A Summary of Mechanistic Hypotheses of Gabapentin Pharmacology

Home , Amino acid transporter, Nipecotic acid

Epilepsy Research 29 (1998) 233–249

A summary of mechanistic hypotheses of gabapentin pharmacology

Charles P. Taylor a,*, Nicolas S. Gee d, Ti-Zhi Su b, Jeffery D. Kocsis e, Devin F. Welty c, Jason P. Brown d, David J. Dooley a, Philip Boden d, Lakhbir Singh d a Department of Neuroscience Therapeutics, Parke–Da6is Pharmaceutical Research, Di6ision of Warner–Lambert Co., Ann Arbor, MI 48105, USA b Department of Molecular Biology, Parke–Da6is Pharmaceutical Research, Di6ision of Warner–Lambert Co., Ann Arbor, MI 48105, USA c Department of Pharmacokinetics and Drug Metabolism, Parke–Da6is Pharmaceutical Research, Di6ision of Warner–Lambert Co., Ann Arbor, MI 48105, USA d Parke–Da6is Neuroscience Research Centre, Cambridge Uni6ersity For6ie Site, Robinson Way, Cambridge, CB22QB, UK e Neuroscience and Regeneration Research Center A127A, Veterans Affairs Medical Center, Building 34, Room 123, 950 Campbell A6e., West Ha6en, CT 06516, USA

Received 28 July 1997; received in revised form 1 October 1997; accepted 8 October 1997

Abstract

Although the cellular mechanisms of pharmacological actions of gabapentin (Neurontin®) remain incompletely described, several hypotheses have been proposed. It is possible that different mechanisms account for anticonvulsant, antinociceptive, anxiolytic and neuroprotective activity in animal models. Gabapentin is an amino acid, with a mechanism that differs from those of other anticonvulsant drugs such as phenytoin, carbamazepine or valproate. Radiotracer studies with [14C]gabapentin suggest that gabapentin is rapidly accessible to brain cell cytosol. Several hypotheses of cellular mechanisms have been proposed to explain the pharmacology of gabapentin: 1. Gabapentin crosses several membrane barriers in the body via a specific amino acid transporter (system L) and competes with leucine, isoleucine, valine and phenylalanine for transport. 2. Gabapentin increases the concentration and probably the rate of synthesis of GABA in brain, which may enhance non-vesicular GABA release during seizures. 3. Gabapentin binds with high affinity to a novel binding site in brain tissues that is associated with an auxiliary subunit of voltage-sensitive Ca2+ channels. Recent electrophysiology results suggest that gabapentin may modulate certain types of Ca2+ current. 4. Gabapentin reduces the release of several monoamine neurotransmitters. 5. Electrophysiol- ogy suggests that gabapentin inhibits voltage-activated Na+ channels, but other results contradict these findings. 6. Gabapentin increases serotonin concentrations in human whole blood, which may be relevant to neurobehavioral

* Corresponding author.

0920-1211/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0920-1211(97)00084-3 234 C.P. Taylor et al. / Epilepsy Research 29 (1998) 233–249 actions. 7. Gabapentin prevents neuronal death in several models including those designed to mimic amyotrophic lateral sclerosis (ALS). This may occur by inhibition of glutamate synthesis by branched-chain amino acid aminotransferase (BCAA-t). © 1998 Elsevier Science B.V. All rights reserved.

1. Introduction gabapentin was originally modeled after the structure of GABA, it does not modulate GABA Gabapentin, 1-(aminomethyl)cyclohexaneacetic receptor function like conventional GABAergic acid (Neurontin®) is a novel anticonvulsant drug drugs, and it is inactive at GABA receptors. This that is active in a variety of animal seizure models review outlines several potential mechanisms of (Taylor, 1995) and prevents partial seizures and pharmacological action of gabapentin. The vari- generalized tonic-clonic seizures in several ous hypotheses are considered in approximate placebo-controlled clinical studies, both in add-on order of their discovery, and each is considered and monotherapy (Andrews et al., 1990; McLean critically, with references to the published litera- et al., 1993; Marson et al., 1996; Beydoun et al., ture. Although a consensus has not yet been 1997; Burgey et al., 1997). Clinical use of reached, it seems likely that several different sites gabapentin has been associated with several side of action may be necessary to account for all of effects, but it is generally well tolerated (Ramsey, the pharmacological actions of gabapentin. 1995). Its pharmacokinetic profile, and use in combination with other medications has been described (McLean, 1995). Gabapentin was origi- 2. Gabapentin and system L amino acid nally synthesized to treat spasticity and to reduce transporters polysynaptic spinal reflexes. It is active in several animal models of spasticity. Gabapentin is an amino acid that exists at Recently, it has been shown in animal models physiological pH as a zwitterion, and since it is that gabapentin prevents nociceptive responses doubly-charged, its native permeability to mem- from hyperalgesia in animal models (Everhart et brane barriers within the body is low. However, al., 1997; Field et al., 1997a,b; Gillin and Sorkin, like several other amino acids, gabapentin is a 1997; Hunter et al., 1997; Hwang and Yaksh, substrate of the so-called system L transporter of 1997; Jun and Yaksh, 1997; Singh et al., 1996; gut (Stewart et al., 1993a) and of neurons and Xiao and Bennett, 1997) and also has analgesic astrocytes (Su et al., 1995). This property allows actions in clinical reports (Rosner et al., 1996; gabapentin molecules to cross membrane barriers Backonja et al., 1997; Mellick and Mellick, 1997; more easily. In addition to the facilitated trans- McGraw and Kosek, 1997). Other studies suggest port across cell membranes, there is a smaller that gabapentin has anxiolytic-like effects in ani- non-saturable component of transport (Stewart et mal models (Singh et al., 1996). Furthermore, al., 1993a; Su et al., 1995) that is likely due to gabapentin treatment prevents motoneuron de- passive diffusion. Due to differences in the influx generation in an in vitro model of amyotrophic and efflux rate of gabapentin via system L in lateral sclerosis (ALS) (Rothstein and Kuncl, cultured cells, it accumulates in cytosol to greater 1995) and delays death in a transgenic animal concentrations than in the bathing medium (Su et model of ALS (Gurney et al., 1996). Results in a al., 1995). These transport properties of preliminary placebo-controlled clinical trial of gabapentin probably account for the access of gabapentin for treatment of ALS were not statisti- gabapentin to brain cytosol (Vollmer et al., 1986), cally significant (Miller et al., 1996), but suggested where it is present at about ten-fold higher con- that additional clinical studies are warranted. centrations than in the brain extracellular space Gabapentin was designed as a structural analog (Welty et al., 1993). The delayed anticonvulsant of the inhibitory neurotransmitter k-aminobutyric action of gabapentin in rats after a bolus intra- acid (GABA) (Satzinger, 1994). Although venous dose (Welty et al., 1993) may be explained C.P. Taylor et al. / Epilepsy Research 29 (1998) 233–249 235

Fig. 1. Schematic diagram of GABAergic inhibitory synapse in brain. In presynaptic ending, glutamate is synthesized primarily from glutamine (by glutaminase) and free GABA is synthesized from glutamate by the enzyme glutamic acid decarboxylase (GAD). GABA is degraded by the enzyme GABA-transaminase. The irreversible GABA-transaminase inhibitor, vigabatrin, is an anticonvulsant. GABA is packaged into synaptic vesicles, where it is released in response to presynaptic calcium influx. GABA also can be released from the cytosol by reversal of the GABA transporter (GABA uptake). Post-synaptically, GABA activates GABAA receptors, which are modulated by anticonvulsant benzodiazepines and barbiturates. GABAB receptors are also activated by synaptic release of GABA or by the GABAB agonist, baclofen. GABA is taken up from the extracellular space by a specific transporter that is blocked by the anticonvulsant tiagabine. Nipecotic acid is both a blocker and substrate for the GABA transporter. in part by delayed distribution of drug to the 3. Gabapentin and function of GABA systems in brain compartment, with maximal concentrations brain reached approximately 60 min after administration. Radiotracer studies confirm this idea (Welty Numerous reports in the literature (Meldrum, et al., 1997), but anticonvulsant results suggest 1985; Tunnicliff and Raess, 1991; Bowery, 1993; that there may be an additional delay caused by Macdonald and Olsen, 1994) indicate that k- biochemical events within brain tissue prior to aminobutyric acid (GABA) is a major inhibitory anticonvulsant action (Welty et al., 1993, 1997). neurotransmitter in mammalian brain and that However, recent data from models of hyperalgesia seizures occur if GABA synapses are impaired in rats (Everhart et al., 1997; Field et al., 1997b) (Tunnicliff and Raess, 1991). A variety of GABA- indicate that the analgesic action of gabapentin is enhancing drugs such as GABAA agonists, attained rapidly after an intrathecal injection of GABAA modulators (e.g. benzodiazepines), drugs drug, suggesting that either analgesia and anticon- converted metabolically to GABA, GABA uptake vulsant actions have different mechanisms, or that inhibitors (e.g. tiagabine (Gram et al., 1989)), and the delay observed in anticonvulsant experiments inhibitors of GABA degradation (e.g. vigabatrin with animals may be an over-estimate. (Meldrum, 1985)) prevent seizures in animal mod- The competition of gabapentin with transport els or in clinical use (Suzdak and Jansen, 1995; of L-leucine, L-valine and L-phenylalanine in vitro Tunnicliff and Raess, 1991) (see Fig. 1). The (Su et al., 1995) raises the possibility that some of similarity of chemical structures between GABA the pharmacological properties of gabapentin and gabapentin also suggests a functional rela- arise from changes in cytosolic concentrations of tionship. endogenous branched-chain amino acids. How- Gabapentin does not alter radioligand binding ever, no studies to date support this hypothesis. at GABAA or GABAB receptors at concentrations 236 C.P. Taylor et al. / Epilepsy Research 29 (1998) 233–249 up to 100 vM (Taylor, 1995) nor does it alter this action is probably not relevant for anticon- [3H]GABA uptake into neuronal or glial cultures vulsant activity (Goldlust et al., 1995). (Su et al., 1995). In addition, gabapentin does not Gabapentin in vitro increases the activity of par- alter neuronal responses to the application of tially-purified glutamic acid decarboxylase GABA in electrophysiological experiments (Rock (GAD), suggesting that gabapentin treatment et al., 1993). These studies indicate that might increase the synthesis of GABA from gluta- gabapentin does not interact directly with either mate in brain tissues (Taylor et al., 1992). GAD

GABAA or GABAB receptors, nor with high- exists in two isoforms with different molecular + afﬁnity Na -dependent GABA transporters. and functional properties. GAD67 has a larger Therefore, gabapentin’s actions are distinct from molecular weight, and exists mostly in neuronal those of several drugs that directly modulate cell bodies; GAD65 is localized more in nerve GABAA receptor function (Macdonald and endings and synaptosomes (Erlander and Tobin, Olsen, 1994) such as benzodiazepines or barbitu- 1991; Erlander et al., 1991; Martin et al., 1991). rates. Furthermore, the actions of gabapentin are The in vitro activity of GAD65 is increased seven- distinct from those of GABA uptake inhibitors fold in saturating concentrations of pyridoxal 5%-

(Suzdak and Jansen, 1995) and from drugs that phosphate, while GAD67 is only modulated 2-fold alter GABAB receptor function (Bowery, 1993). (Erlander and Tobin, 1991). Gabapentin may In vivo results suggest an interaction of modulate GAD activity in vivo. Gabapentin treat- gabapentin with GABA systems. Gabapentin ment (25 mg/kg IP) increases brain concentrations treatment causes decreased firing rates in rat sub- of GABA in rats pretreated with aminooxyacetic stantia nigra pars reticulata GABA neurons acid (AOAA) (Lo¨scher et al., 1991). AOAA in- (Bloms-Funke and Lo¨scher, 1996). Spontaneous hibits GABA transaminase and prevents GABA firing rates are reduced substantially by other degradation. Therefore, changes in brain GABA GABAergic drugs such as benzodiazepines and concentrations after AOAA treatment suggest muscimol. In animals, tonic extensor convulsions that gabapentin enhances the GABA synthesis caused by the GABA synthesis inhibitor thiosemi- rate by 50–100% in several brain regions in vivo. carbazide are blocked more potently than seizures A recent study (Leach et al., 1997) with mice from several other convulsant agents (Bartoszyk given single or twice-daily doses of gabapentin et al., 1986; Taylor, 1995). Thiosemicarbazide is showed some significant changes in brain gluta- an inhibitor of pyridoxyl 5%-phosphate (Tapia and mate and glutamine concentrations and also Salazar, 1991), an enzymatic cofactor needed for changes in GABA-T activity, suggesting alter- GABA synthesis by the cellular enzyme glutamic ations in brain amino acid metabolism. In vivo acid decarboxylase (GAD). NMR spectroscopy indicates that brain GABA Thiosemicarbazide acts to trap pyridoxyl 5%- concentrations are elevated in human patients phosphate by combining with the free carbonyl taking gabapentin, and that elevation of GABA is group to form an inert complex. Clonic convul- related to seizure control (Petroff et al., 1996; sions caused by antagonism of GABA receptors Mattson et al., 1997). Recent results with NMR are prevented by gabapentin less potently than spectroscopy of rat brain tissues indicates that convulsions from inhibition of GABA synthesis. gabapentin treatment elevates brain GABA con- Gabapentin does not prevent clonic convulsions centration and also decreases brain glutamate from antagonism of spinal glycine receptors. concentration (Petroff et al., 1997). These results suggest that gabapentin may selec- Gabapentin increases GABA release from rat tively counteract GAD inhibitors. The results also striatal brain slices in vitro (Go¨tz et al., 1993), and suggest that gabapentin does not interact directly this action is prevented by the GABAA antago- with GABA receptors or with glycinergic in- nist, bicuculline. Although the mechanism of the hibitory synapses of spinal cord. bicuculline-sensitive effect is not clear, GABA can Gabapentin is a mixed-type inhibitor of be released from neuronal tissues by either Ca2+- GABA-transaminase at high concentrations, but dependent or Ca2+-independent mechanisms, the C.P. Taylor et al. / Epilepsy Research 29 (1998) 233–249 237 latter depending upon reversal of the GABA up- gyrus (Xiong and Stringer, 1997). At first glance, take carriers (Szerb, 1982; Pin and Bockaert, decreased paired-pulse inhibition suggests a de- 1989). Gabapentin increases electrophysiological crease in GABA function, but similar results were responses caused by the non-vesicular release of obtained in another study with vigabatrin (Sayin GABA in rat optic nerves in a sucrose-gap ap- et al., 1997) (a known inhibitor of GABA degra- paratus (Kocsis and Honmou, 1994). Very similar dation). The vigabatrin results were partially re- increases in GABA responses were seen upon versed by co-application of a GABAB blocker, inward current in voltage-clamped pyramidal neu- suggesting that higher extracellular GABA con- rons in rat hippocampal slices in vitro (Honmou centrations activate presynaptic GABAB receptors et al., 1995a,b, see Fig. 2. In both experiments, (Sayin et al., 1997). GABA release was triggered by nipecotic acid, a Measurements of GABA release have been substrate and competitive inhibitor of GABA confirmed in studies of [3H]GABA release from transport that is not active at GABA receptors. brain slices (Fichter et al., 1996). Rat striatal Application of nipecotic acid disrupts the normal brain slices were preincubated with either equilibrium of the GABA transporter, and causes [3H]GABA or [3H]glutamine to differentially label the release of cytosolic GABA by hetero-exchange two GABA pools. GABA release was measured (Szerb, 1982; Kocsis and Honmou, 1994). The by scintillation counting (for pre-loaded 3 responses are due to activation of GABAA recep- [ H]GABA) or by cation-exchange chromatogra- tors, and are blocked by bicuculline. phy followed by scintillation counting (for In addition, in vivo, gabapentin treatment de- [3H]GABA synthesized from [3H]glutamine). creases paired-pulse inhibition in the rat dentate [3H]GABA release after preloading with [3H]glutamine was caused by application of nipecotic acid (1 mM) and was enhanced approximately 50% by gabapentin pretreatment. In con- trast, [3H]GABA release after preloading with [3H]GABA was not altered by gabapentin. These results are consistent with the idea that gabapentin increases synthesis of GABA via GAD. Furthermore, since results were obtained in the presence of the GABA-transaminase inhibitor AOAA, the action of gabapentin is not likely to be caused by GABA-T inhibition. The electrophysiological and biochemical results with gabapentin on GABA systems (Kocsis and Hon- mou, 1994; Honmou et al., 1995a,b; Fichter et al., 1996) are consistent with increased GAD activity, since hippocampal tissues (Tapia and Salazar, Fig. 2. Gabapentin enhances the nipecotic acid-induced release 1991), optic nerves (Ochi et al., 1993) and striatal of GABA in rat hippocampal slices in vitro. A whole-cell tissues each contain significant amounts of GAD. voltage clamp recording was made from a single CA1 pyrami- Although nipecotic acid normally is not present dal neuron, and nipecotic acid (10 mM) was applied for 2.0 s by a pressure pulse to a nearby micropipette. The nipecotate in brain, it is a convenient probe for the GABA 3 caused non-vesicular GABA to be released from neighboring uptake transporter. [ H]Nipecotic acid (During et cellular elements, resulting in a bicuculline-sensitive inward al., 1995) or [3H] derivatives of other selective current that was measured at intervals of 5 min. The applica- GABA uptake inhibitors (Suzdak et al., 1994) tion of gabapentin (100 vM, triangles) at time zero caused a have been used to study the localization and significant increase in inward current in comparison to control experiments with no drug present (circles); N=5 for control function of GABA transporters. The stoichiome- experiments, N=7 for gabapentin experiments. Reproduced try of GABA transporters has been described from Honmou et al. (1995a) with permission. (Keynan and Kanner, 1988; Liron et al., 1988) 238 C.P. Taylor et al. / Epilepsy Research 29 (1998) 233–249

Fig. 3. Gabapentin labels a high-affinity binding site in brain tissues. This autoradiograph was made by incubating a frozen section of rat brain tissue with [3H]gabapentin and a film emulsion. After incubation, the film was developed, revealing areas of dense [3H]gabapentin binding sites (shown in white). Abbreviations show brain regions: 1,2 are superficial layers of neocortex; CA3, CA2, CA1 and dg (dentate gyrus) are areas of hippocampus; cg is central gray; ls is lateral septum; gr, mol and w are granule cell layer, molecular layer and white matter of the cerebellum. and changes in the cellular microenvironment 4. Receptor binding studies with [3H]gabapentin would cause reversal of the normal inward flux of GABA via transporters. Either cytosolic Na+ Gabapentin does not affect ligand binding at a loads, cellular depolarization, or particularly acti- wide variety of commonly-studied drug and neu- vation of glutamate receptors (which causes both rotransmitter binding sites and voltage-activated depolarization and Na+ loading) cause net efflux ion channels including GABA, glutamate, and of GABA by reversed transport (Pin and Bock- glycine receptors of several types (Taylor, 1995). aert, 1989). Glutamate release and cellular depo- However, gabapentin itself has been used to larization occur during seizures, and a recent define a novel binding site in brain tissues. report (During et al., 1995) suggests that in hu- Tritiated gabapentin binds with high affinity to man epileptic brain tissues, non-vesicular GABA a single population of sites in rat (KD 38 nM), release is reduced while calcium-dependent GABA mouse (KD 14 nM) and pig (KD 17 nM) synaptic release occurs normally. Therefore, gabapentin plasma membranes prepared from cerebral cortex may compensate for a pathological reduction in (Suman et al., 1993; Thurlow et al., 1993). The transport-mediated GABA release in epileptic maximum binding capacity of [3H]gabapentin re- brain Kocsis and Mattson, 1996). This hypothesis ported for rat brain membranes is 4.6 pmol/mg remains to be tested critically, but experiments (Suman et al., 1993), and similar values have been with gabapentin using microdialysis to measure obtained for mouse and pig tissue (Thurlow et al., synaptic and non-vesicular GABA release might 1993). Mapping of [3H]gabapentin binding sites in help address this idea. rat brain has been achieved by autoradiographic C.P. Taylor et al. / Epilepsy Research 29 (1998) 233–249 239 studies (Hill et al., 1993) (Fig. 3). The highest [3H]gabapentin labels a novel pharmacological levels of specific binding were detected in the site. outer layers of the cerebral cortex. Binding sites in Compounds from two chemical series have the hippocampus were highest in the dendritic been identified as potent inhibitors of region of the CA1 pyramidal cell layer and molec- [3H]gabapentin binding. Several 3-substituted ana- ular layer of the dentate gyrus. Binding appeared logues of GABA, most notably (SR)-3-isobutyl- to be localized to areas of excitatory input com- GABA (IC50 =80 nM), were found to be active monly associated with seizure activity. In the cere- (Suman et al., 1993). The S(+)-enantiomer of bellum, sites were concentrated in the molecular 3-isobutyl-GABA was 10-fold more potent than layer, around regions of excitatory input. While the R(−)-enantiomer (Taylor et al., 1993). The the distribution of [3H]gabapentin binding sites rank order of potency of gabapentin and the two was qualitatively similar to that of the NMDA enantiomers of 3-isobutyl GABA were the same receptor, as defined by strychnine-insensitive both in the [3H]gabapentin binding assay and in [3H]glycine binding, regression analysis yielded a animal seizure models (Taylor et al., 1993). This correlation coefficient of only 0.48. Lesion studies observation suggests that the binding of showed that [3H]gabapentin binding sites were gabapentin to the [3H]gabapentin binding site most probably localized on neurons rather than may be important to the antiepileptic activity of on glia. the drug. Potent and stereospecific displacement A variety of compounds have been tested for of [3H]gabapentin binding was also apparent with inhibition of specific [3H]gabapentin binding to several large neutral amino acids (Thurlow et al., brain membranes. Inactive compounds (IC50 \1 1993). For each amino acid tested, the L-enan- mM) included those that interact with GABA tiomer was 100–1000-fold more potent than the receptors (kojic amine, muscimol, bicuculline, corresponding D-enantiomer. These findings to- isonipecotic acid), NMDA receptors (glutamate, gether with the observation that [3H]gabapentin is glycine, D-serine, N-methyl-D-aspartic acid, 7- transported across membranes by a system L chlorokynurenic acid and trans-crotonic acid) and transporter (Stewart et al., 1993a,b; Su et al., the GABA transporter (nipecotic acid and THPO 1995) led to the hypothesis that [3H]gabapentin (4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol)) may label the recognition site of a neuronal trans- (Suman et al., 1993). The low affinities of GABA, porter similar to system L (Thurlow et al., 1993). baclofen and (+)-MK-801 for the [3H]gabapentin binding site suggest that [3H]gabapentin does not 4.1. Purification and identification of the interact with either GABA or NMDA receptors [ 3H]Gabapentin binding protein (Suman et al., 1993). Mg2+ and the polyamines, spermine and spermidine, which are known al- To clarify the molecular nature of the losteric modulators of NMDA receptors (Foster [3H]gabapentin binding protein (GBP), it was and Fagg, 1987), inhibited [3H]gabapentin binding purified from pig brain. Preliminary biochemical in a non-competitive manner with IC50 values of studies suggested that the GBP was a membrane- 27, 12 and 15 vM, respectively. Maximal inhibi- associated protein. Brain membranes were solubi- tion with Mg2+ or polyamines was only about lized with a non-ionic detergent, Tween 20, and

60%. A combination of 3 mM MgCl2 and 3 mM the GBP was purified to near-homogeneity by spermine did not inhibit [3H]gabapentin binding sequential chromatography over six matrices (Gee to a greater extent than either compound alone, et al., 1996). Electrophoretic analysis revealed the suggesting the possibility of a common site of purified GBP to have a subunit molecular weight action. A range of anticonvulsant drugs including of approximately 130 000. The partial N-terminal diazepam, carbamazepine, phenobarbital, pheny- sequence of the purified protein was identical to toin, pentobarbital, sodium valproate, ethosux- that reported for the h2l subunit of the L-type imide and i-hydroxy-GABA were all inactive voltage-dependent Ca2+ channel from skeletal

(IC50 \1 mM) supporting the idea that muscle (Hamilton et al., 1989). Binding of 240 C.P. Taylor et al. / Epilepsy Research 29 (1998) 233–249

[3H]gabapentin to COS-7 cells transiently express- larly or extracellularly disposed (see Fig. 4). Hy- ing rabbit skeletal muscle h2l cDNA and to par- drophilicity plots suggest that the h2 polypeptide tially purified muscle Ca2+ channel subunits contains two transmembrane domains (Ellis et al., 2+ confirmed that the GBP and the h2l Ca chan- 1988) but biochemical data support a model nel subunit are the same protein (Gee et al., 1996). where the h2 polypeptide is wholly extracellular Gabapentin is the first ligand described that inter- (Jay et al., 1991; Brickley et al., 1995). The pres- 2+ acts with the h2l subunit of a Ca channel. ence of a single transmembrane anchor in the l polypeptide is widely accepted. 4.2. Structure of 6oltage-dependent calcium channels 4.3. Roles of the subunits in calcium channel function Ca2+ channels are multi-subunit complexes found not only in the brain but also in peripheral Only the N-type channel from rabbit brain tissues such as skeletal muscle, heart and lung (Witcher et al., 1993) and the L-type Ca2+ chan- (Catterall, 1995). The distribution of nel from skeletal muscle (Leung et al., 1988; Jay et [3H]gabapentin binding sites in rat peripheral tis- al., 1991) have been purified along with their sues is consistent with an interaction of associated auxiliary subunits. Thus, the particular gabapentin at Ca2+ channels (Gee et al., 1996). subunit combinations that occur in vivo for other Ca2+ channels consist of at least three subunits channels are unclear. This should be borne in

(for review, see Dolphin (1995)): h1 (the pore- mind when considering the results of ‘mix and forming subunit), h2l and i. Skeletal muscle match’ heterologous expression studies with Ca2+ channels also have a k subunit. High- cloned Ca2+ channel subunits. Most of these threshold Ca2+ channels are classified as L-, N-, studies have utilized the Xenopus lae6is oocyte P-, or R-types on the basis of their sensitivity to expression system. Functional channels require various organic ligands and several neurotoxins. only the h1 subunit (Mori et al., 1991). However, Apart from gabapentin, all Ca2+ channel ligands significant changes to the electrophysiology of the appear to bind to h1 subunits. Molecular cloning channel are effected by co-expression with the studies have revealed at least six genes encoding auxiliary subunits. A peak barium current (IBa) of h1 subunits, and a number of splice variants have 31 nA was recorded for the N-type h1B subunit also been identified (for review, see Hofmann et alone, whereas expression with the skeletal muscle al. (1994)). The i subunit is a hydrophilic protein h2lA or skeletal muscle i1 yielded a peak IBa of 89 that interacts with the h1 subunit (Pragnall et al., and 664 nA, respectively. Significant co-operativ- 1994). Four genes encode i subunits and splice ity of the auxiliary subunits was demonstrated by variants for three of them have been described co-expression of all three subunits (h1Bh2lAi1) (Perez-Reyes and Schneider, 1994). A single gene which yielded an IBa of \6 vA (Mori et al., encodes the h2l subunit (DeJongh et al., 1990) 1991). Several other groups also report a co-oper- and a number of splice variants have been found ative effect of h2l and i on h1 (Singer et al., 1991; (Perez-Reyes and Schneider, 1994). The h2l sub- Williams et al., 1992). Shistik et al. (1995) have unit is synthesized as a pre-protein that undergoes dissected the electrophysiological and biochemical extensive post-translational modifications. The mechanisms of modulating Ca2+ channel current membrane targeting signal sequence is removed characteristics by auxiliary subunits. Whole-cell and a second proteolytic cleavage event generates calcium channel currents of h1C (cardiac) were a small C-terminal fragment (l) that remains increased 8–10 fold by either h2l or i2 alone but attached to the larger fragment (h2) by a disulfide 100-fold with both subunits. They found that the bridge. The protein is also heavily glycosylated increase in the current by h2l resulted both from (Sharp and Campbell, 1989; Jay et al., 1991). The a three-fold increase in the amount of h1C present membrane topology of the h2l subunit is unclear, in the membrane and from modulation of the 3 thus the [ H]gabapentin site could be intracellu- gating characteristics of h1C. On the other hand, C.P. Taylor et al. / Epilepsy Research 29 (1998) 233–249 241

2+ Fig. 4. Schematic diagram of the structure of the Ca channel h1, h2 and i subunits. Taken with permission from Gurnett et al. (1996). The cell plasma membrane is shown as a lipid bilayer with intracellular and extracellular media labeled. Small branched structures indicate extracellular sites of glycosylation (sugar polymers attached to protein). ‘S–S’ denotes sites of putative cysteine linkages. The main voltage-sensing and ion-conducting subunit (central part of h1) is shown in its postulated conformation spanning the lipid bilayer. The gabapentin binding protein (h2l) is shown in its presumed conformation anchored in the lipid bilayer and associating with the h1 subunit, but with most amino acids exposed to the extracellular ﬂuid. Recent experiments with site-directed 2+ deletions of h2l Gurnett et al. (1996) indicate that the extracellular region of this protein enhances Ca current amplitude and the intramembrane portion interacts directly with the h1 subunit (arrows). It is not yet known what part of the h2l protein gabapentin binds to.

i2 alone affected only the gating characteristics. in a subtle manner. It is possible that inhibition of Few electrophysiological studies on the effects of monoamine neurotransmitter release (Reimann, gabapentin on Ca2+ channels have been reported 1983; Schlicker et al., 1985; Dooley et al., 1996) is and further work in this area is required. caused by an interaction of gabapentin with Ca2+ However, several observations are consistent channels. Monoamine release is never inhibited by with the idea that gabapentin modulates Ca2+ more than 50% by gabapentin (Reimann, 1983; channels, particularly if channels are modulated Schlicker et al., 1985; Dooley et al., 1996). Re- 242 C.P. Taylor et al. / Epilepsy Research 29 (1998) 233–249 cently, voltage-clamp records of high-threshold it alter sustained firing of action potentials in Ca2+ currents from rat neocortical pyramidal cultured neurons (Rock et al., 1993). However, neurons show that gabapentin (10–100 vM) re- with longer incubation periods in vitro, it de- versibly reduces the nitrendipine-sensitive compo- creases sustained firing of Na+-dependent action nent of current by about 35% (Stefani et al., potentials (Wamill and McLean, 1994). In addi- 1997). However, another study with acutely-iso- tion, a recent report suggests that gabapentin has lated human dentate granule neurons showed no other electrophysiological actions that may ac- effect of gabapentin on total cellular Ca2+ cur- count for reduced excitability (Kawasaki et al., rents (Schumacher et al., 1997). These findings 1995). It is not yet clear whether these in vitro suggest that gabapentin may have subtle actions findings are relevant for anticonvulsant or other on Ca2+ channels that are only apparent in cer- pharmacological actions of gabapentin in vivo. tain sub-populations or experimental protocols.

7. Gabapentin and serotonin concentration in 5. Gabapentin and other neurotransmitters whole blood

Gabapentin has actions on monoamine neuro- Gabapentin given to healthy human volunteers transmitter release in vitro. Gabapentin causes increases the concentration of serotonin in whole significant decreases (10–15% blockade) in the blood (Rao et al., 1988). These authors speculate electrically- or 20–50 mM K+-evoked release of that increased serotonin might be due to changes noradrenaline, dopamine and serotonin from in serotonin metabolism or uptake in platelets. brain slices (Reimann, 1983; Schlicker et al., 1985; However, this has not been studied in vitro. They Dooley et al., 1996). The inhibitory action of also speculate that changes in serotonin could gabapentin on striatal dopamine release is clearly explain changes that were observed in sleep pat- different from that of the GABAB agonist ba- terns (increased duration of stages 3 and 4 with- clofen (Reimann, 1983). Reduced monoamine re- out changes in total sleep time, REM sleep time lease may relate either to an action on Ca2+ or REM latency). channels (see section above) or to changes in monoamine metabolism. Recent results indicate that pretreatment of rats with gabapentin signifi- 8. Gabapentin and neuroprotection (glutamate cantly reduces the augmented noradrenaline and metabolism) dopamine turnover caused by systemic administration of 3,4-diaminopyridine (Pugsley et al., Although seizures induced by glutamate ago- 1997). These results indirectly suggest that nists are not prevented by gabapentin, high doses gabapentin may alter the function of Ca2+ chan- significantly delay such seizures, suggesting that nels involved with monoamine release (see section gabapentin might interact with glutamatergic above). Although changes in monoamine function synapses (Bartoszyk et al., 1986; Taylor, 1995). induced by gabapentin might not relate directly to Studies with gabapentin in an in vitro model anticonvulsant effects, alterations in monoamine meant to mimic some aspects of motor neuron neurotransmission might underlie behavioral ef- disease (amyotrophic lateral sclerosis or ALS) in- fects (anxiolytic-like action or analgesia). dicate that neuronal cell death is prevented by gabapentin treatment (Rothstein and Kuncl, 1995). The model utilizes the application of a 6. Gabapentin and voltage-sensitive Na+ channels selective inhibitor of glutamate uptake, and neuronal death is reduced by selective antagonists of Gabapentin does not alter voltage-clamped the AMPA type of glutamate receptor. Therefore, sodium currents in the manner of phenytoin, car- it is reasonable to postulate that protective effects bamazepine or lamotrigine (Taylor, 1993), nor did of gabapentin might arise from changes in gluta- C.P. Taylor et al. / Epilepsy Research 29 (1998) 233–249 243 mate metabolism or release. Additionally, studies 1990). This hypothesis for the action of with a transgenic mouse model of ALS (Gurney et gabapentin by enhanced degradation of glutamate al., 1994) indicate that chronic treatment with also remains to be tested in vivo. high dosages of gabapentin significantly delays Gabapentin in vitro decreases the tissue content paralysis and death in transgenic mice (Gurney et of glutamine (a metabolic precursor of glutamate al., 1996). and GABA) in isolated hippocampal tissue A recent paper (Welty et al., 1995) details two (Kapetanovic et al., 1995). Although the hypotheses for the neuroprotective action of metabolic pathway involved with this effect has gabapentin. One pathway for glutamate synthesis not been described, changes in glutamine in brain tissues arises from transamination of metabolism or transport might be relevant for h-ketoglutarate by branched-chain amino acids decreased glutamate synthesis. (leucine, isoleucine, valine) by the enzyme branched-chain amino acid aminotransferase (BCAA-T). Gabapentin is a competitive inhibitor 9. Effects in animal models of anxiety of BCAA-T with inhibition constants (Ki =0.6– 1.2 mM) equivalent to the substrate affinity for Gabapentin was investigated in several animal endogenous branched-chain amino acids (Km = models that predict utility for the treatment of 0.4–1.2 mM) (Goldlust et al., 1995). Recent in anxiety. These include the marmoset human vitro studies show that this action of gabapentin threat test, the rat elevated X-maze and the rat is selective for the isoform of BCAA-T found in conflict test (Singh et al., 1996). In all of these brain cytosol (neuronal form), with little effect on models, gabapentin produced anxiolytic-like ef- the mitochondrial form of BCAA-T (astroglial fects with minimum effective doses ranging from 3 form) (Hutson et al., 1995). Since the therapeutic to 30 mg/kg (Singh et al., 1996). In all three range of concentrations for gabapentin (10–100 models, gabapentin produced activity with similar vM) is similar to the endogenous concentrations efficacy to that of benzodiazepines. Most non- of branched-chain amino acids, gabapentin may benzodiazepine drugs produce much weaker activ- significantly inhibit the action of BCAA-T in vivo, ity than gabapentin in the rat elevated X-maze and this may result in decreased cytosolic concen- and the marmoset human threat test. For exam- trations of glutamate that would reduce gluta- ple, gabapentin’s activity is more pronounced mate-dependent cell death. Decreased synthesis of than that of buspirone or other experimental com- glutamate from labeled L-leucine in the presence pounds such as cholecystokinin CCKB antago- of gabapentin has been demonstrated in vitro, nists (Singh et al., 1991) or serotonin 5-HT3 although total tissue content of glutamate was not receptor antagonists (Broekkamp et al., 1989). A altered (Kapetanovic et al., 1995). Decreases in recent study of the antidepressant, phenylzine whole-brain glutamate of 20% after gabapentin (Paslawski et al., 1996) suggests that elevation of treatment have been demonstrated by NMR spec- whole brain GABA concentration (see above) troscopy of rat brain extracts (Petroff et al., could be related to antianxiety effects. 1997). A decrease in the synthesis of glutamate from L-leucine after administration of gabapentin in vivo remains to be demonstrated. 10. Effects in animal models of pain In addition, gabapentin in vitro stimulates the activity of the catabolic enzyme glutamate dehy- Gabapentin administration to rats or mice does drogenase (GDH) at high concentrations (Gold- not alter acute responses to thermal or chemical lust et al., 1995). It is possible that gabapentin stimulation. However, delayed pain responses in administration would enhance GDH activity in several animal models are reduced. Formalin and vivo, as has been proposed for the treatment of carrageenan are two chemical irritants that are ALS with high-dose administration of endoge- widely used in studies of tonic pain and hyperal- nous branched-chain amino acids (Plaitakis, gesia from peripheral inflammation. The subcuta- 244 C.P. Taylor et al. / Epilepsy Research 29 (1998) 233–249 neous administration of formalin into the plantar glutamate receptors on dorsal horn neurons of the surface of the rodent paw produces a biphasic spinal cord. It remains to be seen whether nocifensive behavioral response. The early phase gabapentin alters NMDA responses in the spinal consists of intense licking and biting of the in- cord, but NMDA antagonists have similar actions jected paw and lasts up to 10 min but a second to gabapentin in the formalin test. late phase of licking and biting occurs from 10 to The antihyperalgesic action of gabapentin does 60 min after injection (Dubuisson and Dennis, not depend on activation of opiate receptors, and 1977). The late phase is a state of facilitated pain is not altered by the opiate antagonist, naloxone response (hyperalgesia) associated with inflamma- (Field et al., 1997b). Unlike morphine, gabapentin tion. This behavioral response has been shown to does not reduce gut motility in rats. Sedation and correlate with a biphasic increase in the activity of ataxia are caused by gabapentin only at doses ten C-fiber primary afferent neurons after formalin times higher than those preventing pain responses. injection (McCall et al., 1996). Carrageenan elicits Repeated administration of gabapentin does not little or no immediate pain response (Wheeler- result in tolerance to antihyperalgesia. Morphine Aceto et al., 1990), but causes hyperalgesic re- tolerance does not cross-generalize to gabapentin sponses to thermal or mechanical stimuli with a (Field et al., 1997b). Therefore, gabapentin’s anal- maximum 3–4 h after injection into the footpad gesic actions are distinct from those of opiate (Hargreaves et al., 1988). The formalin and car- analgesics. rageenan behavioral tests involve sensitization of Results of an open-label clinical study in pa- sensory neurons of the spinal dorsal horn in re- tients with reflex sympathetic dystrophy (RSD) sponse to injury or intense artificial activation of suggest that gabapentin may reduce neuropathic C-fiber afferents (Woolf and Wall, 1986). pain (Mellick et al., 1995). RSD is characterized Gabapentin (30–300 mg/kg) selectively blocks the by burning pain, allodynia, hyperpathia, vasomo- tonic phase of formalin nociception without tor and sudomotor disturbances, edema, and changing the peripheral swelling caused by car- trophic changes to bone, skin and soft tissues. rageenan, but gabapentin reduces the mechanical Satisfactory (scored good to excellent) pain relief and thermal hyperalgesia from carrageenan was obtained in all eight patients given 300 or 600 (Singh et al., 1996). Therefore, gabapentin may mg gabapentin daily. Gabapentin treatment cor- act within the spinal cord or brain to reduce rected skin temperature and color, and reduced sensitization of dorsal horn sensory neurons. allodynia, hyperalgesia and hyperpathia in most Gabapentin reduces pain responses from neu- patients. These results suggest that a placebo-con- ropathy produced by chronic constriction of the trolled clinical study of gabapentin for RSD is sciatic nerve (Hunter et al., 1997; Xiao and Ben- warranted. In addition, results of a double- nett, 1997) or by ligation of spinal nerves at the blinded clinical study of gabapentin for pain from L5 and L6 levels (Hunter et al., 1997; Hwang and diabetic neuropathy showed a significant reduc- Yaksh, 1997). These results in various animal tion of pain scores in comparison to placebo models show that gabapentin reduces mechanical (Backonja et al., 1997). hyperalgesia (pin prick response), mechanical al- In summary, gabapentin is active in animal lodynia (Von Frey nylon monofilament), thermal models that require sensitization of pain responses hyperalgesia (from radiant heat) and thermal allo- but is not active in transient models of pain. dynia (from cold water). The intrathecal adminis- Therefore, it may not reduce immediate pain from tration of gabapentin blocked thermal and injury, but it appears to reduce abnormal hyper- mechanical hyperalgesia (Hwang and Yaksh, sensitivity (allodynia and hyperalgesia) induced by 1997; Xiao and Bennett, 1997), suggesting that inflammatory responses or nerve injury. When gabapentin may work from a spinal site of action. considered with gabapentin’s relative lack of un- Thermal hyperalgesia is mediated primarily by desirable side effects, gabapentin may eventually C-fiber sensory afferents, which produce their ac- be shown to improve treatment for several tions in the spinal cord mainly via NMDA-type chronic pain syndromes; three additional placebo- C.P. Taylor et al. / Epilepsy Research 29 (1998) 233–249 245 controlled clinical studies for various pain syn- dogenous amino acids) interact with an auxiliary dromes are presently underway. subunit of voltage-gated Ca2+ channels. Addi- tional studies are needed to establish which of the various potential mechanisms account for activity 11. Electrophysiology studies of glutamate of gabapentin in anticonvulsant, antinociceptive, responses anxiolytic and neuroprotective models.

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