0.2 p m o l / m g protein, K D = 46.0 + 8.6 nM). c a p a c i t y Bmax,

TABLE 3 Affinities of GABA derivatives substituted at the 2-, 4- and primary amine positions of GABA for the [3H]gabapentin binding site. Data shown represent the geometric mean of ICs0 values determined in 2-4 separate experiments (using 20 nM [3H]gabapentin) as described in the Materials and methods section. Compound

R

R'

2-substituted

H 2N ~

IC50 (~M)

3.2.4. Ions Magnesium ions inhibited [3H]gabapentin binding in a concentration-dependent manner, with an IC50 value of 27/zM. The pattern of inhibition was similar to that of the polyamines spermine and spermidine, i.e. maximum displacement was only about 50% of specific binding as defined by 100 ~ M gabapentin and the inhibition appeared to be due mainly to a reduction in Bmax although a small change in binding affinity was noted (control Bmax = 4.6 + 0.4 p m o l / m g protein, K D = 38.0 + 2.8 nM; plus 3 mM MgC12 Bmax = 2.0 +__0.3 p m o l / m g protein, K o = 51.4 + 4.9 nM). A combination of 3 mM MgC12 and 3 mM spermine did not inhibit [3H]gabapentin binding to a greater

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Affinities of polyamines and magnesium ions for the gabapentin binding site. Binding assays were performed using 20 nM [3H]gabapentin. Each value is the geometric mean (and range) from 2-3 separate experiments. Compound

IC 50 (/x M)

Spermine Spermidine Putrescine NS-Acetyl-spermidine N1-Acetyl-spermidine N l_Acetyl_spermine Arcaine Agmatine DFMO Ifenprodil Magnesium chloride

12 (8-14) 15 (13 - 16) > 1,000 > 1,000 > 1,000 > 1,000 > 1,000 > 1,000 > 1,000 > 1,000 27 (16-46)

299

extent than either compound alone (data not shown), suggesting the possibility of acting at a common site. Zinc ions weakly displaced [3H]gabapentin binding (ICs0 value approximately 100 /xM), but sodium, calcium, potassium, chloride and thiocyanate ions were without effect (ICs0 values > 100 mM)

3.2.5. Anticonvulsants A number of known anticonvulsants with different mechanisms of action were examined for ability to displace [3H]gabapentin binding. Diazepam, carbamazepine, phenobarbitone, phenytoin, pentobarbitone, sodium valproate, ethosuxemide and/3-hydroxy-GABA weakly inhibited binding, with ICs0 values of greater than 1 mM. 3.2.6. Amino acids and miscellaneous compounds A wide variety of neuroactive compounds were evaluated in the [3H]gabapentin binding assay. Dopamine, sulpiride, apomorphine, adenosine, desimipramine, pargyline, neomycin, picrotoxin, the guanine nucleotide analogues GMP-PNP and AMP-PNP, galanin and aminooxyacetic acid were inactive or possessed very low affinity (> 1 mM) at the gabapentin binding site. The amino acids L-aspartic acid, L-arginine, Lhistidine, L-asparagine, L-threonine, L-glutamine, quinolinic acid, kainic acid, taurine, /3-alanine and L-alanine were also tested for activity. Most of these possessed relatively low affinity for this binding site, although two, L-glutamine and L-threonine were active with ICs0 values of 20/xM and 50/xM, respectively. 3.3. Activity at GABA receptors Because of the structural similarity of the gabapentin analogues and the 3-substituted compounds to the inhibitory amino acid GABA, their affinity for GABA A and GABA B receptors was measured. GABA A and GABAB receptor binding to well washed crude cerebro-cortical synaptic membranes was measured according to the method of Bowery et al. (1983), using [3H]GABA (60 Ci/mmol) but with filtration as a means of terminating the assay rather than centrifugation. All gabapentin analogues possessed ICs0 values of > 1 mM in both the GABA A and GABA B receptor binding assays (data not shown). The activity of the 3-substituted GABA analogues in the different binding assays is summarized in table 2. Of the 3-substituted analogues showing significant (ICs0 < 30 /xM) inhibition at the gabapentin site, only 3methyl-GABA was active at the GABA A receptor. The GABA B receptor was slightly more tolerant of substitution at the 3-position with -methyl, -n-butyl, -isopentyl and -phenyl derivatives all showing some inhibition at concentrations over 10 /zM. Nonetheless, the

TABLE 5 Comparison of [3H]gabapentin binding in rat brain and a variety of peripheral tissues. Tissue

Specific binding (dpm/mg protein)

Cerebral cortex Liver Kidney Heart Lung Pancreas Spleen

71,000 100 0 1,800 900 300 100

most potent inhibitors at the gabapentin site were inactive at both GABA receptors.

3.4. Tissue distribution of [ 3H]gabapentin binding In order to determine whether the gabapentin binding site was unique to the central nervous system, [3H]gabapentin was incubated with crude membranes prepared from a variety of peripheral tissues. The results of these experiments are summarized in table 5. Negligible levels of specific [3H]gabapentin binding were measured in crude membranes prepared from pancreas, liver, heart, spleen, lung and kidney. By contrast, in parallel incubations using brain membranes, high levels of specific binding were obtained.

4. Discussion

Gabapentin (CI-945) is a novel anticonvulsant with an as yet, unknown mechanism of action. Gabapentin does not exhibit anticonflict or analgesic activity although may potentiate morphine-induced analgesia and haloperidol catalepsy (Dooley et al., 1986). Gabapentin appears to be a remarkably specific compound, showing little or no activity in a wide range of in vitro binding assays including benzodiazepine, GABAA, GABA B or dihydropyridine-sensitive calcium channels (Dooley et al., 1986). Indeed in these early studies no specific [3H]gabapentin binding was detected to homogenates of rat brain. Despite this, the data contained in this report show that [3H]gabapentin may be used to label specific sites in homogenates of rat brain using in vitro binding assays. Binding to this novel site is most sensitive to inhibition by gabapentin itself and derivatives of the major inhibitory amino acid transmitter GABA substituted at the 3-position. The structure-activity relationship determined from each group of compounds indicates that both the amino and the carboxylic acid groups, their relative spatial location and hence ionic interactions, are essential to binding at the [3H]gabapentin site. In

300 addition, activity of the GABA derivatives is enhanced by the presence of an alkyl substituent at the 3-position, thereby differentiating ligands active at this site from those binding to GABA A and GABA B receptors. The gabapentin binding site appears to be specific to the central nervous system with very little binding being measurable in a range of peripheral tissues. Slight differences in the way central and peripheral tissues were prepared (homogenisations in sucrose or HEPES, respectively) are unlikely to account for the low levels of binding in peripheral tissues as crude homogenates of brain prepared in HEPES yielded high binding (data not shown). This observation suggests that the binding site may not be associated with any general aspect of cellular physiology such as an ion channel or enzyme system. Moreover, within the brain, the binding site is widely but unevenly distributed with high levels present in regions known to be associated with seizure activity such as the cerebral cortex and hippoeampus (Hill et al., submitted). Although recent behavioural (Oles et al., 1990) and electrophysiological (Sprosen, 1990) studies have suggested the involvement of the NMDA/glycine complex in the mechanism of action of gabapentin, the binding studies described here provide little evidence for a direct interaction between gabapentin and the excitatory amino acid complex. Compounds known to active at the glycine co-agonist site or the NMDA receptor did not exert any direct effect on [3H]gabapentin binding under the conditions employed in this study. Nonetheless, the possibility of an indirect interaction with the NMDA complex cannot be eliminated, as the polyamines spermine and spermidine inhibit [3H]gabapentin binding with surprisingly high affinity. Polyamines have been implicated in a wide variety of cellular functions, and their presence in brain tissue is well documented in the literature (see Williams et al., 1991). Recent studies have shown that spermine and spermidine increase the binding of [3H]MK-801 to the NMDA receptor (Ransom and Stec, 1988). This enhancement appears to be due to an allosterically-induced increase in ligand affinity rather than a direct activation of the NMDA receptor by the polyamines (Reynolds, 1990). It is.unlikely however, that [3H]gabapentin labels the allosteric polyamine binding site on the NMDA receptor as in our own preliminary studies gabapentin did not affect the potentiation of [3H]MK-801 binding induced by spermidine. Furthermore, no correlation was observed between the activity of a range of polyamine analogues in the [3H]gabapentin binding assay and their activity in electrophysiological studies (Sprosen, 1990). For example, Sprosen (1990) showed arcaine, Nl-acetylspermidine and N 1acetylspermine to be competitive antagonists at the NMDA/polyamine site, whereas NS-acetylspermidine acted as an agonist. Although agonist or antagonist

activity cannot be differentiated in binding assays, none of these compounds showed any significant activity at the gabapentin binding site at concentrations up to 1 mM. Another circumstantial, yet intriguing indication of an association between the gabapentin binding site and the NMDA receptor complex is the ability of magnesium ions to affect [3H]gabapentin binding. The importance of magnesium ions in NMDA receptor-mediated activity is again well documented (Mayer et al., 1984; Foster and Fagg, 1987) and recent reports have suggested that the polyamines and magnesium ions may in fact share a common binding site (Sacaan and Johnson, 1990). In keeping with this report, magnesium ions, spermine and spermidine show similar patterns of displacement of [3H]gabapentin binding, with approximately 50% maximum inhibition of specific binding. All other compounds tested in this assay to date fully displaced all specific [3H]gabapentin binding. Of the range of other compounds tested for activity at the gabapentin site, only 3-substituted GABA analogues showed any activity even though baclofen and GABA itself were only weakly active, thus precluding binding to either of the two major classes of GABA receptor. The series of GABA analogues tested here, which were originally designed in a rational attempt to alter endogenous GABA levels by interfering with its metabolism (Andruszkiewicz and Silverman, 1990), have been shown to be anticonvulsants (Silverman et al., 1991). However, high concentrations (in excess of 1 mM) are required to alter either GABA transaminase (GABA-T) or glutamate decarboxylase (GAD) activity in vitro whilst anticonvulsant effects are seen at relatively modest doses that are unlikely to result in brain concentrations of this magnitude. This suggests that the antiseizure activity of these compounds may be mediated by a mechanism other than via GABA-T or GAD. One possibility is a direct action at GABA receptors, particularly the GABA A receptor (Meldrum, 1985). Again this is unlikely as none of the 3-alkyl-substituted analogues displayed high affinity at the GABA A receptor. In contrast, all 3-substituted compounds were potent inhibitors at the gabapentin site, with 3-isobutyl GABA showing comparable activity to gabapentin itself. Moreover, this compound is the most potent anticonvulsant of the series (Silverman et al., 1991), suggesting that activity at the binding site may indeed relate to anticonvulsant activity. Other clinically effective anticonvulsants from a variety of differing classes interacted only weakly with this binding site. It is unlikely therefore that gabapentin labels a general 'anticonvulsant' binding site. However, the possibility that gabapentin is labelling a specific site for the action of a particular type of anticonvulsant cannot be eliminated.

301

In conclusion, the data presented in this report provide strong evidence for a novel and hitherto undescribed binding site that exhibits high affinity for the anticonvulsant gabapentin. The site does not appear to be related to either the inhibitory or the excitatory amino acid system yet is specific to the central nervous system. This finding together with the high affinity displayed by certain GABA analogues that display anticonvulsant properties strongly suggests that the recognition site may play an important role in controlling the excitability of neurones.

Note added in proof

Since submitting this and our companion paper (Hill et al., 1993) we have found that a number of neutral L-amino acids such as L-leucine (IC50 = 77 nM), L-isoleucine (IC50 = 72 nM), L-methionine (IC50 = 50 nM) and also the system-L inhibitor BCH (2-(-)-endoamino-bicycloheptane-2-carboxylic acid: IC5o = 40 nM) are potent inhibitors of [3H]gabapentin binding. This raises the possiblity that [3H]gabapentin labels a site in brain that closely resembles the large neutral amino acid transporter (system-L) that has been characterised elsewhere.

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