Analgesia with Gabapentin and Pregabalin May Involve N-Methyl-d-Aspartate Receptors, Neurexins, and Thrombospondins.

The gabapentinoid drugs gabapentin and pregabalin (Neurontin and Lyrica) are mainstay treatments for neuropathic pain and preventing focal seizures. Both drugs have similar effects to each other in animal models and clinically. Studies have shown that a protein first identified as an auxiliary subunit of voltage-gated calcium channels (the α 2 δ-subunit type 1 [α 2 δ-1], or Ca V a2d1) is the high-affinity binding site for gabapentin and pregabalin and is required for the efficacy of these drugs. The α 2 δ-1 protein is required for the ability of gabapentin and pregabalin to reduce neurotransmitter release in neuronal tissue, consistent with a therapeutic mechanism of action via voltage-gated calcium channels. However, recent studies have revealed that α 2 δ-1 interacts with several proteins in addition to voltage-gated calcium channels, and these additional proteins could be involved in gabapentinoid pharmacology. Furthermore, gabapentin and pregabalin have been shown to modify the action of a subset of N-methyl-d-aspartate-sensitive glutamate receptors, neurexin-1α, and thrombospondin proteins by binding to α 2 δ-1. Thus, these effects may contribute substantially to gabapentinoid therapeutic mechanism of action. SIGNIFICANCE STATEMENT: It is widely believed that gabapentin and pregabalin act by modestly reducing the membrane localization and activation of voltage-gated calcium channels at synaptic endings in spinal cord and neocortex via binding to the α 2 δ-1 protein. However, recent findings show that the α 2 δ-1 protein also interacts with N-methyl-d-aspartate-sensitive glutamate receptors, neurexin-1α, thrombospondins (adhesion molecules), and other presynaptic proteins. These newly discovered interactions, in addition to actions at calcium channels, may be important mediators of gabapentin and pregabalin therapeutic effects.

Anxiolytic-like activity of pregabalin in the Vogel conflict test in α2δ-1 (R217A) and α2δ-2 (R279A) mouse mutants.

The α(2)δ auxiliary subunits (α(2)δ-1 and α(2)δ-2) of voltage-sensitive calcium channels are thought to be the site of action of pregabalin (Lyrica), a drug that has been shown to be anxiolytic in clinical trials for generalized anxiety disorder. Pregabalin and the chemically related drug gabapentin have similar binding and pharmacology profiles, demonstrating high-affinity, in vitro binding to both α(2)δ-1 and α(2)δ-2 subunits. Two independent point mutant mouse strains were generated in which either the α(2)δ-1 subunit (arginine-to-alanine mutation at amino acid 217; R217A) or the α(2)δ-2 subunit (arginine-to-alanine mutation at amino acid 279; R279A) were rendered insensitive to gabapentin or pregabalin binding. These strains were used to characterize the activity of pregabalin in the Vogel conflict test, a measure of anxiolytic-like activity. Pregabalin showed robust anticonflict activity in wild-type littermates from each strain at a dose of 10 mg/kg but was inactive in the α(2)δ-1 (R217A) mutants up to a dose of 320 mg/kg. In contrast, pregabalin was active in the α(2)δ-2 (R279A) point mutants at 10 and 32 mg/kg. The positive control phenobarbital was active in mice carrying either mutation. These data suggest that the anxiolytic-like effects of pregabalin are mediated by binding of the drug to the α(2)δ-1 subunit.

Anxiolytic profile of pregabalin on elicited hippocampal theta oscillation.

Previous published work with the novel anticonvulsant, analgesic and anti-anxiety medication, pregabalin (Lyrica), has shown that it has anxiolytic-like actions in several animal behavioral models. However, pregabalin is structurally and pharmacologically different from other classes of known anxiolytic drugs, and the mechanisms that alter brain activity to produce anxiolytic-like actions are not well understood. In an effort to determine more about the cellular mechanisms of pregabalin, we studied its effects on hippocampal theta activity of urethane-anesthetized rats that was elicited by electrical stimulation of the nucleus pontis oralis (nPO) in the brainstem. We found that systemic administration of pregabalin significantly reduced the frequency of stimulation-induced hippocampal theta activity similarly to the effects of diazepam. In addition, pregabalin (but not diazepam) significantly altered the stimulus intensity/frequency relationship, and increased slow delta oscillation (<3.0Hz) in spontaneous hippocampal EEG in a dose-dependent manner. Our findings suggest that pregabalin may alter evoked theta frequency activity in the hippocampus by reducing neurotransmitter-mediated activation of either the septal nucleus or the hippocampus, and that its actions are unlikely to be mediated by direct activation of GABA neurotransmitter systems. These observations provide further insight to the action of pregabalin, and support the utilization of stimulation-induced theta model in discovery of novel anxiolytic drugs.

Oxadiazolone bioisosteres of pregabalin and gabapentin.

A series of oxadiazolone bioisosteres of pregabalin 1 and gabapentin 2 were prepared, and several were found to exhibit similar potency for the alpha(2)-delta subunit of voltage-gated calcium channels. Oxadiazolone 9 derived from 2 achieved low brain uptake but was nevertheless active in models of osteoarthritis. The high clearance associated with compound 9 was postulated to be a consequence of efflux by OAT and/or OCT, and was attenuated on co-administration with cimetidine or probenecid.

Ca2+ channel alpha2delta ligands: novel modulators of neurotransmission.

The term 'Ca2+ channel alpha2delta ligands' has recently been applied to an evolving drug class that includes gabapentin (Neurontin) and pregabalin (Lyrica), and reflects significant progress over the past decade in elucidating the mechanism of action of these drugs: a novel, specific action at one of the subunits constituting voltage-sensitive Ca2+ channels. Binding of these ligands to the alpha2delta subunit is considered to explain their usefulness in treating several clinical disorders, including epilepsy, pain from diabetic neuropathy, postherpetic neuralgia and fibromyalgia, and generalized anxiety disorder. The evidence indicates a relationship between alpha2delta subunit binding and the modulation of processes that subserve neurotransmission. This modulation is characterized by a reduction of the excessive neurotransmitter release that is observed in certain neurological and psychiatric disorders.

Pharmacology and mechanism of action of pregabalin: the calcium channel alpha2-delta (alpha2-delta) subunit as a target for antiepileptic drug discovery.

Pregabalin (Lyrica) is a new antiepileptic drug that is active in animal seizure models. Pregabalin is approved in US and Europe for adjunctive therapy of partial seizures in adults, and also has been approved for the treatment of pain from diabetic neuropathy or post-herpetic neuralgia in adults. Recently, it has been approved for treatment of anxiety disorders in Europe. Pregabalin is structurally related to the antiepileptic drug gabapentin and the site of action of both drugs is similar, the alpha2-delta (alpha2-delta) protein, an auxiliary subunit of voltage-gated calcium channels. Pregabalin subtly reduces the synaptic release of several neurotransmitters, apparently by binding to alpha2-delta subunits, and possibly accounting for its actions in vivo to reduce neuronal excitability and seizures. Several studies indicate that the pharmacology of pregabalin requires binding to alpha2-delta subunits, including structure-activity analyses of compounds binding to alpha2-delta subunits and pharmacology in mice deficient in binding at the alpha2-delta Type 1 protein. The preclinical findings to date are consistent with a mechanism that may entail reduction of abnormal neuronal excitability through reduced neurotransmitter release. This review addresses the preclinical pharmacology of pregabalin, and also the biology of the high affinity binding site, and presumed site of action.

Calcium channel alpha2-delta type 1 subunit is the major binding protein for pregabalin in neocortex, hippocampus, amygdala, and spinal cord: an ex vivo autoradiographic study in alpha2-delta type 1 genetically modified mice.

Pregabalin is a synthetic amino acid compound effective in clinical trials for the treatment of post-herpetic neuralgia, diabetic peripheral neuropathy, generalized anxiety disorder and adjunctive therapy for partial seizures of epilepsy. However, the mechanisms by which pregabalin exerts its therapeutic effects are not yet completely understood. In vitro studies have shown that pregabalin binds with high affinity to the alpha(2)-delta (alpha(2)-delta) subunits (Type 1 and 2) of voltage-gated calcium channels. To assess whether alpha(2)-delta Type 1 is the major central nervous system (CNS) binding protein for pregabalin in vivo, a mutant mouse with an arginine-to-alanine mutation at amino acid 217 of the alpha(2)-delta Type 1 protein (R217A mutation) was generated. Previous site-directed mutagenesis studies revealed that the R217A mutation dramatically reduces alpha(2)-delta 1 binding to pregabalin in vitro. In this autoradiographic analysis of R217A mice, we show that the mutation to alpha(2)-delta Type 1 substantially reduces specific pregabalin binding in CNS regions that are known to preferentially express the alpha(2)-delta Type 1 protein, notably the neocortex, hippocampus, basolateral amygdala and spinal cord. In mutant mice, pregabalin binding was robust throughout regions where the alpha(2)-delta Type 2 subunit mRNA is abundant, such as cerebellum. These findings, in conjunction with prior in vitro binding data, provide evidence that the alpha(2)-delta Type 1 subunit of voltage-gated calcium channels is the major binding protein for pregabalin in CNS. Moreover, the distinct localization of alpha(2)-delta Type 1 and mutation-resistant binding (assumed to be alpha(2)-delta Type 2) in brain areas subserving different functions suggests that identification of subunit-specific ligands could further enhance pharmacologic specificity.

Pregabalin action at a model synapse: binding to presynaptic calcium channel alpha2-delta subunit reduces neurotransmission in mice.

Pregabalin, ((S)-3-(aminomethyl)-5-methylhexanoic acid, also known as (S)-3-isobutyl GABA, Lyricatrade mark) is approved for treatment of certain types of peripheral neuropathic pain and as an adjunctive therapy for partial seizures of epilepsy both the EU and the USA and also for generalized anxiety disorder in the EU. Though pregabalin binds selectively to the alpha(2)-delta (alpha(2)-delta) auxiliary subunit of voltage-gated calcium channels, the cellular details of pregabalin action are unclear. The high density of alpha(2)-delta in skeletal muscle fibers raises the question of whether pregabalin alters excitation-contraction coupling. We used the mouse soleus neuromuscular junction from mice containing an artificially mutated alpha(2)-delta Type 1 protein (R217A) as a model to examine the effect of pregabalin. Pregabalin reduced nerve-evoked muscle contractions by 16% at a clinically relevant concentration of 10 muM in wildtype mice. When acetylcholine receptors were blocked with curare, pregabalin had no effect on contraction from direct stimulation of muscle, suggesting a lack of drug effects on contraction coupling. Our data are consistent with pregabalin having no effect on striated muscle L-type calcium channel function. However, in mice expressing mutant (R217A) alpha(2)-delta Type 1, there was no significant effect of pregabalin on nerve-evoked muscle contraction. We propose that pregabalin reduces presynaptic neurotransmitter release without altering postsynaptic receptors or contraction coupling and that these effects require high affinity binding to alpha(2)-delta Type 1 auxiliary subunit of presynaptic voltage-gated calcium channels.

Activity profile of pregabalin in rodent models of epilepsy and ataxia.

Pregabalin (Lyrica) is a novel amino acid compound that binds with high affinity to the alpha2-delta (alpha2-delta) auxiliary protein of voltage-gated calcium channels. In vivo, it potently prevents seizures, pain-related behaviors and has anxiolytic-like activity in rodent models. The present studies were performed to determine the profile of pregabalin anticonvulsant activity in a variety of mouse and rat models. In the high-intensity electroshock test, pregabalin potently inhibited tonic extensor seizures in rats (ED50 = 1.8 mg/kg, PO), and low-intensity electroshock seizures in mice. It prevented tonic extensor seizures in the DBA/2 audiogenic mouse model (ED50 = 2.7 mg/kg, PO). Its time course of action against electroshock induced seizures in rats roughly followed the pharmacokinetics of radiolabeled drug in the brain compartment. At higher dosages (ED50 1= 31 mg/kg, PO), pregabalin prevented clonic seizures from pentylenetetrazole in mice. In a kindled rat model of partial seizures, pregabalin prevented stages 4-5 behavioral seizures (lowest effective dose = 10 mg/kg, IP), and also reduced the duration of electrographic seizures. Pregabalin was not active to prevent spontaneous absence-like seizures in the Genetic Absence Epilepsy in Rats from Strasbourg (GAERS) inbred Wistar rat strain. Pregabalin caused ataxia and decreased spontaneous locomotor activity at dosages 10-30-fold higher than those active to prevent seizures. These findings suggest that pregabalin has an anticonvulsant mechanism different from the prototype antiepileptic drugs and similar to that of gabapentin except with increased potency and bioavailability. In summary, our results show that pregabalin has several properties that favor treatment of partial seizures in humans.

Pharmacology of dextromethorphan: Relevance to dextromethorphan/quinidine (Nuedexta®) clinical use.

Dextromethorphan (DM) has been used for more than 50years as an over-the-counter antitussive. Studies have revealed a complex pharmacology of DM with mechanisms beyond blockade of N-methyl-d-aspartate (NMDA) receptors and inhibition of glutamate excitotoxicity, likely contributing to its pharmacological activity and clinical potential. DM is rapidly metabolized to dextrorphan, which has hampered the exploration of DM therapy separate from its metabolites. Coadministration of DM with a low dose of quinidine inhibits DM metabolism, yields greater bioavailability and enables more specific testing of the therapeutic properties of DM apart from its metabolites. The development of the drug combination DM hydrobromide and quinidine sulfate (DM/Q), with subsequent approval by the US Food and Drug Administration for pseudobulbar affect, led to renewed interest in understanding DM pharmacology. This review summarizes the interactions of DM with brain receptors and transporters and also considers its metabolic and pharmacokinetic properties. To assess the potential clinical relevance of these interactions, we provide an analysis comparing DM activity from in vitro functional assays with the estimated free drug DM concentrations in the brain following oral DM/Q administration. The findings suggest that DM/Q likely inhibits serotonin and norepinephrine reuptake and also blocks NMDA receptors with rapid kinetics. Use of DM/Q may also antagonize nicotinic acetylcholine receptors, particularly those composed of α3ß4 subunits, and cause agonist activity at sigma-1 receptors.

Structure-activity relationships of pregabalin and analogues that target the alpha(2)-delta protein.

Pregabalin exhibits robust activity in preclinical assays indicative of potential antiepileptic, anxiolytic, and antihyperalgesic clinical efficacy. It binds with high affinity to the alpha(2)-delta subunit of voltage-gated calcium channels and is a substrate of the system L neutral amino acid transporter. A series of pregabalin analogues were prepared and evaluated for their alpha(2)-delta binding affinity as demonstrated by their ability to inhibit binding of [(3)H]gabapentin to pig brain membranes and for their potency to inhibit the uptake of [(3)H]leucine into CHO cells, a measure of their ability to compete with the endogenous substrate at the system L transporter. Compounds were also assessed in vivo for their ability to promote anxiolytic, analgesic, and anticonvulsant actions. These studies suggest that distinct structure activity relationships exist for alpha(2)-delta binding and system L transport inhibition. However, both interactions appear to play an important role in the in vivo profile of these compounds.

Novel cyclopropyl beta-amino acid analogues of pregabalin and gabapentin that target the alpha2-delta protein.

As part of a program aimed at generating compounds with affinity for the alpha(2)-delta subunit of voltage-gated calcium channels, several novel beta-amino acids were prepared using an efficient nitroalkane-mediated cyclopropanation as a key step. Depending on the ester that was chosen, the target amino acids could be prepared in as few as three steps. The cyclopropyl amino acids derived from ketones proved to be potent binders of the alpha(2)-delta subunit of voltage-gated calcium channels, but did not interact with the large neutral amino acid system L (leucine) transporter. Anticonvulsant effects were observed in vivo with compound 34 but only after intracerebroventricular (icv) administration, presumably due to inadequate brain concentrations of the drug being achieved following oral dosing. However, pregabalin 1 was active in the DBA/2 model after oral (and icv) dosing, supporting a hypothesis that active transport is a prerequisite for such zwitterionic species to cross the blood-brain barrier.

Pregabalin and gabapentin reduce release of substance P and CGRP from rat spinal tissues only after inflammation or activation of protein kinase C.

Gabapentin and pregabalin are amino acid derivatives of gamma-amino butyric acid that have anticonvulsant, analgesic, and anxiolytic-like properties in animal models. The mechanisms of these effects, however, are not well understood. To ascertain whether these drugs have effects on sensory neurons, we studied their actions on capsaicin-evoked release of the sensory neuropeptides, substance P and calcitonin gene-related peptide from rat spinal cord slices in vitro. Although release of immunoreactive peptides from non-inflamed animals was not altered by either drug, prior in vivo treatment by intraplantar injection of complete Freund's adjuvant enhanced release from spinal tissues in vitro, which was attenuated by gabapentin and pregabalin. These drugs also reduced release of immunoreactive neuropeptides in spinal tissues pretreated in vitro with the protein kinase C activator, phorbol 12,13-dibutyrate. Our results suggest that gabapentin and pregabalin modulate the release of sensory neuropeptides, but only under conditions corresponding to significant inflammation-induced sensitization of the spinal cord.

The diverse therapeutic actions of pregabalin: is a single mechanism responsible for several pharmacological activities?

Pregabalin is a specific ligand of the alpha2-delta (α2-δ) auxiliary subunit of voltage-gated calcium channels. A growing body of evidence from studies of anxiety and pain indicate that the observed responses with pregabalin may result from activity at the α2-δ auxiliary protein expressed presynaptically, in several different circuits of the central nervous system (CNS). The disorders that appear to be effectively treated with pregabalin are thematically linked by neuronal dysregulation or hyperexcitation within the CNS. This review proposes how binding to the α2-δ protein target in different regions of the CNS may contribute to the observed clinical activity of pregabalin, as well as to the adverse event profile of the compound. Whether this compound regulates synaptic function via α2-δ in additional conditions is yet to be discovered. The potential of pregabalin to regulate neuronal hyperactivity involving other CNS circuits will require further exploration.

Pregabalin is a potent and selective ligand for α(2)δ-1 and α(2)δ-2 calcium channel subunits.

Pregabalin, a synthetic branched chain γ-amino acid with anticonvulsant, anxiolytic, and analgesic activities, has been shown to bind with high affinity to the voltage-gated calcium channel α(2)δ subunit. Given the broad therapeutic utility of pregabalin, a series of experiments was undertaken to determine the potency, selectivity, and specificity of pregabalin's receptor-binding profile at α(2)δ-1 and α(2)δ-2 subunits of voltage-gated calcium channels along with 38 widely studied receptors and channels. Receptor autoradiography was used to assess regional-binding density of pregabalin throughout the rat spinal cord and brain. In addition, a series of studies using in vivo electrophysiological recordings of γ-aminobutyric acid (GABA)(A)- and GABA(B)-evoked currents was undertaken to determine the interaction of pregabalin with GABAergic receptor subtypes. Together, the results of these studies demonstrate potent and selective binding of pregabalin to α(2)δ-1 and α(2)δ-2 subunits in native and recombinant human and porcine systems. Pregabalin did not interact with any of the 38 receptors and ion channels evaluated, and a variety of central nervous system (CNS)-targeted therapeutic drugs did not show activity at the α(2)δ subunits of voltage-gated calcium channels. Receptor autoradiography demonstrated extensive [(3)H]-pregabalin binding throughout the CNS, with high-level binding in the cortex, hippocampus, cerebellum, dorsal horn of the spinal cord, and amygdala. Finally, receptor-binding and electrophysiological techniques failed to show evidence of an interaction between pregabalin and GABA(A) or GABA(B) receptors. These studies suggest that the clinical effects of pregabalin are likely due to direct and selective interactions with α(2)δ-1 and α(2)δ-2 subunits of voltage-gated calcium channels.

Central sensitization and Ca(V)α2δ ligands in chronic pain syndromes: pathologic processes and pharmacologic effect.

UNLABELLED: Central sensitization is one form of long-term plasticity in the central nervous system. Sustained activation of primary sensory fibers supplying dorsal horn can induce long-lasting increases in the discharge amplitude of primary afferent synapses. This is similar to the long-term potentiation that occurs in many other CNS regions. Drugs that limit the short-duration wind-up component of central sensitization include sodium channel blockers, NMDA antagonists, fast-acting opioids and the calcium-channel ligands gabapentin and pregabalin (S-3-(aminomethyl)-5-methylhexanoic acid). Pregabalin, like gabapentin, binds selectively to the Ca(V)α2δ auxiliary subunit of presynaptic voltage-gated calcium channels. The conformational changes induced by this binding inhibit abnormally intense neuronal activity by reducing the synaptic release of glutamate and other neurotransmitters. Recent identification in animal models of increased Ca(V)α2δ protein expression in chronic pain, allodynia, and hyperalgesia have drawn additional interest to drugs that bind the Ca(V)α2δ site. Experimental studies with animal models and healthy human volunteers have shown that pregabalin reduces nociceptive responses, particularly in conditions involving central sensitization. Since these actions occur with relatively modest effects on physiological and cognitive functions, pregabalin may be an important consideration in the pharmacotherapy of otherwise difficult-to-treat pain syndromes. PERSPECTIVE: This focus article discusses how the central nervous system plasticity phenomenon, central sensitization, is established in the induction and maintenance of chronic pain, allodynia, and hyperalgesia. In addition, it explores the neurophysiologic actions of the calcium-channel ligands gabapentin and pregabalin in limiting pathological manifestations of central sensitization.

Pregabalin reduces the release of synaptic vesicles from cultured hippocampal neurons.

Pregabalin [S-[+]-3-isobutylGABA or (S)-3-(aminomethyl)-5-methylhexanoic acid, Lyrica] is an anticonvulsant and analgesic medication that is both structurally and pharmacologically related to gabapentin (Neurontin; Pfizer Inc., New York, NY). Previous studies have shown that pregabalin reduces the release of neurotransmitters in several in vitro preparations, although the molecular details of these effects are less clear. The present study was performed using living cultured rat hippocampal neurons with the synaptic vesicle fluorescent dye probe FM4-64 to determine details of the action of pregabalin to reduce neurotransmitter release. Our results indicate that pregabalin treatment, at concentrations that are therapeutically relevant, slightly but significantly reduces the emptying of neurotransmitter vesicles from presynaptic sites in living neurons. Dye release is reduced in both glutamic acid decarboxylase (GAD)-immunoreactive and GAD-negative (presumed glutamatergic) synaptic terminals. Furthermore, both calcium-dependent release and hyperosmotic (calcium-independent) dye release are reduced by pregabalin. The effects of pregabalin on dye release are masked in the presence of l-isoleucine, consistent with the fact that both of these compounds have a high binding affinity to the calcium channel alpha(2)-delta protein. The effect of pregabalin is not apparent in the presence of an N-methyl-d-aspartate (NMDA) antagonist [D(-)-2-amino-5-phosphonopentanoic acid], suggesting that pregabalin action depends on NMDA receptor activation. Finally, the action of pregabalin on dye release is most apparent before and early during a train of electrical stimuli when vesicle release preferentially involves the readily releasable pool.

Carboxylate bioisosteres of pregabalin.

Several beta-amino tetrazole analogs of gabapentin 1 and pregabalin 2 were prepared by one of two convergent, highly efficient routes, and their affinity for the alpha(2)-delta protein examined. Two select compounds with potent affinity for alpha(2)-delta, 8a and 16a, were subsequently tested in vivo in an audiogenic seizure model and found to elicit protective effects.

Carboxylate bioisosteres of gabapentin.

A series of carboxylate bioisosteres of structures related to gabapentin 1 have been prepared. When the carboxylate was replaced by a tetrazole, this group was recognized by the alpha2-delta protein. Further characterization of alpha2-delta binding compounds 14a and 14b revealed a similar pattern of functional in vitro and in vivo activity to gabapentin 1.

The design and synthesis of human branched-chain amino acid aminotransferase inhibitors for treatment of neurodegenerative diseases.

The inhibition of the cytosolic isoenzyme BCAT that is expressed specifically in neuronal tissue is likely to be useful for the treatment of neurodegenerative and other neurological disorders where glutamatergic mechanisms are implicated. Compound 2 exhibited an IC50 of 0.8 microM in the hBCATc assays; it is an active and selective inhibitor. Inhibitor 2 also blocked calcium influx into neuronal cells following inhibition of glutamate uptake, and demonstrated neuroprotective efficacy in vivo. SAR, pharmacology, and the crystal structure of hBCATc with inhibitor 2 are described.