Mechanisms of the Antinociceptive Action of Gabapentin

Home , GABA analogue, Nicardipine

Jen-Kun Cheng1,2,3 and Lih-Chu Chiou1,4,* 1Institute and 4Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan 2Department of Anesthesiology, Mackay Memorial Hospital, Taipei, Taiwan 3Department of Anesthesiology, Taipei Medical University, Taipei, Taiwan

Received October 12, 2005

Abstract. Gabapentin, a γ-aminobutyric acid (GABA) analogue anticonvulsant, is also an effective analgesic agent in neuropathic and inflammatory, but not acute, pain systemically and intrathecally. Other clinical indications such as anxiety, bipolar disorder, and hot flashes have also been proposed. Since gabapentin was developed, several hypotheses had been proposed for its action mechanisms. They include selectively activating the heterodimeric GABAB receptors consisting of GABAB1a and GABAB2 subunits, selectively enhancing the NMDA current at GABAergic interneurons, or blocking AMPA-receptor-mediated transmission in the spinal cord, binding to the L-α-amino acid transporter, activating ATP-sensitive K+ channels, activating hyperpolarization-activated cation channels, and modulating Ca2+ current by selectively binding 3 2+ to the specific binding site of [ H]gabapentin, the α2δ subunit of voltage-dependent Ca channels. Different mechanisms might be involved in different therapeutic actions of gabapentin. In this review, we summarized the recent progress in the findings proposed for the antinociceptive 2+ action mechanisms of gabapentin and suggest that the α2δ subunit of spinal N-type Ca channels is very likely the analgesic action target of gabapentin.

Keywords: gabapentin, GABAB receptor, NMDA receptor, KATP channel, 2+ α2δ subunit of Ca channels

1. Introduction actively studied. Several hypothesis were raised, which have been summarized in the comprehensive review of Gabapentin, 1-(aminomethyl)cyclohexaneacetic acid Taylor et al. in 1998 (1). The cellular action mechanisms (Neurontin)®, was originally developed as a chemical of gabapentin were also recently reveiwed (7 – 9). analogue of γ-aminobutyric acid (GABA) (Fig. 1) to Different mechansims might be involved in the different reduce the spinal reflex for the treatment of spasticity actions of gabapentin (10). The clinical uses of and was found to have anticonvulsant activity in various gabapentin in the management of chronic neuropathic seizure models (1). In addition, it also displays anti- pain have been previoulsy reviewed (11, 12). In this nociceptive activity in various animal pain models. review, we summarized and discussed the recent Clinically, gabapentin is indicated as an add-on medica- progresses in the studies investigating the antinociception for the treatment of partial seizures (2) and neuropathic pain (3). It was also claimed to be beneficial in several other clinical disorders such as anxiety (4), bipolar disorder (5), and hot flashes (6). The possible mechanisms or targets involved in the multiple therapeutic actions of gabapentin have been

*Corresponding author. [email protected] Published online in J-STAGE: DOI: 10.1254/jphs.CR0050020 Fig. 1. Chemical structures of γ-aminobutyric acid (GABA) and Invited article gabapentin.

1 2 J-K Cheng and L-C Chiou tive action machanisms of gabapentin to elucidate the Table 1. Antinociceptive effects of gabapentin in various animal most likely analgesic action target(s) of gabapentin. pain models Pain model Dose (route) Reference 2. Analgesic effects of gabapentin Gabapentin ineffective 2-1. Animal studies Acute nociception After gabapentin was found to have an analgesic Hot plate test 0.1 – 100 nmol (i.t.) 168 effect in patients with intractable neuropathic pain (13), 10 – 100 mg/kg (i.p.) 168 its antinociceptive effects had been reported in several Tail-flick test 200 µg (i.t.) 15 animal pain models, including the phase 2, but not phase 30 – 300 mg/kg (i.p.) 24 1, of formalin inflammatory (14 – 19), neuropathic Paw pressure test 30 – 300 mg/kg (s.c.) 19 (17, 20 – 26), postoperative (27, 28), lumbar adhesive Plantar thermal test 30 – 300 mg/kg (s.c.) 19 arachnoiditis (29), and cancer-induced bone (30) pain 10 – 300 µg (i.t.) 14 models (Table 1). It seems that gabapentin is effective Warm water tail 100 mg/kg (i.p.) 17 in inflammatory- and tissue-injury induced pain models withdrawal test but not in acute physiological pain models (14, 17, 19) Gabapentin effective (Table 1). Gabapentin is effective when given systemically (10 – Inflammatory pain 300 mg/kg) or intrathecally (10 – 300 µg) (Table 1). Formalin test (phase 2) 100 mg/kg (i.p.) 17 However, it seems that much higher efficacy can be 10 – 300 mg/kg (s.c.) 19 achieved for gabapentin via intrathecal administration. 6–200µg (i.t.) 15 In the postoperative pain model, only 19% of the 10 – 300 µg (i.t.) 14 maximal possible antiallodynic effect was observed at 6–600µg (local) 16 the highest tested dose of gabapentin when it was given Carrageenan-induced 10 – 100 mg/kg (s.c.) 19 intraperitoneally (31). However, intrathecal gabapentin 1–100µg (i.t.) 19 could produce up to 90% – 100% the maximal possible Freund’s complete 30 – 250 mg/kg (s.c.) 21 antiallodynic effect in the same model (28). Similar adjuvant test findings were also reported in certain neuropathic pain Neuropathic pain models such as partial sciatic nerve ligation (21) and Spinal nerve ligation 34 mg/kg (i.p.) a 24 chronic constriction injury (25) models. Takasaki et al. 10 – 100 mg/kg (i.p.) 17 (32) reported that gabapentin was effective when given 10 – 300 µg (i.t.) 26 orally and intrathecally, but not intraventricularlly, intra- Partial sciatic nerve ligation 10 – 250 mg/kg (s.c.) 21 cisternally, or intraplantarlly in relieving the nociceptive 10 – 300 µg (i.t.) 21 behaviors induced by transdermal infection with herpes Chronic constriction injury 103 mg/kg (i.p.)a 24 simplex virus. It seems that the spinal cord is an impor- 30 – 300 mg/kg (i.p.) 25 tant site for the systemic analgesic action of gabapentin. 1–4µmol (i.t.) 25 However, since gabapentin is also effective in the Diabetic neuropathy 10 – 100 mg/kg (p.o.) 22 management of migraine and trigeminal neuralgia (see 1–100µg (i.t.) 22 section 2-2), the contribution of the supraspinal site to Postherpetic neuralgia 30 – 60 mg/kg (i.p.) 23 the systemic analgesic action of gabapentin cannot be µ ruled out. 10 – 30 g (i.t.) 23 Trigeminal neuralgia 30 – 50 mg/kg (i.p.) 20 2-2. Clinical studies Postoperative pain 3 – 30 mg/kg (s.c.) 27 Clinically, gabapentin has been reported to be effec- 10 – 100 µg (i.t.) 28 tive in the following pain status, including reflex Cancer-induced bone pain 30 mg/kg (s.c.) 30 sympathetic dystrophy (33), trigeminal neuralgia (34), postherpetic neuralgia (35, 36), diabetic neuropathy (36, Lumbar adhesive 60 nmol (i.t.) 29 arachnoiditis pain 37), migraine (38), acute pain in herpes zoster infection a (39), and other neuropathic pain status (40, 41) : ED50 (Table 2). Gabapentin is also effective in the postoperative pain management in different surgeries such as breast (42, 43), hysterectomy (44, 45), cholecystectomy opioid consumption was found to be reduced in several (46), spinal (47), and knee (48) surgeries (Table 2). The pain states when gabapentin was co-administered clini- Antinociceptive Mechanisms of Gabapentin 3

Table 2. Clinical studies in the analgesic actions of gabapentin Pain status Dosage Patient No. Side effects Reference

Neuropathic pain Diabetic neuropathy 900 – 3600 mg/day 165 dizziness 37 somnolence Diabetic neuropathy a 900 – 3200 mg/day 35 sedation 36 dry mouth Lyme borreliosis (late stage) 500 – 1200 mg/day 10 40 Postherpetic neuralgia up to 3600 mg/day 229 somnolence 35 dizziness ataxia Postherpetic neuralgia a 900 – 3200 mg/day 22 sedation 36 dry mouth Trigeminal neuralgia 2 34

Postoperative pain a Radical mastectomy 1200 mg (pre-operative) 70 42 Breast cancer surgery 1200 mg/day 75 43 Hysterectomy 1200 mg (initial) 80 44 600 mg (maitaining) Laparoscopic cholecystectomy 300 mg (pre-operative) 459 sedation 46 nausea vomiting Spinal surgery 1200 mg (pre-operative) 50 47 Knee ligament repair 1200 mg (pre-operative) 40 48

Herpes zoster (acute stage) 900 mg 26 39

Migraine prophylaxis 400 – 1200 mg/day 63 somnolence 38 dizziness tremor fatigue ataxia

a: reducing opioids comsumption cally (Table 2). On the other hand, gabapentin might 4. Mechanisms of actions of gabapentin also be helpful in reducing the dependence and tolerance of opioids, as suggested by animal studies (49, 50). It 4-1. GABAergic system seems that gabapentin is relatively a pretty safe drug in 4-1-1. GABAA receptors terms of its tolerable effective doses with minor un- Although being developed as a brain penetrant wanted or side effects clinically (Table 2). analogue of GABA, gabapentin showed little affinity at GABAA or GABAB receptors in the initial binding 3. Other clinical implications of gabapentin study (57). The antinociceptive effects of intrathecal gabapentin in the formalin test (58) and L5/6 nerve In addition to the management of seizures and pain, ligation pain model (26) were unaffected by the several clinical indications for gabapentin have also GABAA-receptor antagonist bicuculline. Therefore, it is been implied, including anxiety (4), bipolar depression generally believed that the GABAA receptor is not (5), hot flashes (6), social phobia (51), essential tremor involved in the actions of gabapentin (1). In the post- (52), ataxia in cortical cerebellar atrophy (53), hiccup operative pain model, where the GABAA-receptor (54), post cardiac surgery diaphragmatic spasm (55), and agonist isoguvacine is ineffective, bicuculline also failed antipsychotic-induced akathisia (56). to reverse the antinociceptive effect of intrathecal gabapentin (59).

4-1-2. GABAB receptors GABAB receptors are functional in a heterodimeric 4 J-K Cheng and L-C Chiou form consisting of GABAB1 and GABAB2 subunits (60). GABAB receptors might be involved in the anti- Several splicing variants of GABAB1 receptors were convulsant but not the anxiolytic action of gabapentin found, but the majority of GABAB heterodimers are (10). Parker et al. (72) found that gabapentin inhibit 3 3 consisted of GABAB1a and GABAB2 or GABAB1b and [ H]glutamate release, but not [ H]GABA release, in rat GABAB2 subunits (60). By coexpressing the GABAB2 neocortical brain slices in a manner blocked by GABAB- subunit with different GABAB1 variants to form hetero- receptor antagonist. Therefore, they suggested that dimeric functional GABAB receptors in Xenopus oo- gabapentin selectively activates presynaptic GABAB cytes, the group of Ng (61, 62) claimed that gabapentin heteroreceptors on glutamatergic terminals, but not selectively activated heterodimeric GABAB1a-B2 recep- GABAB autoreceptors on GABAergic terminals. It is not tors, but not GABAB1b-B2 or GABAB1c-B2 receptors. They known if the subunit composition of the presynaptic also reproted that gabapentin could activate the native GABAB receptors located on glutamatergic terminals is GABAB receptors that mediate G-protein-coupled different from that on GABAergic terminals. However, inwardly rectifying K+ (GIRK) channel activation in their finding is unexpected since gabapentin did not pyramidal neurons of rodent hippocampal slices, in a affect the presynaptic GABAB receptors on glutamater- way not similar to baclofen (61 – 64). gic terminals in hippocampus slices (67). Nevertheless, this conclusion was disproved by several studies in vitro and in vivo. First, gabapentin was 4-1-3. GABA level proved to be completely inactive at the recombinant In addition to interacting with GABA receptors, GABAB1a-B2 or GABAB1b-B2 heterodimeric receptors gabapentin has been reported to increase brain GABA expressed in Xenopus oocytes or mammalian cells levels. It seems that gabapentin does not inhibit GABA (65 – 67). Second, gabapentin failed to displace uptake directly through inhibiting GABA transporter 3 [ H]GABA from GABAB-receptor sites in rat synaptic (73) unless the concentration of gabapentin is high (74). membranes (66). Third, gabapentin had no effect in the On the contrary, gabapentin may enhance promoted GTP-γ-S binding assay, suggesting its effect is not a G- release of GABA, which is thought to be due to reverse protein coupled event (67). Fourth, gabapentin, unlike operation of the GABA transporter (75), through baclofen, was ineffective in activating the GABAB increasing GABA uptake by a redistribution of GABA receptors that mediate GIRK channel activation in transporter protein from intracellular locations to the ventrolateral neurons of rat periaqueductal gray (PAG) plasma membrane (73). Gabapentin also did not affect slices (68) and that in pyramidal neurons of rat hippo- the enzymes and intermediates of the GABA shunt in campal slices (67), the same preparations used by Ng’s rat brain and retina (76). group. It was also ineffective in inducing GABAB- In healthy volunteers and epileptic patients, brain receptor-mediated inhibition of transient lower esopha- GABA level was reported to be increased after oral geal sphincter relaxation in vivo (66). Fifth, the GABAB intake of high doses of gabapentin (77, 78). This may be antagonist saclofen failed to affect the gabapentin due to the ability of gabapentin to increase glutamic acid inhibition of the peak whole-cell Ca2+ current in cultured decarboxylase activity (79) or to promote non-vesicular rat dorsal root ganglion (DRG) neurons (69). release of GABA (75). However, using proton magnetic In the superficial laminae of the spinal cord where resonance spectroscopy, Errante and Petroff (80) failed nociceptive primary afferent fibers terminate, the pre- to observe elevated GABA level in gabapentin-treated dominant subtype of GABAB receptors is the GABAB1a-B2 rats. heterodimeric type (70), which was proposed to be Elevated GABA level in the brain has been proposed selectively activated by gabapentin (61, 62). It is reason- to contribute to the antiepileptic and anxiolytic actions able to suggest that spinal GABAB receptors might be of gabapentin (1, 81). So far, there is no report indicating the target of the antinociceptive effect of intrathecal that the analgesic action of gabapentin is attributed to gabapentin (61). However, GABAB-receptor antago- an elevation of GABA level. Since GABA-receptor nists, at the doses that effectively antagonize the antagonists did not attenuate the antinociceptive effect antinociceptive effect of baclofen, failed to affect the of gabapentin in several animal pain models (see effect of gabapentin in L5/6 nerve ligation (26, 71), sections 4-1-1 and 4-1-2), it is unlikely that the anti- partial sciatic nerve ligation (21), and postoperative (68) nociceptive effect of gabapentin is mediated through pain models. Therefore, it is unlikely that the GABAB elevated GABA level. receptor is the analgesic action target of gabapentin. The possibility remains that GABAB-receptor acti- 4-2. Glutamatergic system vation is involved in the therapeutic actions of gabapen- 4-2-1. AMPA receptors tin other than analgesia. It has been reported that Gabapentin was found to inhibit both the excitatory Antinociceptive Mechanisms of Gabapentin 5 synaptic transmission in vitro (82) and the neuronal nists of NMDA or GABA receptors did not reverse the response to noxious electrical and mechanical stimula- antinociceptive effects of gabapentin in animal pain tion in vivo (83) mediated by α-amino-3-hydroxy-5- models (21, 26, 58, 59, 68). In addition, NMDA-receptor methyl-4-isoxazolepropionic acid (AMPA), but not antagonists shared the same actions of gabapentin in those mediated by N-methyl-D-asparate (NMDA) recep- inhibiting morphine tolerance (49, 93) and potentiating tors. It is, therefore, suggested that gabapentin acts as morphine analgesia (94, 95). Therefore, it is unlikely an AMPA-receptor antagonist in the rat spinal cord to that gabapentin acts as an NMDA-receptor enhancer to exert its spinal antinociceptive effect. However, this exert its analgesic effect. possibility could be neglected by the following findings: First, intrathecal gabapentin had a synergistic analgesic 4-2-3. Glutamate level effect with CNQX, a non-NMDA antagonist, in the Gabapentin has been reproted to decrease the nerve ligation neuropathic pain model (84). Second, in glutamate level in the brain (80). This effect of gaba- the formalin test, intrathecal gabapentin worked syner- pentin was suggested to contribute to its neuroprotective gistically with MK-801 and NBQX, NMDA- and effect observed in animal models of amyotrophic lateral AMPA-receptor antagonists, respectively (85). There- sclerosis (96), possibly via an inhibition of glutamate fore, the AMPA receptor might not be the action target synthesis by branched-chain amino acid aminotrans- of gabapentin, at least for its spinal analgesic effect. ferase (1, 97). Nevertheless, clinical trials failed to demonstrate the effectiveness of gabapentin in the 4-2-2. NMDA receptors treatment of amyotrophic lateral sclerosis (98). A recent D-Serine, an agonist at the glycine binding site of study using a double-isotope technique also proved NMDA receptors (86), was found to reverse the anti- that gabapentin did not affect glutamate metabolism in nociceptive effects of gabapentin in the formalin test the rat brain (99). (16) and thermal- or substance P-induced hyperalgesia (87, 88). It, therefore, has been suggested that gaba- 4-3. Gabapentin and system L-amino acid trans- pentin exerts its analgesic action through a negative porter interaction with the glycine binding site of NMDA Gabapentin is a spiral compound with both amine receptors. However, gabapentin does not interact and acid terminals mimicking an α-amino acid (Fig. 1). directly with the glycine/NMDA-receptor complex Therefore, it is susceptible to the system L-α-amino acid (57, 89). transporter that is responsible for the entry of α-amino Suarez et al. (90) reported that gabapentin depressed, acids into cells (100) and their intestinal absorption but NMDA enhanced, the presynaptic fiber volley (the (101). L-Leucine, a branched-chain L-amino acid that presynaptic terminal action potentials recorded extra- could inhibit the L-amino acid transporter, did not affect cellularly) in the CA1 region of rat hippocampal slices. the antinociceptive effects of intrathecal gabapentin in The depressant effect of gabapentin was occluded by the postoperative pain model (28) and the formalin test NMDA-receptor antagonists, and the enhancing effect (58). This suggests that the analgesic effect of intrathecal of NMDA was blocked by lowering extracellualr Na+ gabapentin is independent of the L-amino acid trans- concentration, but not affected by increasing K+ concen- porter. Similarly, the inhibition by gabapentin on K+- trations. Therefore, they suggested that the effect of evoked norepinephrine release from rat neocortical gabapentin might be mediated via a decrease of Na+ slices was also proved to be independent of the L-amino influx through presynaptic NMDA receptors (90). acid transporter (102). Therefore, it might not be In the postoperative pain model, which is insensitive necessary for gabapentin to be transported into cells to to NMDA-receptor antagonists (91), intrathecal gaba- exert its cellular actions. pentin produces an effective and consistent antiallodynic Several gabapentin analogues with high binding 2+ effect (27, 28). Therefore, a negative interaction with affinity to the α2δ subunit of Ca channels, a specific NMDA-receptor channels might not be the main binding site of gabapentin (see section 4-4-4-1), have mechanism contributing to the analgesic effect of been developed and shown to have similar analgesic gabapentin. and anticonvulsant activity as gabapentin (103 – 105). On the other hand, Gu and Huang (92) found that One of these compounds, compound 34 which did not gabapentin enhanced the NMDA current selectively in bind to the system L-amino acid transporter, had an the GABAergic neurons of spinal dorsal horn in anticonvulsant effect in vivo when given intracere- inflamed rats. This finding raises the possibility that broventricularly but not orally (103). Therefore, the L- gabapentin might exert its antinociceptive effect through amino acid transporter may be important for the active enhancing spinal GABA release. However, the antago- transport of gabapentin through the blood-brain barrier 6 J-K Cheng and L-C Chiou or intestinal absorption but may not be directly involved ing Ih currents. in its cellular actions. 4-4-3. Voltage-dependent Na+ channels 4-4. Ionic channels Gabapentin has been shown to limit repetitive firing + 4-4-1. KATP channels of Na -dependent action potentials of cultured spinal Glibenclamide, a blocker of adenosine triphosphate- cord and neocortical neurons (117). Systemic gaba- + sensitive K (KATP) channels, was found to antagonize pentin and its analogue pregabalin, which is also effec- gabapentin-induced inhibition of [3H]-noradrenaline tive in the management of neuropathic pain, have been release in rat hippocampal slices (106) as well as its found to suppress ectopic discharges from injured sciatic spinal analgesic effect in the nerve ligation neuropathic nerves in neuropathic rats (118, 119). Kanai et al. (120) pain model (107). Therefore, the KATP channel was reported that gabapentin decreased the number of action suggested to be the action target of gabapentin. How- potentials during depolarization in injured DRG neurons ever, in the postoperative pain model, glibenclamide from neuropathic rats. Since voltage-dependent Na+ failed to reverse the antiallodynic effect of intrathecal channels are implicated in the generation of ectopic gabapentin and the KATP channel openers pinacidil and discharge activity of injured nerves (121, 122), it is diazoxide are ineffective in the same model (59). In suggested that gabapentin might exert its analgesic primary afferent neurons, gabapentin also failed to action by affecting the Na+ channels at the injured nerves affect the KATP current that was sensitive to both (118). However, gabapentin was found to have no effect + glibenclamide and KATP channel openers (108). The on neuronal Na channels in cultured DRG, nodose discrepancy among studies may be due to that the ganglia, and cortical neurons (123) or isolated cortical + contribution of KATP channels in different assay systems neurons (124). It remains to be clarified if Na channels is different. Alternatively, glibenclamide might also play a role in its antinociceptive action. affect other ionic channels (109 – 112). It is not know if this nonspecific effect of glibenclamide would 4-4-4. Voltage-dependent Ca2+ channels 2+ contribute to the positive effect of glibenclamide 4-4-4-1. α2δ Subunit of Ca channels: In 1993, observed in the above studies (106, 107). Suman-Chauhan et al. (57) identified a high affinity binding site for gabapentin in rat brain homogenate 4-4-2. Hyperpolarization-activated cation current (Ih) using [3H]gabapentin as the radioligand. Gee et al. (125) Surges et al. (113) reported that gabapentin at clini- later identified this specific binding site of gabapentin to 2+ cally relevant concentrations (50 – 100 µM) increased be the α2δ subunit of voltage-dependent Ca channels. the hyperpolarization-activated cation current (Ih) in The α2δ subunit as well as the β subunit are the auxiliary rat CA1 pyramidal cells through a cAMP-independent subunits of all types of Ca2+ channels and their coexpres- mechanism. This effect, resulting in a 2.4 mV depolar- sion with the pore-forming α1 subunit results in a ization of the neuronal membrane potential, was pro- significant increase in whole cell Ca2+ current (126). The 2+ posed to contribute to the antiepileptic effect of gaba- α2δ subunit also plays a role in Ca channel membrane pentin by decreasing the sensitivity to excitatory inputs incorporation (127, 128). By binding to the α2δ subunit, (114). However, we did not observe membrane depolar- gabapentin might affect Ca2+ currents to modulate ization induced by gabapentin at concentrations up to neurotransmitter release (7, 129) or neuronal excitability 300 µM in ventrolateral neurons of rat PAG slices (68). (130). Scott et al. (9) have summarized the effect of Up to now, there is no study investigating the role of gabapentin on Ca2+ currents in cultured DRG neurons Ih channels in the antinociceptive effect of gabapentin. and its relationship with the expression of α2δ and β Nevertheless, it seems that activating Ih channels would subunits. result in pronociceptive but not antinociceptive action. Synthetic analogues of gabapentin with high binding First, the amplitude of Ih in DRG neurons isolated affinity at the α2δ subunit have been developed and from the nerve-ligated neuropathic rats was significantly shown to have anticonvulsant and antinociceptive increased and the Ih channel blocker ZD7288 reversed activities in vivo (103, 105, 131, 132). However, the the nociceptive response in this pain model (115), in functional consequence of gabapentin binding to the α2δ which gabapentin is an effective analgesic (17, 84). subunit is still not well established. In the postoperative Recently, Sun et al. (116) found that ZD7288, by inhi- pain model (133), intrathecal injection of magnesium biting Ih current, suppressed the ectopic discharges of chloride and ruthenium red, which both modulated the 3 injured DRG neurons in a gabapentin-sensitive neuro- [ H]gabapentin binding at the α2δ subunit (57, 89, 134), pathic pain model. Therefore, it is unlikely that gaba- attenuated the antiallodynic effect of intrathecal gabapentin exerts its antinociceptive effect through enhanc- pentin. Pregabalin, a gabapentin analogue also with α2δ Antinociceptive Mechanisms of Gabapentin 7 binding affinity, had less analgesic effect in R217A (a1G), the pore-forming α1 subunit of R-, P/Q-, and T- 2+ mutant mice, in which the amino acid (217th) of the α2δ type Ca channels, respectively (142). subunit critical for gabapentin binding was mutated from It remains to be further elucidated if the α2δ subunits arginine to alanine (135). These findings suggested that other than α2δ-1, especially the α2δ-2, are also changed the α2δ subunit is involved in the antinociceptive action in animal pain models since it was noted that the of gabapentin. gabapentin-sensitive cultured DRG neurons, compared Cunningham et al. (136) found that the inhibitory to the insensitive ones, have lower α2δ-2 and higher β effect of gabapentin on the frequency of miniature levels in the mRNA expression (148). excitatory postsynaptic currents (mEPSCs) was pre- 4-4-4-3. Types of Ca2+ channels involved in gaba- vented by L-isoleucine, an L-amino acid transporter pentin actions: Given that the α2δ subunit was proposed inhibitor. This effect was suggested to be an intracellular to be the action target of gabapentin and all types of 2+ effect of gabapentin rather than an action via the α2δ Ca channels consist of α2δ subunits, it is not surprising subunit that is mostly located extracellularly (137) since to see that gabapentin affects various types of Ca2+ mEPSC is an event mainly dependent on the Ca2+ channels, depending on the tissues or preparations coming from the intracellular stores rather than that used. Stefani et al. (124, 130) reported that gabapentin from extracellular Ca2+ entry (138). However, Sutton decreased predominantly L-type Ca2+ currents in neo- et al. (69) found that L-isoleucine attenuated gabapentin- cortical and, to a lesser extent, in striatal and globus induced inhibition of whole-cell Ca2+ current in DRG pallidus neurons. Gabapentin decreased the current neurons. Hence, L-isolucine might have a direct action sensitivity to both dihydropyridine antagonists and the on the α2δ subunit since it also competed with gaba- agonist Bay K 8644 in cortical pyramidal neurons. pentin in α2δ subunit binding (102). Scott et al. (9) also noted that Bay K 8644 enhanced the 2+ 4-4-4-2. Upregulation of α2δ-1 subunit in pain current flow through L-type Ca channels in cultured models: Four members of the α2δ subunit family, α2δ-1 DRG neurons treated with gabapentin. Alden and Garcia (139), α2δ-2 (140), α2δ-3 (140), and α2δ-4 (141), have (149) found that gabapentin inhibited the excitation- been identified so far. Northern analysis has shown that contraction coupling in skeletal myotubes, which is 2+ α2δ-1 is ubiquitously expressed; α2δ-2 is found in several mediated by L-type Ca channels. Oka et al. (150) + tissues including brain and heart; α2δ-3 is brain-specific; found that gabapentin inhibited K -evoked nitric oxide 2+ and α2δ-4 has limited distribution in special cell types synthase activation via blocking L- and P/Q-type Ca of the pituitary, adrenal gland, colon, and fetal liver. channels in cultured murine cortical neurons. However, Primary sequence comparison suggests that α2δ-3 and it exerts neuroprotection through inhibiting hypoxia- α2δ-4 lack the gabapentin binding motifs characterized induced nitric oxide synthesis in rat cerebrocortical 2+ for α2δ-1 and α2δ-2 (141 – 143). Gabapentin has higher slices via blocking N- and P/Q-type Ca channels + affinity at α2δ-1 than that at α2δ-2 (KD: 59 nM vs (151). Gabapentin inhibited K -evoked norepinephrine 153 nM) (142). release in rat neocortical slices (102) and glutamate The α2δ-1 subunits, but not the pore-forming α1 or β release in entorhinal cortical slices (136) through subunits, of Ca2+ channels in the spinal cord and DRG inhibiting presynaptic P/Q-, but not L- and N-, type Ca2+ were found to be upregulated in the gabapentin-sensitive channels. Chronic treatment with gabapentin in the pain models such as the mechanical- and diabetic- PC12 cell line resulted in significant decrease in the neuropathic models (144 – 146) and the postoperative number of membrane N-type Ca2+ channels (152). pain model (147), but not in the gabapentin-insensitive Sutton et al. (69) found that gabapentin decreased the chemical neuropathic pain model (146). Intrathecal whole cell Ca2+ current mainly gated by N-type Ca2+ pretreatment with antisense oligonucleotide against the channels in cultured DRG neurons. The IC50 of 2+ α2δ-1 subunit diminished the tactile allodynia in neuro- gabapentin in inhibiting Ca current is simialr to its KD pathic rats (145). The upregulation of the α2δ-1 subunit value in the α2δ binding (142) and the inhibitiory effect has a time course parallel to the duration of nerve injury- was saturated at a concentration close to the therapeutic induced allodynia, which could be alleviated by intra- plasma concentration of gabapentin in the treatment of thecal gabapentin (144). It is, therefore, suggested that epilepsy and neuropathy (10 – 100 µM) (62, 153). 2+ 2+ the α2δ-1 subunit of Ca channels may be the anti- Therefore, they suggested that the α2δ subunit of Ca nociceptive target of intrathecal gabapentin. The α2δ-1 channels is the therapeutic action target of gabapentin. subunit is known to enhance dihydropyridine binding to The effects of gabapentin and all types of Ca2+ L-type Ca2+ channels and ω-conotoxin GVIA to N-type channel blockers on nociceptive behaviors in vairous 2+ Ca channels, and α2δ-2 and α2δ-3 may preferentially animal pain models are summerized in Table 3. It 2+ interact with Cav2.3 (a1E), Cav2.1 (a1A), and even Cav3.1 demonstrates that intrathecal L-type Ca channel 8 J-K Cheng and L-C Chiou

Table 3. Comparison of the effectiveness of gabapentin and Ca2+ channel blockers in various animal pain models Ca2+ channel blockers (Reference) Pain model Route Gabapentin L-type P/Q-type T-type N-type Acute pain Tail-flick test i.t. + verapamil (169) + ω-AGA (170) − mibefradil (171) + ω-CTX (172) − gabapentin (15) (300 – 500 µg) (0.33–33pmol)* (3–20µg)* (16–64pmol) (200 µg) + diltiazem (169) + neomycin (173) (300 – 500 µg) (10–80µg)* i.p. − nimodipine (174) N.D. + ethosuximide (175) + gentamicin (176) − gabapentin (24) (1 mg/kg) (30–600mg/kg) (11.5 µg/kg)a (30 – 300 mg/kg) − lercanidipine(174) + trimethadione (175) (0.3 mg/kg) (100 – 600 mg/kg) i.c. N.D. N.D. − mibefradil (175) N.D. N.D. (3–30µg) Hot plate test i.t. − verapamil (177) − ω-AGA (177) N.D. + SNX111 (178) − gabapentin (168) (200 nmol) (0.006 nmol) (0.03 nmol/h)b (0.1 – 100 nmol) + ziconotide (179) (0.03 µg/h)b i.p. N.D. N.D. N.D. + gentamicin (176) − gabapentin (168) (147.9 µg/kg) *,a (10 – 100 mg/kg) Plantar thermal test i.t. N.D. N.D. N.D. N.D. − gabapentin (14) (10 – 300 µg) i.p. + verapamil (180) N.D. + mibefradil (181) N.D. N.D. (6–9mg/kg) (3–9mg/kg) Paw pressure test i.t. N.D. N.D. N.D. + ziconotide (179) N.D. (0.03 – 1 µg) s.c. N.D. N.D. N.D. N.D. − gabapentin (19) (30 – 300 mg/kg) Von Frey hair test i.p. + verapamil (180) N.D. + mibefradil (181) N.D. N.D. (3–18mg/kg) (3–9mg/kg)

Neuropathic pain Nerve injury- i.t. − nimodipine (154) − ω-AGA (154) − mibefradil (182) + SNX111 (154) + gabapentin (26) induced (500 µg) (0.3 µg) (10–40µg) (3 µg) (10 – 300 µg) − verapamil (154) − ethosuximide (182) + SNX159 (154) + gabapentin (21) (250 µg) (10–40µg) (1 µg) (10 – 300 µg) − diltiazem (154) + SNX239 (154) (500 µg) (3.3 µg) + SNX111 (154) (30 – 300 ng) i.p./s.c. N.D. N.D. + mibefradil (182) N.D. + gabapentin (21) (5–20mg/kg) (10 – 250 mg/kg) + ethosuximide (182) + gabapentin (17) (50–300mg/kg) (10 – 100 mg/kg) i.pl. − nifedipine (183) − SNX-230 (183) + mibefradil (182) + SNX111 (183) N.D. (0.25 nmol) (0.25 nmol) (30–300µg) (0.25 nmol) − ethosuximide (182) (500 µg) Chemotherapy- i.t.* − calciseptine (184) − ω-AGA (184) N.D. + ω-CTX (184) N.D. induced (1–33pmol) (0.33–10pmol) (0.33–1pmol) i.p. N.D. N.D. + ethosuximide (185) N.D. − gabapentin (146) (300 – 450 mg/kg) (50 – 300 mg/kg) p.o. N.D. N.D. N.D. N.D. + gabapentin (186) (400 µmol/kg)a

Postoperative pain i.t. − nimodipine (59) − ω-AGA (59) − mibefradil (59) + SNX111 (155) + gabapentin (28) (500 µg) (0.3 µg) (600 µg) (0.3 µg) (10 – 100 µg) − verapamil (59) + ω-CTX (59) (300 µg) (0.1 – 3 µg) − diltiazem (59) (500 µg) s.c. N.D. N.D. N.D. N.D. + gabapentin (27) (3–30mg/kg) Antinociceptive Mechanisms of Gabapentin 9

Table 3. (continued) Ca2+ channel blockers (Reference) Pain model Route Gabapentin L-type P/Q-type T-type N-type Inflammatory pain Formalin test i.t. − nifedipine (177) + ω-AGA (177) + mibefradil (187) + SNX111 (156) + gabapentin (15) (24 nmol) (0.2–8pmol) (50–500µg) (100 ng)c (6 – 200 µg)c

− nimodipine (177) + NiCl2 (187) + SNX111 (177) + gabapentin (14) (29 nmol) (1–10µg) (0.003 nmol) (10 – 300 µg)c − verapamil (177) − ethosuximide (187) (200 nmol) (100 – 1200 µg) − diltiazem (177) (220 nmol) i.p./s.c. + nifedipine (188) N.D. + ethosuximide (175) N.D. + gabapentin (19) (1–30mg/kg) (100 – 450 mg/kg) (10 – 300 mg/kg)c + nimodipine (188) + trimethadione (175) + gabapentin (17) (1–30mg/kg) (600 mg/kg)c (100 mg/kg)c + verapamil (188) (1–30mg/kg) + diltiazem (188) (1–30mg/kg) i.c. N.D. N.D. + mibefradil (175) N.D. N.D. (10–30µg) i.pl. N.D. N.D. N.D. N.D. + gabapentin (16) (6c, 60c, 600 µg) Chemicals other i.t. − nifedipine (158) + ω-AGA (158) N.D. + SNX111 (158) + gabapentin (19) than formalin (0.01–1mM)d (0.01 – 1 µM)d (0.001 – 0.1 mM)d (1 – 100 µg) + AM336 (157) (0.11 nmol) i.p. N.D. N.D. + ethosuximide (175) + gentamicin (176) N.D. (30–600mg/kg) (200 – 800 µg/kg) + trimethadione (175) (100 – 600 mg/kg) i.c. N.D. N.D. + mibefradil (175) N.D. N.D. (30 µg) p.o. N.D. N.D. N.D. N.D. + gabapentin (189) (10 – 100 mg/kg) + gabapentin (190) (10 – 300 mg/kg)

Visceral pain Colorectal distension i.t. + verapamil (169) N.D. N.D. N.D. N.D. (300 – 500 µg) + diltiazem (169) (300 – 500 µg)

Acetic acid i.t.* + nicardipine (191) N.D. − NiCl2 (191) + gentamicin (191) N.D. writhing test (0.5 – 80 µg) (2.5 – 10 µmol) (5–40µg) + diltiazem (191) (0.5 – 80 µg) + verapamil (191) (0.5 – 80 µg) i.p. N.D. N.D. N.D. N.D. + gabapentin (159) (50 – 200 mg/kg) All experiments were conducted in rats except that denoted by * was performed in mice. −: ineffective, +: effective, N.D.: not determined, i.t.: intrathecal, i.p.: intraperitoneal, s.c.: subcutaneous, i.pl.: intraplantar, i.c.: intracisternal, p.o.: per oral, ω-AGA: ω-agatoxin IVA, ω-CTX: a b c d ω-conotoxin GVIA. : ED50, : intrathecal infusion, : phase 2 only, : microdialysis fiber infusion. blockers are ineffective in most of the pain models Ca2+ channel blockers, when administered intrathecally, except the acute and visceral pain models. The P-type are exclusively effective in the formalin test, while they Ca2+ channel blocker, ω-agatoxin IVA, is effective in are effective when given systemically in both acute and certain inflammatory and acute pain models. The T-type inflammatory pain models, but not in neuropathic, 10 J-K Cheng and L-C Chiou postoperative, and visceral pain models. However, N- the spinal tissues only after inflammation or protein type Ca2+ channel blockers are effective in almost all kinase activation. Patel et al. (161) reported that the models. Gabepentin is also effective in many models inhibition of EPSCs by gabapentin was only observed (Table 3). It is interesting to note that Bay K 8644, the in the spinal dorsal horn neurons of diabetic rats, but L-type Ca2+ channel opener, reversed the antiallodynic not that of normal rats. Similarly, gabapentin depressed effect of gabapentin in the postoperative pain model Aδ fiber-evoked polysynaptic EPSCs in substantia (59). The finding of Scott et al. (9) that Bay K 8644 gelatinosa neurons from carrageenan-inflamed, but not enhanced L-type Ca2+ current in the presence of normal, rats (162). Fehrenbacher et al. (163) found that gabapentin also suggests that Bay K 8644 interacts with gabapentin reduced the release of substance P and gabapentin in cutured DRG neurons. calcitonin-gene related peptide from rat spinal tissues Comparing the effectiveness of gabapentin in various only after inflammation or activation of protein animal pain models (Table 3), it is found that intrathecal kinase C. Gabapentin decreased K+-evoked glutamate administration of N-type, but not L-, T-, and P/Q-type, release in caudal trigeminal nuclear slices isolated Ca2+ channel blockers produced antinociceptive effects from diabetic rats (164) but not that from normal rats in the gabapentin-sensitive models, including the nerve (165). The inhibition of gabapentin was also observed in ligation neuropathic (26, 154), postoperative (28, 59, slices of normal rats where glutamate release had been 155), formalin (14, 156), and other inflammatory (19, facilitated by substance P (165) or by the activator of 157, 158) pain models (Table 3). It is, therefore, protein kinase C or adenylyl cyclase (166). These suggested that spinal N-type Ca2+ channels may be findings are in agreement with the reports that gaba- involved in the antinociceptive effect of intrathecal pentin exerts its antinociceptive effects only in inflam- gabapentin. This does not mean that gabapentin selec- matory or neuropathic pain models but not in acute tively interacts with N-type Ca2+ channels but that the N- physiological pain models (Table 1). Taken together, it type Ca2+ channels are important analgesic targets in the is suggested that only under pathological status such as spinal cord. The possibility remains that other types of inflammation or certain protein phosphorylation (7, Ca2+ channels are involved in other therapeutic actions 167), does gabapentin exert its antinociceptive actions. of gabapentin. 5. Conclusions 4-4-5. Synaptic transmission The consequence of inhibiting Ca2+ channels by gaba- Gabapentin, with the merit of minor side effects at pentin, through binding to the α2δ subunit, would result clinical effective doses, has make itself a promising drug in a decrease of neuronal excitatibility and synaptic clinically in several CNS disorders even though the transmission. Indeed, gabapentin was reported to inhibit mechanism(s) of action is still unclear. Among the syantpic transmission presynaptically through reducing proposed targets of its analgesic action, the α2δ subunit Ca2+ influx in several brain slices or synaptosome pre- of N-type Ca2+ channels is the most promising one. It parations. remains to be elucidated how gabapentin exerts its Gabapentin inhibited glutamatergic synaptic trans- analgesic action through binding at the α2δ subunit of N- mission presynaptically in the superficial, but not deep, type Ca2+ channels. Further studies are needed to clarify lamina of the spinal dorsal horn in normal rats (82). The if conditional protein phosphorylation or inflammatory in vivo studies also showed that gabapentin reduced changes are required for gabapentin to exert its thera- excitatory amino acid release in the spinal cord in peutic effects and if these changes are mediated through several pain models: Feng et al. (159) found that sys- intracellular signalling pathways. temic administration of gabapentin decreased the release of glutamate and aspartate in the spinal cord, which was Acknowledgments elicited by intraperitoneal injection of acetic acid. Coderre et al. (25) demonstrated that gabapentin reduced This work was supported by grants NSC94-2314-B- paw formalin-injection-induced spinal glutamate release 195-006 (J.K.C.) and NSC94-2320-B002-034 (L.C.C.) in both naive and neuropathic rats. Lin et al. (160) found from National Science Council, Taiwan; MMH-9416 that the intrathecal gabapentin attenuated morphine (J.K.C.) and MMH-9507 (J.K.C.) from Mackay tolerance possibly through a suppression of morphine- Memorial Hospital; NHRI-EX94-9005NC (L.C.C), evoked excitatory amino acid release in the rat spinal NHRI-EX95-9506NI (L.C.C.) from National Health cord. Research Institutes, Taiwan; and DOH94-NNB-1019 Several lines of evidence indicate that gabapentin (L.C.C.) and DOH95-NNB-1025 (L.C.C.) from inhibits the excitaory transmission in sensory neurons of National Bureau of Controlled Drugs, Department of Antinociceptive Mechanisms of Gabapentin 11

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