Modulation of Glycine-Mediated Spinal Neurotransmission for the

Perspective pubs.acs.org/jmc

Modulation of Glycine-Mediated Spinal Neurotransmission for the Treatment of Chronic Pain Christopher L. Cioffi* Departments of Basic and Clinical Sciences and Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, 106 New Scotland Avenue, Albany, New York 12208 United States ABSTRACT: Chronic pain constitutes a significant and expanding worldwide health crisis. Currently available analgesics poorly serve individuals suffering from chronic pain, and new therapeutic agents that are more effective, safer, and devoid of abuse liabilities are desperately needed. Among the myriad of cellular and molecular processes contributing to chronic pain, spinal disinhibition of pain signaling to higher cortical centers plays a critical role. Accumulating evidence shows that glycinergic inhibitory neurotransmission in the spinal cord dorsal horn gates nociceptive signaling, is essential in maintaining physiological pain sensitivity, and is diminished in pathological pain states. Thus, it is hypothesized that agents capable of enhancing glycinergic tone within the dorsal horn could obtund nociceptor signaling to the brain and serve as analgesics for persistent pain. This Perspective highlights the potential that pharmacotherapies capable of increasing inhibitory spinal glycinergic neurotransmission hold in providing new and transformative analgesic therapies for the treatment of chronic pain.

1. INTRODUCTION Pain, as defined by The International Association for the Study of Pain (IASP), is “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage.”1 Pain constitutes a critical somatosensory warning mechanism designed to alert us to the presence of actual or impending dangerous stimuli that could lead to tissue injury. Although pain taxonomy is multidimensional and complex,1,2 it can most broadly be classified into two temporally distinct categories: acute and chronic. Acute nociceptive pain results from activation of nociceptive pathways in response to noxious stimuli that may lead to tissue damage. It is a protective and transitory response occurring only in the presence of a noxious stimulus. Acute inflammatory pain results from tissue injury, and the associated pain sensations generally abate in conjunction with resolution of inflammation and healing of the damaged tissue. This type of pain is typically managed via administration of nonsteroidal anti-inflammatory drugs (NSAIDs) and/or opioid analgesics. Chronic pain is defined as pain that lasts or recurs for more than 3−6 months.1 Unlike acute pain, chronic pain does not confer a protective biological function as it can be decoupled from noxious stimuli and persist well beyond tissue repair and healing. Indeed, chronic pain is considered a pathological process or disease state in itself.1,3 In cases of pathological pain, the pain experienced may be heightened and protracted in response to a noxious stimulus (hyperalgesia), may occur spontaneously, or may arise from a normally innocuous stimuli (allodynia). Chronic pain can be associated with injury to the somatosensory system (neuropathic pain), degenerative © 2017 American Chemical Society

processes and chronic inflammation (i.e., osteoarthritis and rheumatoid arthritis), disease (i.e., cancer pain), or poorly managed acute pain that has transitioned into chronic pain (i.e., postsurgical and posttraumatic pain). In cases of chronic dysfunctional pain (i.e., chronic widespread pain, irritable bowel syndrome, and fibromyalgia), contributing etiological mechanisms remain poorly understood. Chronic pain is highly prevalent, as an estimated 20% of the worldwide population, including approximately 116 million adults in the U.S., are afflicted.9 Furthermore, the condition is accompanied by comorbidities that include poor sleep,4 depression,5 anxiety,6 and cognitive dysfunction,7 leading to functional impairment and significantly reduced quality of life. The 2008 Medical Expenditure Panel Survey (MEPS) approximates that the total U.S. annual costs attributed to pain (direct health care costs and indirect costs associated with lost productivity) ranges from a staggering $560 to $635 billion.10 Despite the high level of prevalence and the enormous socioeconomic burdens incurred, pharmacological treatment for chronic pain remains limited. The condition is often refractory to currently available analgesics, which include NSAIDs, anticonvulsants (e.g., gabapentin and pregabalin), antidepressants, topical agents (e.g., capsaicin and lidocaine), N-methyl-Daspartate (NMDA) receptor antagonists, and opioids. These agents do not reduce pain in all treated individuals, provide ineffective pain relief to responsive individuals, induce severe dose-limiting side effects, or present significant risks of tolerance and abuse.8 In addition, there has been a dearth of new Received: June 29, 2017 Published: September 6, 2017 2652

DOI: 10.1021/acs.jmedchem.7b00956 J. Med. Chem. 2018, 61, 2652−2679

Journal of Medicinal Chemistry

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Figure 1. (A) Simplified depiction of ascending and descending pain signaling pathways. Ascending pain pathway = red; modulatory descending tracts = blue. Afferent nociceptive input enters the spinal cord via the DRG. Secondary order projection neurons ascend in the contralateral spinothalamic and spinorecticular tracts that relay the signal to cortical centers. Descending pathways projecting from the periaqueductal gray (PAG) in the midbrain and the rostral ventromedial medulla (RVM) to the dorsal horn and modulate pain transmission. (B) The Rexed laminae of the spinal cord gray matter.

2. BACKGROUND Several comprehensive reviews detailing the pain matrix and pathophysiological changes to pain processing are available.12 The following sections of our review will provide a brief overview for readers unfamiliar with these topics, which will establish the basis for subsequent sections that focus on the role of glycinergic neurotransmission in the pain signaling process. 2.1. The Pain Signaling Pathway. The experience of pain involves the transduction, conduction, and transmission of noxious stimuli to the CNS via peripheral afferent sensory neurons called nociceptors.13 Nociceptors express high-threshold transducing ion-channel receptors at their terminals (i.e., transient receptor potential (TRP), acid sensing (ASIC), and purinoceptor (P2X) ion channels) that are capable of detecting noxious thermal, mechanical, and chemical stimuli. Voltagegated ion channels (i.e., Nav, K+, Ca2+) mediate the initiation and propagation of action potentials. Nociceptors fall within two main classes: thinly myelinated Aδ fibers and nonmyelinated group C (chemosensitive) fibers.14 Aδ fibers conduct action potentials in response to stimuli related to pressure and temperature, and they are responsible for the perception of localized sharp pain, termed as first pain. C fibers are smaller in diameter, slower in conduction velocity, and are largely polymodal. These nociceptors transduce diffuse, slow burning pain, known as second pain. Moreover, C fibers account for approximately 70% of all nociceptors and are classified as either peptidergic (expressing the neuropeptide modulators substance P and calcitonin gene related peptide (CGRP)) or nonpeptidergic.13 A third major class of sensory neurons includes mechanosensor Aβ fibers, which are heavily myelinated and larger in diameter than the nociceptors.15 These fibers conduct action potentials at a higher velocity than Aδ and C fibers, have a low activation threshold, and are normally only involved in transmitting non-noxious tactile stimuli (touch and pressure sensations) except in pathological chronic pain states.

analgesics for several years despite recent advances in the understanding of pain mechanisms and the discovery of multiple, novel antinociceptive targets. Poor preclinical to clinical translation coupled with design challenges inherent with pain trials (i.e., high clinical heterogeneity and high placebo response rates) has resulted in exceptionally high clinical failure rates. Therefore, many pharmaceutical companies have abandoned the field of pain drug discovery.11 However, the need for more effective analgesics will continue to escalate with the projected growth of aging populations and diseases associated with chronic pain such as arthritis, cancer, and diabetes. Thus, the discovery of novel therapeutics that provide improved safety, reduced side effect liabilities, and meaningful relief for a broader patient population constitutes a significant unmet medical need that requires urgent attention. Dysfunction of spinal inhibitory input onto central pain circuits plays a crucial role in the facilitation and maintenance of chronic pain. Specifically, several research groups have shown that glycine-mediated fast synaptic inhibitory neurotransmission within the spinal cord dorsal horn suppresses pain signaling to the higher CNS and is impaired in pathological pain states. Moreover, inhibition of glycine reuptake and GlyR positive allosteric modulation have been shown to augment spinal glycinergic tone and ameliorate pain behaviors in various in vivo rodent models of acute, inflammatory, and neuropathic pain. In this Perspective, we will review studies that have contributed to a better understanding of the role spinal glycinergic neurotransmission plays in the modulation of pain signaling and the potential mechanisms by which it can become impaired in pathological pain states. We will also present recent advances in the areas of glycine transporter (GlyT) inhibitors and GlyR positive allosteric modulators (PAMs) toward the development of novel analgesics. 2653



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The cell bodies of sensory neurons cluster at either the dorsal root ganglion (DRG) or in the trigeminal ganglia and their respective central processes terminate within specific layers of the spinal cord dorsal horn or the trigeminal nucleus of the brainstem, respectively.13 Within these regions, primary afferent input is transmitted to secondary order neurons (projection neurons) that then relay the signals through ascending pathways (i.e., spinothalamic and spinorecticular tracts) to higher cortical centers (Figure 1A).13 2.2. The Spinal Dorsal Horn. Spinal cord gray matter is grossly divided into two major segments: the dorsal horn (posterior component receiving afferent sensory input from the DRG) and the ventral horn (anterior component projecting efferent motor control circuits).16 The gray matter is further subdivided into 10 separate layers (Rexed laminae I−X)17 of which laminae I−VI and X receive afferent sensory input. The laminae of the dorsal horn (laminae I−VI) can be further categorized as the marginal zone (lamina I), superficial layer (laminae I−II), the substantia gelatinosa (laminae II inner (IIi) and II outer (IIo)), and the deep dorsal horn (laminae III−VI) (Figure 1B). The spinal dorsal horn presents a nexus whereby the integration and relay of afferent somatosensory input to the brain is modulated via a complex interplay between three primary neuronal components: ascending secondary order projection neurons, local inhibitory and excitatory interneurons, and descending modulatory supraspinal tracts. C and Aδ pain fibers largely innervate lamina I and II,18 while Aδ and Aβ fibers predominantly innervate lamina III−VI.19 Lamina I and II house a high concentration of nociceptive-specific (NS) secondary order projection neurons, which receive synaptic input from C and Aδ fibers, segmental excitatory and inhibitory interneurons, and descending supraspinal modulatory tracts.18,20 Many, though not all, NS projection neurons express the neurokinin 1 receptor (NK1R) and are therefore responsive to substance P released by peptidergic C fibers.21 Aδ and Aβ fibers arborize within the deep dorsal horn where they synapse with excitatory and inhibitory interneurons and wide dynamic range (WDR) projection neurons that respond to both nociceptive and non-nociceptive input (Figure 2).19

Figure 3. Excitatory neurotransmitter 1 and inhibitory neurotransmitters 2 and 3.

in laminae I−III are excitatory and release 1 (glutamate) (Figure 3).22 Interneurons that release 2 (γ-aminobutyric acid) (GABA) and 3 (glycine) provide local inhibitory input and are widely distributed throughout the dorsal horn, comprising ∼30% of the neuronal content found in laminae I−II and ∼40% in lamina III. The highest densities of GABAergic interneurons are within laminae I−II, whereas glycinergic interneurons are most heavily concentrated within deeper layers of laminae III−VI.23 Inhibitory GABAergic and glycinergic interneurons within the dorsal horn attenuate nociceptive input in order to maintain a physiological response to painful stimuli.24 This includes preventing spontaneous activity of both excitatory interneurons and secondary order projection neurons innervated by afferent nociceptors. Furthermore, inhibitory interneurons integrate and separate different signaling modalities in order to prevent innocuous stimuli from driving nociceptive pathways. Lastly, inhibitory interneurons limit projection neuron receptive fields and prevent the spatial spread of sensory inputs, thereby maintaining somatotopic pain sensations. Loss of inhibitory control of these processes leads to symptoms such as hyperalgesia, spontaneous pain, allodynia, and radiating pain, respectively. GABAergic and glycinergic inhibitory interneurons also work in concert with descending inhibitory supraspinal tracts to maintain physiological levels of pain sensitivity. The main descending projections from the brain are monoaminergic (i.e., serotonergic and noradrenergic), which emanate from midbrain and brainstem regions (e.g., periaqueductal gray (PAG), dorsolateral pontomesencephalic tegmentum (DLMT), locus coeruleus).25 GABAergic and glycinergic supraspinal tracts descend from the rostral ventromedial medulla (RVM).26

3. MECHANISMS UNDERLYING CHRONIC PAIN 3.1. Chronic Pain Classifications. Chronic pain is broadly categorized as either nociceptive or neuropathic.1−3 Factors contributing to chronic nociceptive pain may include degenerative processes, chronic inflammation, ischemia, or disease.27 Neuropathic pain results from injury to, or dysfunction of, the somatosensory system. Peripheral neuropathic pain is caused by lesions to the peripheral nervous system (PNS) that may result from mechanical trauma (e.g., radiculopathy, postoperative neuralgia, trigeminal neuralgia, complex regional pain syndrome), metabolic diseases (e.g., diabetic neuralgia), cancer, toxins (e.g., chemotherapy-induced neuropathy), or infection (e.g., postherpetic neuralgia).28,29 Central neuropathic pain is commonly associated with spinal cord injury, multiple sclerosis, and stroke.28,29 An emerging third category for chronic pain, dysfunctional pain, is characterized by chronic regional or widespread pain for which no identifiable noxious stimulus, inflammatory response, or discernible neural damage can be identified.30 This type of pain appears to derive from autonomous amplification of central pain signaling and is associated with clinical disorders that include fibromyalgia, irritable bowel syndrome, primary erythermalgia, and interstitial cystitis.

Figure 2. Sensory neuron innervation of the spinal dorsal horn. C and Aδ nociceptive fibers innervate the superficial layers of the dorsal horn (laminae I and II) where the synapse with NS secondary order projection neurons and interneurons. Aβ fibers arborize throughout the deep dorsal horn (laminae III−VI). Aβ and Aδ fibers innervate WDR projection neurons in the deep dorsal horn (lamina V).

The neuronal content of the dorsal horn is largely interneurons (∼95%), which are the major postsynaptic targets for primary afferents. Approximately 60−70% of the interneurons 2654



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3.2. Peripheral Sensitization. Damaged peripheral tissue release damage-associated molecular patterns (DAMPs), which activate innate and adaptive inflammatory and reparatory processes. Local and migrating immune cells together with damaged tissue release a complex milieu of mediators referred to as an “inflammatory soup” at the site of injury.31 Components of this soup include a vast array of algogens (i.e., adenosine triphosphate (ATP), K+ and H+ ions, bradykinin, histamine, and serotonin (5-HT)), which directly activate receptors expressed on nociceptor peripheral terminals causing depolarization and action potential generation. Pro-algesic inflammatory mediators are also released (i.e., prostaglandin E2 (PE2), interleukin-1β (IL-1β), interleukin-6 (IL-6), nerve growth factor (NGF), and tumor necrosis factor-alpha (TNF-α)) that sensitize local peripheral nociceptors by activating intracellular secondary messenger systems (i.e., protein kinase C (PKC) and protein kinase A (PKA)), leading to phosphorylation and altered activity of existing transducer and voltage-gated ion channels.31 Subsequent transcriptional changes within the DRG result in upregulation of existing receptors and voltage-gated ion channels as well as production of new pro-inflammatory proteins.31−33 Aberrant peripheral C-fiber terminals also release agents that drive neurogenic inflammation (i.e., substance P, CGRP, neurokinin A, neuropeptide Y, and nitric oxide), which further exacerbates nociceptor sensitization.34 The summation of these processes leads to a reduction in threshold and an increase in peripheral nociceptor transduction activity, resulting in abnormal pain hypersensitivity at the site of injury and inflammation (primary hyperalgesia). Peripheral nerve damage confers significant transcriptional changes within the DRG.31−33 Upregulation of neuronal injury markers (i.e., growth associated protein 43 (GAP43) and cyclic AMP-dependent transcription factor (ATF3)), neurotrophic factors (i.e., NGF and brain-derived neurotrophic factor (BDNF)), neuromodulators, and pro-inflammatory mediators occur in order to induce neuronal growth and promote axon regeneration.35 Increased transducer and voltage-gated ion channel expression within the neuroma and DRG leads to increased membrane excitability and altered neurotransmitter release.35 Sensitization may produce ectopic discharge and spontaneous action potentials, which can originate from the DRG or at other points along the injured nerve.36 The changes incurred by injured neurons can also lead to excitation of adjacent uninjured neurons, including non-nociceptive fibers, within the DRG. Furthermore, collateral spouting of local sympathetic neurons into the DRG may induce ephaptic conduction and facilitate sympathetically maintained pain. Nerve injury can also cause atrophic changes to neurons (Wallerian degeneration) that lead to cell death, which may manifest as loss of sensation or deafferentated pain. Lastly, Schwann cells and DRG satellite cells undergo dramatic phenotypic changes in response to peripheral nerve injury and release a myriad of factors that also contribute to peripheral nociceptor sensitization.37 3.3. Central Sensitization. Intense, repetitive, and sustained afferent bombardment from nociceptors in response to peripheral tissue or neural injury induces neuroplastic changes within the dorsal horn that lead to central sensitization. Central sensitization is characterized by an enhanced facilitation and amplification of pain signaling throughout the neuraxis and it is responsible for the temporal, spatial, and threshold changes in pain sensitivity that occur in acute and chronic pain states.38

The process is responsible for an increased gain in pain sensitivity (hyperalgesia), for producing pain sensitivity in an uninjured area surrounding a site of peripheral tissue damage and inflammation (secondary hyperalgesia), and for co-opting normally innocuous input onto nociceptive pathways (allodynia).38 Central sensitization is a form of activity-dependent longterm potentiation (LTP) that progresses from an early onset to a late-onset phase.38 The early onset phase is adaptive, reversible, and serves a protective function by motivating recuperative behaviors that limit the use of an injured area of the body until tissue repair and healing is complete. This phase involves enhanced responsiveness of spinal postsynaptic secondary order projection neurons to intense glutamate release from afferent nociceptors (homosynaptic strengthening). The initiation and maintenance of central sensitization is dependent upon activation of postsynaptic N-methyl-D-aspartate (NMDA) receptors. Indeed, NMDA receptor antagonists such as (5R,10S)(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10imine (MK-801) and (R)-4-[(E)-3-phosphonoprop-2-enyl]piperazine-2-carboxylic acid (midafotel, D-CCP) (structures not shown) reverse or inhibit hyperexcitability of nociceptive neurons induced by conditioning inputs and NR1 conditional knockdown prohibits acute activity-dependent central sensitization.39,40 Stimulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and metabotropic glutamate (mGluR) receptors facilitates long-lasting membrane depolarization of the postsynaptic neuron, which is required to remove a magnesium block from the NMDA receptor channel pore prior to activation.41 Once fully activated, NMDA receptors confer increases in intracellular Ca2+ concentrations that activate secondary messenger systems (i.e., PKC, PKA, phospholipase C (PLC), calcium/calmodulin-dependent protein kinase II (CAM-KII), phosphoinositide 3-kinase (PI3 kinases), extracellular signal-regulated kinase (ERK), and Src-family kinases) and concomitant processes that initiate and subsequently strengthen LTP.38 Some of these processes include (1) phosphorylation of cell surface NMDA, AMPA, and other ion channel receptors that alter their activation threshold and kinetics, and (2) increased trafficking of AMPA and NMDA receptors to the synapse. The release of additional modulators from afferent fibers, such as CGRP, BDNF, and bradykinin, also contributes to the activation of intracellular cascades that reinforce membrane depolarization and maintain LTP.38 Furthermore, NMDA receptor-mediated production of retrograde neurotransmitter nitric oxide enhances LTP by diffusing to the presynaptic terminal and inducing an increased release of synaptic glutamate, substance P, and CGRP.38 Enhanced synaptic efficacy lowers activation thresholds of nociceptive signaling and enables stronger excitation of supra-threshold excitatory input. Continuous afferent input under conditions such as chronic inflammation or nerve damage may cause central sensitization to progress to the late phase. This phase involves maladaptive transcriptional changes leading to long-lasting and profound neuroplastic alterations within the dorsal horn and throughout the CNS that underlie pathological pain states. Phenotypic changes include increased expression levels of existing receptors and production of new, pro-algesic proteins (i.e., prostaglandin E (PE) receptors, cyclooxygenase enzymes (COX-1 and COX-2), IL1-β, TNF-α).38 In addition to possessing lowered activation thresholds, sensitized dorsal horn neurons at this phase may also exhibit spontaneous activity and present expanded receptive fields that co-opt normally non-nociceptive Aβ 2655



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Figure 4. Inhibitory strychnine sensitive glycine receptor architecture. (A) The membrane topology of Cys loop ion channel subunits. Cys loop family ion channels contain a disulfide linkage within the NH2 terminal ligand-binding domain. The NH2 terminal ligand-binding domain connects to a four transmembrane domains (TM1−TM4), which present a large intracellular loop between TM3 and TM4. (B) Five subunits come together to form the GlyR pentameric structure, which contains a central chloride pore. The orthosteric glycine binding sites are formed as a cleft between adjacent subunits located midway between the top and bottom of the receptor (denoted as green circles; two additional sites are obstructed from view). (C) Structure of the GlyRα1 receptor with glycine bound obtained by single particle electron cryomicroscopy (PDB 3JAE).49 View of the receptor−glycine complex down the 5-fold symmetric pore axis from the extracellular side of the membrane.

fiber-mediated input. Lastly, resident spinal microglia and astrocytes activated upon nerve damage migrate to primary afferent neuron terminals where they stimulate the complement component of the immune system and release pro-inflammatory mediators into the synaptic cleft that further exacerbate sensitization.38,42 In addition to increased excitatory drive and enhanced synaptic efficacy, central sensitization is also facilitated by reduced spinal GABAergic and glycinergic inhibitory control of nociceptive pathways. Preclinical studies have shown that pharmacologically induced loss of either GABAergic or glycinergic neurotransmission in the dorsal horn can lead to hyperalgesic and/or allodynic pain behaviors that typically accompany central sensitization. In the following sections, we will focus solely on spinal inhibitory glycinergic neurotransmission and present recent studies that demonstrate the crucial role it plays in the modulation of pain signaling within the dorsal horn and the potential mechanisms by which it may be impaired in chronic pain states.

sequesters action potential initiation and propagation and generates inhibitory postsynaptic potentials (IPSPs).44 Glycine also modulates glutamatergic fast synaptic excitatory neurotransmission as an obligatory coagonist that binds to the GluN1 subunit glycine-B site of voltage and ligand-gated ionotropic NMDA receptors.45 Activation of NMDA receptors relies on two synergistic processes; simultaneous binding of L-glutamate at the GluN2 subunit and coagonist glycine at GluN1 to open the channel and membrane depolarization to alleviate a magnesium block from the channel pore. Activation of NMDA receptors results in calcium influx, which triggers neuronal excitation and intracellular signaling cascades involved in synaptic plasticity processes such as LTP.46 GlyR-mediated fast-synaptic inhibitory neurotransmission is a key regulator of spinal inhibitory neurotransmission and GlyRs are highly involved in the modulation of motor and sensory reflex activity, muscle tone, respiratory rhythms, and sensory and pain sensation.47 GlyR architecture incorporates a pentameric arrangement of homologous membrane-spanning subunits symmetrically arranged to form a central chloride channel (Figure 4).44 Five subunit isoforms have been identified: four α subunits (designated α1−4) and one β subunit. The α subunits share a >90% homology and the α4 gene is a pseudo gene in humans due to a premature stop codon. Each subunit contains a large, extracellular NH2-terminal ligand-binding domain that presents a distinguishing loop incorporating a disulfide linkage between two cysteine residues. The extracellular domain is connected to an array of four α-helical transmembrane domains (TM1−TM4), which present a large intracellular domain between TM3 and TM4.44 The orthosteric glycine binding sites are formed as a cleft at the interface between adjacent subunits approximately midway between the top and bottom of the receptor.44

4. GLYCINERGIC NEUROTRANSMISSION The nonessential amino acid 3 serves as both an inhibitory and excitatory neurotransmitter within the CNS. Glycine facilitates fast synaptic inhibitory neurotransmission via binding at the glycine-A binding site at strychnine-sensitive GlyRs, which are ligand-gated and chloride-permeable ion channels belonging to the cysteine-loop (Cys-loop) receptor family (group I).43,44 This family of receptors also includes the cation-selective nicotinic acetylcholine receptor (nAChR) and the serotonin type-3 receptor (5-HT3R) and the anion-selective GABA type A receptor (GABAAR). Activation of GlyRs results in chloride influx and subsequent neuronal hyperpolarization, which 2656



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GlyRs can form α-subunit homomeric channels (GlyRα1, GlyRα2, and GlyRα3) or heteromeric channels (GlyRα1β, GlyRα3β) in 3:2 or 2:3 α/β subunit ratios. The majority of GlyRs in the adult CNS are heteromeric and colocalize with the postsynaptic scaffold protein gephyrin via β-subunit anchoring.44,48 While inhibitory GABA receptors are diffusely expressed throughout the CNS, GlyR distribution is more circumscribed. Autoradiographic and immunohistochemical studies have shown that GlyR expression is largely restricted within caudal regions (i.e., cerebellum,50 brainstem,51 and spinal cord52) and the retina.53 GlyRα1β is the predominant subtype uniformly expressed throughout the CNS, while GlyRα3β is spatially restricted to the superficial dorsal horn.54 GlyRα2 homomers are largely expressed throughout the CNS during embryonic development, however, their numbers sharply decline postnatally and they persist only in selective areas of the adult CNS such as the retina and auditory brain stem.55

monosynaptic nociceptive C and Aδ input, whereas lamina III NK1R-positive neurons received input mainly from mechanosensor Aβ fibers. NK1R-negative neurons in lamina I received polysynaptic A-fiber input. Importantly, GABAergic or glycinergic inhibition with bicuculline or strychnine, respectively, permitted significant Aβ-mediated input onto NK1Rpositive neurons in lamina I.59 This observation suggests that a polysynaptic connection exists between non-nociceptive Aβ fibers in the deep dorsal horn and lamina I NS projection neurons, which is gated by segmental inhibitory interneurons. Inhibition of either GABAergic or glycinergic activity also led to enhanced control-evoked responses for both NK1R-positive and NK1R-negative neurons. In vivo electrophysiological recordings conducted by Miraucourt and co-workers demonstrate that strychninemediated segmental disinhibition of glycinergic transmission in the rat trigeminal brainstem permits Aβ fiber input onto lamina I NS projection neurons and results in tactile allodynia.60 Anatomical experiments using Fos expression as a surrogate for activity-dependent changes were employed to map a polysynaptic circuit containing lamina II protein kinase γ (PKCγ) excitatory interneurons that are innervated by Aβ fibers and inhibitory glycinergic interneurons in lamina III. Accordingly, inhibition of PKCγ excitatory interneurons via the selective peptide antagonist KIG31-1 (10100 and 500 pmoles, intracisternal (ic)) administered to rats 30 min prior to ic application of strychnine (10 μg) dose-dependently reduced allodynic scores of up to 68%.60 These findings demonstrate that inhibition of glycine input opens a gate that allows lowthreshold Aβ-mediated stimuli to activate NS projection neurons and produce allodynic behaviors. Additional findings concerning these polysynaptic connections was presented in a subsequent study published by Lu and co-workers.61 Their work confirmed that glycinergic interneurons and Aβ-fibers innervate PKCγ excitatory interneurons and form a feed-forward inhibitory circuit that prevents nonnociceptive input from the deep dorsal horn from activating the nociceptive pathway (Figure 6). Immunohistochemical and simultaneous paired patch-clamp studies with sagittal rat spinal cord slices revealed both lamina III glycinergic and lamina IIi PKCγ interneurons receive input from Aβ afferents. In addition, glycinergic and PKCγ interneurons form a unitary inhibitory connection. A unitary excitatory connection between PKCγ and nociceptive transient central (TC) excitatory interneurons was also established, and these interneurons are normally silent upon Aβ fiber stimulation.61 However, under conditions of spinal nerve injury (SNI) or upon administration of strychnine, disinhibition of Aβ afferent input onto PKCγ excitatory interneurons allows low-threshold innocuous stimuli to drive the nociceptive pathway. The authors also show that intrathecal (i.t.) administration of glycine attenuated mechanical allodynia in SNI rats and that this effect was antagonized by strychnine.61 Petitjean et al. have recently shown that the inhibitory interneurons gating Aβ input onto PCKγ excitatory neurons are parvalbumin (PV)-expressing.62 Using PV::Cre; tdTom mice, the authors determined that the PV-expressing interneurons are both GABAergic and glycinergic and that they do not die after SNI. They also observed that nerve injury induces a detachment of PV neuron processes from PKCγ interneurons, which may contribute to disinhibition of Aβ afferent drive onto nociceptive pathways. In addition, clozapine-N-oxide (CNO)induced pharmacogenetic activation of PV-expressing neurons in SNI mice infected with AAV-M3D significantly attenuated

5. SPINAL GLYCINERGIC CONTROL OF PAIN SIGNALING In 1965, Melzack and Wall originally postulated that lowthreshold mechanosensitive fibers, interacting with inhibitory interneurons within the substantia gelatinosa of the dorsal horn, override or “gate” high-threshold nociceptive input when activated by innocuous tactile stimuli.56 Although this model, known as the gate theory of pain, has undergone numerous modifications over the years, the underlying concepts remain highly relevant toward our understanding of central pain mechanisms. Recent work from several laboratories has revealed the pivotal role that local inhibitory interneurons play in controlling the propagation of nociceptive signaling from the dorsal horn to the brain.57 Importantly, recently compelling evidence has provided direct proof that glycine-mediated neurotransmission segregates innocuous and pain signal processing in the dorsal horn by exerting inhibitory control over polysynaptic Aβ-fiber inputs onto NS secondary order projection neurons. Early studies involving the selective pharmacological blockade of GlyRs with 4 (strychnine) (Figure 5), an alkaloid derived

Figure 5. Chemical structure of strychnine.

from the plant Strychnos nux vomica, have provided extensive information about the physiological function of spinal glycinergic neurotransmission, specifically with regard to pain modulation. Spinal application of strychnine has been widely shown to induce recurring stereotypic behaviors (i.e., scratching, biting, and coordinated grooming), mechanical allodynia, and thermal hyperalgesia.58 A study published by Torsney and MacDermott revealed that blockade of GABAergic and glycinergic-mediated inhibition induces changes in NK1R-positive and NK1R-negative neuron activity.59 Using whole-cell patch-clamp recordings of excitatory postsynaptic currents (EPSCs) within rat spinal cord slices, the studies showed lamina I NK1R-positive neurons receive 2657



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Figure 6. Proposed polysynaptic connections by which glycinergic interneurons gate nociceptive signaling.59−63 (A) Glycinergic interneurons within the deep dorsal horn are part of a feed-forward inhibitory circuit that prevents innocuous Aβ fiber input from driving nociceptive pathways. (B) Loss of segmental glycinergic inhibitory control in the deep dorsal horn via application of strychnine, nerve injury, or targeted ablation or silencing “opens the gate” and allows low threshold Aβ mechanosensor input to stimulate NS secondary order projection neurons in the superficial lamina, resulting in allodynia.

(RVM) to superficial and deep dorsal horn layers. Using in vivo patch-clamp techniques in anesthetized rats, Kato and coworkers recorded whole-cell IPSC frequencies within the substantia gelatinosa after evoked chemical (glutamate injection) or electrical stimulation of the RVM.64 Spinal application of bicuculline or strychnine suppressed RVM stimulationinduced IPSCs, whereas 5-HT receptor antagonists had no effect. Furthermore, action potentials in the substantia gelatinosa elicited by pinch stimuli to the ipsilateral hind limb were suppressed by RVM-ES.64 Collectively, these results demonstrate that partially overlapping, monosynaptic GABAergic and glycinergic descending inhibitory input from the RVM to the substantia gelatinosa may preferentially suppresses action potentials elicited by noxious mechanical stimuli.

mechanical allodynia. Conversely, targeted ablation of PV interneurons via spinal administration of the Cre-dependent ribosome inactivating protein saporin produced mechanical allodynia, which was attenuated by blocking PCKγ interneuron activity via i.t. injection of γ-isozyme-specific peptide PKC inhibitor γV5-3.62 Zeilhofer and co-workers recently reported a series of elegant studies that provide highly compelling evidence for the crucial role of spinal glycinergic neurotransmission in the control of nociceptive signaling.63 Using a transgenic mouse expressing a BAC-Cre driven by the glycine transporter-2 promoter (GlyT-2, vide infra), the team initially conducted a morphological mapping study using green fluorescence protein (GFP) GlyT-2 positive glycinergic neurons. The study revealed preferential localization of glycinergic neurons within the deep dorsal horn of lamina III, and these interneurons are highly innervated by myelinated non-nociceptive sensory neurons. The GlyT-2::Cre mouse also afforded precise spatial ablation and silencing of glycinergic interneurons using AAV virus-mediated delivery of diphtheria and tetanus toxins, respectively. Neuronal ablation led to significant losses of glycinergic interneurons in the deep dorsal horn and, to a much lesser extent, within the superficial laminae. Electrophysiological and optogenetics experiments with horizontal spinal cord slices from cell-ablated mice showed a 65% reduction in inhibitory post synaptic currents (IPSCs) in presumptive laminae I/II excitatory dorsal horn neurons relative to GlyT-2::Cre-negative controls.63 In addition, long-lasting mechanical allodynia, thermal hyperalgesia, and spontaneous pain behaviors were observed in cell-ablated mice, which were recapitulated in the tetanus toxin-mediated synaptic silenced mice. Conversely, exogenous pharmacogenetic activation of glycinergic neurons with CNO effectively alleviated neuropathic hyperalgesia by prolonging withdrawal latencies upon heat and noxious cold stimulation, reducing the number of escape responses upon pinprick stimulation of the hind paw and reversing sensitization to mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve. Lastly, pharmacogenetic activation of glycinergic neurons also reversed histamine- or chloroquine-induced itch.63 Significant glycinergic innervation of the dorsal horn also arises from descending supraspinal tracts. GABAergic and glycinergic neurons project from the rostral ventromedial medulla

6. MECHANISMS CONTRIBUTING TO IMPAIRED GLYCINERGIC NEUROTRANSMISSION 6.1. Activity-Dependent Nociceptor Stimulation. Heterosynaptic processes leading to reduced glycine and GABA quantal release in the dorsal horn may contribute to loss of inhibitory pain control. Intense glutamate release from C-fiber nociceptors leads to concomitant activation of mGluRs 1 and 5 with subsequent production and release of endocannabinoids. Endocannabinoids serve as diffusible messengers that activate cannabinoid 1 (CB1) receptors expressed on inhibitory interneurons (Figure 7).65,66 Stimulation of CB1 receptors results in reduced presynaptic release of GABA and glycine. The CB1 agonist 5 (WIN 55,212−2) shown in Figure 8 was found to reduce presynaptic GABA and glycine release, which was reversed by via administrations of the CB1 receptor antagonist 6 (AM251).65,66 Furthermore, global CB1−/− knockout or conditional knockout mice (CB1 receptors deleted from inhibitory neurons) do not undergo capsaicin-induced pain sensitization.65,66 A separate study reported by Radhakrishman and Sluka revealed that intramuscular injections of acidic saline (100 μL of either pH = 4.0 or pH = 7.2 saline) into the gastrocnemius muscle of rats produced long-lasting hyperalgesia.67 Microdialysis experiments showed that two sequential injections of acidic saline led to a significant increase in glutamate and decrease in glycine release in the RVM. 6.2. Glycinergic Disinhibition Resulting from Nerve Damage. A reduction in synaptic GlyRs could lead to diminished 2658



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excitatory lamina II radial interneurons in parasagittal spinal cord slices extracted from partial nerve ligated (PNL) rats received diminished glycinergic, but not GABAergic, input. A 64.8 ± 8.4% reduction in the amplitude of the glycinergic component of the total eIPSC was observed in radial lamina II neurons following nerve injury relative to a sham group. eIPSCs were generated by stimulating electrodes in the deeper laminae (lamina III or IV). An increase of 310.0 ± 38.2% in the decay time constant of eIPSCs was also observed.71 PNL increased the paired-pulse ratios by 218.0 ± 38.6%, suggesting that the probability of presynaptic glycine quantal release is reduced after nerve injury. A reduction in the frequency (73.9 ± 6.23%) and an increase in decay time constant (124.6 ± 3.4%) of glycinergic sIPSCs were also observed in PNL radial neurons. Similarly, the effect of nerve injury also resulted in a reduction in glycinergic mIPSC frequencies (78.4 ± 5.0%) and increase in decay time constant (177.9 ± 22.0%). The increases in eIPSC, sIPSC, and mIPSC frequencies suggested postsynaptic adaptations to glycinergic signaling had also occurred. Indeed, Western blot analysis revealed that a reversion to homomeric GlyRα2 postsynaptic expression occurred upon nerve injury.71 Furthermore, the presence of the selective GlyRα2 antagonist cyclothiazide (100 μM) did not alter eIPSC amplitudes in lamina II radial neurons of control animals, but a significant reduction (37.9 ± 10.3%) was observed after nerve injury. Homomeric GlyRα2 channels exhibit slower activation and deactivation times and have a lower probability of opening in response to rapid and brief applications of glycine compared to heteromeric Glyα1β and GlyRα3β channels. Thus, the increase in GlyRα2 expression level is consistent with the observed eIPSC, sIPSC, and mIPSC prolonged decay constants.71 6.3. Altered Chloride Extrusion Capacity. Reduced expression of the potassium−chloride cotransporter (KCC2) is another mechanism potentially contributing to spinal disinhibition and neuropathic pain (Figure 9). KCC2 maintains neuronal chloride homeostasis by keeping chloride levels low, which allows for chloride influx and hyperpolarization when GABAA and GlyR receptors are stimulated. Upon nerve damage, afferent nociceptive fibers release the cytokine chemokine (C−C motif) ligand 2 (CCL2) within the dorsal horn, which binds to CCR2 receptors on microglia.72 CCR2 receptor stimulation, together with co-obligatory adenosine triphosphate (ATP)gated purinoceptor activation (e.g., P2X4 and P2X7 receptors), activate microglia, which then release BDNF.73 BDNF binds to tropomyosin receptor kinase (trkB) receptors expressed on postsynaptic projection neurons, resulting in reduced expression of KCC2 and altered chloride extrusion capacity.73,74 The net effect of these changes is an increase in intracellular chloride concentration, which lessens the impact of GABAA and GlyR inhibition. Local spinal administration of ATP or the KCC2 inhibitor [(dihydroindenyl)-oxy]alkanoic acid (DIOA) (structure not shown) induces tactile allodynia in rats.75 In vivo recorded spontaneous bursts of activity in lamina I projection neurons treated with ATP or DIOA were comparable to those observed in lamina I projection neurons from SNI rats.75 Conversely, i.t. KCC2 gene transfer (lentiviral vectors engineered to express KCC2-GFP) to spinal nerve ligated (SNL) rats restored Cl− homeostasis in the dorsal horn and DRG and produced complete and long-lasting relief of neuropathic pain.76 Interestingly, Schwale et al. have recently shown that short hairpin RNA (shRNA)-mediated KCC2 knockdown during in vitro maturation of cultured spinal cord neurons significantly reduces the number and sizes of dendritic clusters of Glyα1β

Figure 7. CB1 receptor-mediated reduction of glycine and GABA release.65,66 Intense glutamate input from afferent C-fibers leads to the production and release of endocannabinoids from the postsynaptic neuron. These endocannabinoids diffuse and bind to presynaptic CB1 receptors expressed on inhibitory interneurons, which leads to suppression of glycine and GABA release.

Figure 8. CB1 receptor agonist 5 and CB1 receptor antagonist 6.

glycinergic neurotransmission in chronic pain states. Immunohistochemical studies conducted by Simpson et al. showed that sciatic nerve constriction (SNC) in rats leads to a bilateral reduction in the number of glycine receptors expressed within the dorsal horn.68 GlyR staining (diaminobenzadine tetrachloride) in ipsilateral to injury laminae II−IV dorsal horn neurons of nerve injured rat spinal cord slices was significantly attenuated (clearly stained cells reduced by approximately 80%) relative to sham controls. A more modest reduction of ∼20% was observed in laminae II−IV of the contralateral dorsal horn region.68 Altered extracellular glycine concentrations in response to nerve injury may also contribute to diminished spinal glycinergic tone. Miyazato reported glycine concentrations within the lumbosacral cord of chronic spinal cord injury (SCI) female Sprague−Dawley rats was approximately 54% lower relative to intact rats.69 No changes in glutamate or GlyRα1 mRNA levels were detected. Reduced spinal glycine concentrations were also observed in streptozotocin (STZ)-induced diabetic neuropathic pain (DNP) rats.70 Microdialysis experiments conducted by Chiu and co-workers revealed that persistent hyperglycemia induced by STZ lowered CSF glycine levels after an initial, transient increase. Whole-cell patch-clamp recordings using isolated DNP rat spinal cord slices revealed a statistically significant reduction in mean GlyR-mediated mIPSC frequencies in the ensemble of the spinal lamina I neurons relative to controls. Furthermore, i.t. administration of glycine (10 and 100 μg) to DNP rats reduced tactile pain hypersensitivity.70 Recent evidence reported by Imlach et al. suggest reduced inhibitory glycinergic neurotransmission in response to nerve injury is mediated by pre- and postsynaptic maladaptive changes.71 In vitro whole-cell patch-clamp electrophysiology studies conducted by Imlach and co-workers showed that 2659



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Figure 9. Reduced chloride extrusion capacity contributes to loss of inhibitory control.72−75 CCL2 released by nociceptors and ATP activate resident spinal microglia. BDNF released by activated microglia stimulates postsynaptic trkB receptors. This leads to reduced KCC2 expression and chloride extrusion capacity. Increased concentration of intracellular chloride diminishes the inhibitory capacity of GlyRs and GABAAR.

Figure 10. Inhibition of glycinergic neurotransmission via phosphorylation of GlyRα3β.79,80 (A) GlyRα3β receptors expressed on NS secondary order projection neurons within the superficial lamina become phosphorylated upon PE2 activation of prostaglandin E2 receptor 2 (EP2) and PKA. (B) Localization of the S346 in the primary sequence of the mouse GlyRα3 subunit.83 Activation of EP2 triggers PKA, leading to phosphorylation of S346 in the α3 TM3−TM4 intracellular domain. The resulting conformational changes induced by phosphorylation propagate throughout the receptor and affect the orthosteric glycine-binding site, which significantly diminish glycine affinity for the receptor.

mIPSCs amplitudes in GABAergic lamina II neurons extracted from formalin-treated mice were markedly higher than salinetreated mouse controls (148.7% ± 12.4 increase in mIPSC amplitude relative to controls).78 Collectively, these data show that peripheral inflammation leads to GlyR-mediated LTP on lamina II GABAergic neurons. The authors argue that activation of microglia and astrocytes via stimulation of P2X7 receptors largely drives the secretion of IL-1β that manifests as GlyR LTP.78 Indeed, mouse spinal cord slices exposed to the P2X7 receptor agonist 2′(3′)-O-(4-benzoylbenzoyl)adenosine5′-triphosphate tri(triethylammonium) salt (Bz-ATP) (structure not shown) increased ISPC amplitudes (135.0% ± 13.0 increase relative to controls), whereas pre-exposure to the IL-1β scavenger protein IL-1Trap prevented potentiation induced by Bz-ATP.78 6.4. Inhibition of GlyRα3β Receptors. GlyRα3β receptor distribution is distinctly restricted to the superficial layer of the dorsal horn where it is expressed on NS secondary order projection neurons and plays a critical role in modulating nociceptive signaling. Accordingly, diminished glycinergic signaling via inhibition of GlyRα3β is implicated in certain inflammatory pain states.79,80 Inhibition of GlyRα3β occurs via a prostaglandin type E2 (PGE2) and PKA-dependent phosphorylation (Figure 10).79,80 Using voltage-clamp fluorometry with subsequent in vitro pharmacological assessment, Lynch and co-workers determined that PKA-induced phosphorylation of serine 364 (S346) in the α3 subunit TM3−TM4 intracellular domain induces global conformational changes that propagate to the glycine-binding site structure within the extracellular domain.81 Furthermore, Lynch and co-workers also propose

channels and gephyrin but not of neonatal homomeric GlyRα2.77 Whole-cell patch clamp studies revealed that the changes GlyR subtype expression corresponded with reduced frequencies and amplitudes of glycinergic mIPSCs but had no impact on GABAergic neurotransmission.77 In addition to causing KCC2 downregulation, activated microglia may also be contributing to IL-1β-induced GlyR LTP at glycinergic synapses on inhibitory GABAergic interneurons. Kauer and co-workers hypothesize that IL-1β release from activated microglia or astrocytes potentiates glycine synapses on GABAergic interneurons, thus inducing disinhibition of nociceptive pathways.78 Bath application of IL-1β to mouse spinal cord slices rapidly potentiated lamina II glycinergic IPSCs (IPSC amplitudes: 160.8 ± 11.6% of pre-IL-1β values). Conversely, potentiation of GABAergic or glutamatergic synapses was not observed upon IL-1β application. Glycinergic IPSC potentiation persisted for the duration of the recordings, suggesting initiation of LTP had occurred, which was attributed to an increase in the number and/or biophysical properties of postsynaptic GlyRs. It was postulated that increased GlyR expression or function results from IL-1β downstream activation of the p38 MAPK pathway.78 In vivo peripheral inflammation in formalin-treated mice was also found to correlate with GlyR LTP. Lamina II neurons from spinal cord slices of formalin-treated animals did not show GlyR LTP potentiation in response to IL-1β application. This was in contrast to significant potentiation observed for spinal neurons of saline-treated animals (IPSC amplitudes increased 176.6% ± 16.7), which is consistent with the hypothesis that inflammation-induced release of IL-1β had already induced GlyR LTP.78 In addition, 2660



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that their findings suggest S346 phosphorylation occludes an intracellular binding site for an expression system-specific signaling molecule.81 Application of PGE2 inhibits glycinergic IPSCs in spinal cord slices of wild-type but not GlyRα3β−/− knockout mice.79 Furthermore, EP2−/− and GlyRα3β−/− knockout mice demonstrate a reduction in pain hypersensitivity in response to spinal application of PGE2 and recover more quickly from inflammation-induced pain sensitization relative to wild-type controls. GlyRα3β−/− knockout mice exhibited similar levels of complete Freund’s adjuvant (CFA)-induced thermal pain sensitization relative to wild-type controls, but they recovered more quickly. However, GlyRα3β−/− knockout mice do not show altered pain sensitivity to capsaicin, carrageen, kaolin/carrageen (model of rheumatoid arthritis), or monosodium iodoacetate injection (model of osteoarthritis), suggesting that GlyRα3β inhibition does not contribute to all forms of inflammatory pain.79 GlyRα3β inhibition via glutamate−nitric oxide−cyclic GMP signaling may present a mechanism by which platelet-activating factor (PAF) induces tactile allodynia in mice.82 PAF is a potent phospholipid mediator released by a variety of cells and involved in numerous processes, including inflammation and the facilitation of neuropathic pain. PAF (0.19 fmol, i.t.) failed to induce thermal hyperalgesia and tactile allodynia in mice treated with GlyRα3 siRNA. In addition, siRNA GlyRα3 knockdown significantly diminished antiallodynia activity of PAF antagonists relative to wild-type controls.82

Figure 11. Glycinergic synapse. At inhibitory glycinergic neurons, GlyT-1 expressed on glial astrocytes clears glycine from the synapse, leading to a reduction in GlyR activity. GlyT-2 also clears glycine from the synapse and transports it back into the presynaptic cytosol, where it is repackaged into vesicles via VGAT/VIAAT for rerelease into the synapse. Glycine binds to postsynaptic GlyRs leading to Cl− influx, hyperpolarization, and suppression of neuronal firing.

the retina and has recently been identified within the DRG.89 GlyT-1 transport is bidirectional and likely operating close to equilibrium, requiring a symporter stoichiometry of two Na+ and one Cl− for every glycine transported.90 Unlike GlyT-1, GlyT-2 is exclusively colocalized with GlyRs and its expression is restricted to presynaptic glycinergic axon terminals within the brain stem, cerebellum, and spinal cord.91 The transporter is abundantly expressed in the spinal cord with highest densities within lamina III of the dorsal horn. This distribution pattern has led to the wide use of GlyT-2 as a reliable marker for glycinergic neurons.91 GlyT-2 regulates inhibitory glycinergic neurotransmission via two interdependent mechanisms: (1) clearance of synaptic glycine and uptake into presynaptic terminals and (2) repackaging glycine into vesicles via coordination with the vesicular GABA transporter/ vesicular inhibitory amino acid transporter (VGAT/VIAAT) for synaptic rerelease (Figure 12).92 GlyT-2-mediated transport requires three Na+ and one Cl− with the higher Na+ transport stoichiometry, conferring more powerful glycine transport relative to GlyT-1 that is also unidirectional from extracellular to intracellular space.90 This feature allows GlyT-2 to maintain very high intracellular glycine concentrations (10−20 mM) in order to effectively compete with GABA at VGAT/VIAAT and ensure sufficient glycine loading into presynaptic vesicles. Thus, inhibition of GlyT-2 can lead to increases synaptic glycine concentration, resulting in augmented GlyR-mediated fast inhibitory neurotransmission. 7.2. Inhibition of GlyT-2. Several comprehensive reviews have been written about the therapeutic potential of glycine transporter inhibitors.93 Numerous potent and selective inhibitors of GlyT-1 have been extensively studied for their pharmacotherapeutic potential to treat various neuropsychiatric and neurological disorders. However, inhibition of GlyT-2 has been more thoroughly investigated for the treatment of pain. The combination of high colocalization with GlyRs, restricted distribution within the CNS, and high expression density in the dorsal horn make GlyT-2 a very attractive target for the treatment of chronic pain. Multiple distinct chemotypes capable of inhibiting GlyT-2 with high affinity and selectivity have been reported (e.g., 7 (ALX-1393),94 8 (Org-25543),95 9 (NAGly),96 10 (GT-0198),97 11,98 12,99 (±)-13,100 14,101 15102)

7. GLYCINE TRANSPORTER INHIBITION Intrathecal administration of glycine has proven effective at ameliorating nociceptive behaviors in several preclinical rodent pain models.84 In light of these observations, efforts to identify pharmacological agents capable of increasing spinal glycinergic tone ensued. Several groups have focused on the inhibition of glycine uptake as a means by which to increase spinal extracellular glycine concentrations and enhance GlyR-mediated neurotransmission in the dorsal horn. In addition, efforts focused on identifying subtype selective GlyR PAMs have also begun to emerge, and the approach is beginning to garner significant interest. We will present recent advances and challenges faced for these two key areas. 7.1. The Glycine Transporters. Homeostatic extracellular glycine concentrations within the CNS are tightly regulated by two high-specificity, solute carrier family 6 (SLC6) transporters; glycine transporter-1 (GlyT-1) and glycine transporter2 (GlyT-2).85 GlyT-1 is broadly expressed throughout the CNS with highest levels of distribution found in caudal areas (cerebellum, brainstem, and spinal cord) and lower levels in the forebrain (hippocampus, striatum, and prefrontal cortex (PFC)).86 The transporter is localized on glial cells (largely astrocytes) within the hindbrain and spinal cord, where it serves to clear glycine from the GlyR synaptic cleft, ultimately leading to termination of inhibitory glycinergic neurotransmission (Figure 11). Therefore, inhibition of GlyT-1 in these regions results in increased extracellular glycine concentrations and enhanced glycinergic neurotransmission.86 Within the forebrain, GlyT-1 is expressed on glial cells as well as on preand postsynaptic glutamatergic neurons where it is highly colocalized with NMDA receptors and carefully maintains synaptic glycine concentrations below saturation levels at the glycine-B binding site (less than 1 μM).87,88 Thus, inhibition of GlyT-1 may also lead to increased NR1 glycine-B site occupancy and enhanced NMDA receptor function. In addition, GlyT-1 is also expressed on amacrine and ganglion cells within 2661



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Figure 12. Reported GlyT-2 inhibitors.

Table 1. Reported Preclinical Pain Models for Which GlyT-2 Inhibitors Have Demonstrated Analgesic Efficacy GlyT-2 inhibitor(s) studied

pain models

routesa

ref

rat tail-flick, hot plate, paw pressure rat and mouse formalin test

7 7, 8, 9, 11, 16

i.t., sc, icv

rat and mouse CFA rat CCI rat and mouse PSNL rat SNL rat SCI mouse herpetic and postherpetic pain mouse STZ mouse femur bone cancer

7 and 8 7 7, 8, 9, 10 16 7 7

i.t. i.t., icv, sc i.t., iv, po iv, po i.t. iv

latency of tail removal from radiant heat source; response to pressure to the applied paw measurement of spontaneous biphasic flinching/shaking of the injected paw, quantification of paw lick duration mechanical allodynia (von Frey), thermal hyperalgesia (Hargreaves) mechanical allodynia, cold hyperalgesia, thermal hyperalgesia mechanical allodynia, thermal hyperalgesia mechanical allodynia intercontraction interval measurement dynamic and static mechanical allodynia

7, 8 7, 8

i.t., iv i.t., iv, po

mechanical allodynia allodynia, guarding behavior, limb use

a

i.t.

nociceptive behaviors assessed

104 97, 104, 112, 113, 116 105 105, 107 96, 105, 114 117 110 108 105 111

Routes of administration; i.t. = intrathecal, icv = intracerebroventricular, sc = subcutaneous, iv = intravenous, po = oral.

GlyT-2 inhibitor 8 selectively inhibits [3H]-glycine uptake in CHO cells stably expressing hGlyT-2 (IC50 = 16 nM) with no ancillary activity at GlyT-1 (IC50 > 100 μM).95 Unlike 7, benzamide 8 exhibits moderate brain penetrance (Kp,uu = 0.5, mouse).98 Whole-cell voltage clamp recordings from lamina X neurons in rat spinal cord slices exposed to a bath of 8 (10 μM) revealed that the compound induced slowly developing GlyRmediated inward currents and significantly increased decay time constants of mIPSCs, eIPSCs, and spontaneous inhibitory post synaptic currents (sIPSCs).104 These effects were recapitulated in a concentration-dependent manner in a separate experiment using bath applications of low concentrations of glycine (1−3 μM), confirming that the effects of 8 on IPSCs were the result of increased extracellular glycine concentrations. In addition, in vivo microdialysis perfusion of 8 (10 μM) induced a statistically significant increase in extracellular glycine within the rat spinal cord relative to controls (337 ± 76% increase relative to basal levels).104 7.4. In Vivo Analgesic Efficacy of GlyT-2 Inhibitors 7 and 8. GlyT-2 inhibitor 7 exhibited antinociceptive effects in

(Figure 12), and many of them have shown considerable potential as novel analgesics by demonstrating broad efficacy in several rodent models of acute, inflammatory, and neuropathic pain (Table 1). Furthermore, GlyT-2 inhibitors have demonstrated efficacy in preclinical studies involving both acute and chronic dosing regimens. The two most extensively studied GlyT-2 inhibitors to date are compounds 7 and 8, which have been widely used as tools for gaining a better understanding of GlyT-2 pharmacology. 7.3. In Vitro and in Vivo Profiles of Benchmark GlyT-2 Inhibitors 7 and 8. Lipophilic amino acid 7 inhibits [3H]-glycine uptake in stably transfected HEK-293 cells expressing hGlyT-2 with an IC50 = 100 nM. The compound exhibits a 40-fold selectivity over GlyT-1 (hGlyT-1 IC50 = 4 μM) and poor CNS permeability (Kp,uu = 0.036, mouse).98 Inhibitor 7 prolonged the decay phase of GlyR-mediated evoked inhibitory postsynaptic currents (eIPSCs) and significantly increased the frequency of GlyR-mediated tonic inward miniature inhibitory post synaptic currents (mIPSCs) in the substantia gelatinosa of mouse spinal cord slices.103 2662



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series of acute and inflammatory rat pain models.105 Spinal administration (4, 20, and 40 μg, i.t.) dose-dependently modulated thermal pain and prolonged latencies in the hot plate and tail-flick tests, and the effects were completely reversed by strychnine (10 μg, i.t.). Spinal application (40 μg, i.t.) also significantly increased the vocalization threshold in rats subjected to a paw pressure test, and the effects could again be antagonized by strychnine (10 μg, i.t.).105 Administration of 7 (20 and 40 μg, i.t.) also suppressed flinching/shaking behaviors in the rat formalin test. The test produces a biphasic pain response to formalin injection in the hind paw. Phase I occurs 10 min post formalin injection and is indicative of acute nociceptive pain, whereas phase II begins approximately 60 min after formalin injection and develops in response to central sensitization. Only the 40 μg dose decreased phase I pain behaviors, however, significant dose-dependent decreases in pain behaviors for the 20 and 40 μg doses were observed during phase II. In addition, no motor effects were observed in the rotarod test (20 and 40 μg, i.t.).105 However, severe respiratory depression and motor impairment was observed at a higher 60 μg i.t. dose, which is a phenotype consistent with excessive glycinergic neurotransmission associated with prolonged GlyT-1 inhibition and may be attributed to the compound’s ancillary activity at that transporter. A comprehensive battery of studies were conducted by Morita and colleagues to examine the analgesic efficacy of 7 and 8 against mechanical allodynia in a variety of mouse neuropathic pain models.106 These studies revealed that administration of 7 (10 ng, i.t.; 10 μg/kg, iv) or 8 (3, 30, and 300 ng, i.t.; 0.3 mg/kg, iv) significantly reduced paw withdrawal thresholds (von Frey) in partial sciatic nerve ligation (PSNL) mice, and the effects observed for 8 were dose-dependent. In addition, these effects for 8 were more potent and longer lasting than gabapentin (0.1, 1, and 10 μg, i.t.; 75 mg/kg, iv) (8 ED50 = 11.0 ng/mouse; gabapentin ED50 = 8200 ng/mouse).106 Paw withdrawal thresholds were also significantly and dose-dependently reduced in STZ-induced diabetic mice with administration of 7 (1, 3, and 10 ng, i.t.; 10 μg/kg and 0.1 mg/kg, iv) or 8 (3, 30, and 300 ng, i.t.; 10 μg/kg and 0.1, 1, and 10 mg/kg, iv). Antiallodynia effects for 7 (10 ng, i.t.) and 8 (300 ng, i.t.) were also observed in the CFA chronic inflammation model, and the analgesic effects were comparable to a 10 μg, i.t. dose of gabapentin.106 Furthermore, the effects observed for 7 and 8 in the aforementioned pain models were antagonized by either i.t. administration of strychnine or siRNA GlyRα3β knockdown, confirming that their mechanism of action is via augmentation of GlyR-mediated neurotransmission. Importantly, no adverse effects on locomotor activity, motor behavior, or the righting reflex were observed in any of these studies with 7 or 8.106 Hermanns and co-workers reported only observing analgesic effects with 7 at the highest dose administered (10, 50, and 100 μg, i.t.) in the rat CCI model of neuropathic pain.107 Furthermore, they also reported observing severe respiratory depression and motor effects with some of the animals in the efficacious dose cohort, which may be due ancillary GlyT-1 inhibition, causing an excessive increase in extracellular glycine concentrations. However, a separate study reported by Barthel indicated that chronic administration of 7 administered to CCI rats over 14 days via subcutaneous (sc) osmotic infusion pump (0.2, 2, 20, and 200 μg/kg/day) both dose- and timedependently reduced thermal hyperalgesia and mechanical allodynia without inducing adverse respiratory or neuromotor effects.108 Western blot analysis also revealed that the chronic

application of 7 had no effect on GlyT-2 or GlyT-1 expression levels in the ipsilateral spinal cord.108 The observed sustained analgesic efficacy upon chronic systemic administration of 7 suggests that the compound did not induce tolerance at GlyT-2 nor did it promote GlyR tachyphylaxis or desensitization. GlyT-2 inhibitor 7, but not the GlyT-1 inhibitor sarcosine (vide infra), attenuated dynamic and static allodynia in a mouse herpetic and postherpetic pain model.109 Spinal application of 7 (1, 3, and 5 μg, i.t.) to mice that underwent percutaneous inoculation with herpes simplex virus type-1 (HSV) dosedependently ameliorated dynamic allodynia at the herpetic (day 7) and postherpetic stages (days 35−40). Administration of 7 (10 and 30 μg, i.t.) also produced statistically significant and dose-dependent inhibition of static allodynia at the postherpetic stage. A 10 μg i.t. dose of 3 also attenuated mechanical allodynia at the postherpetic stage. Furthermore, pretreatment with 7 (5 μg, i.t.) inhibited both strychnine and NMDA-induced dynamic allodynia.109 Recent data also suggest that GlyT-2 inhibition may present a potential treatment option for overactive bladder, bladder pain, or interstitial cyctitis.110 Spinal application of 7 (3 and 10 μg, i.t.) significantly increased the intercontraction interval and the micturition pressure threshold during cystometry in cyclophosphamide (CYP)-treated adult female Sprague− Dawley rats.110 These effects could be reversed by strychnine. Furthermore, the 10 μg i.t. dose strongly suppressed the micturition reflex in some of the rats. 7 (3, 10, and 30 μg, i.t.) also dose-dependently suppressed licking and freezing nociceptive behaviors after bladder irritation via treatment with intravesical resininferatoxin.110 Intrathecal application of 7 (3, 10, and 30 μg) dosedependently reduced the amplitude and frequency of nonvoiding contractions (NVC) in SCI female Sprague−Dawley rats.111 Significant reductions were observed at the 10 μg (26% NVC amplitude reduction and 65% frequency reduction) and 30 μg doses (37% NVC amplitude reduction and 76% frequency reduction). Interestingly, RT-PCR analysis revealed SCI rats exhibited significantly higher GlyT-2 mRNA expression levels with no changes to GlyT-1 or GlyR levels in the L6-S1 spinal cord compared to spinal intact rats.109 These findings may indicate that inhibitory glycinergic neurotransmission in SCI rats is compromised due in part to increased expression of GlyT-2, which could enhance glycine uptake and reduce synaptic glycine concentrations. 111 Importantly, these studies provide further evidence that GlyT-2 inhibition may potentially treat overactive bladder with neurogenic detrusor overactivity by suppressing sensory inputs from the bladder to the dorsal horn. Motoyama and co-workers showed that administration of 7 or 8 provided dose-dependent and multiday improvements in allodynia scores in a murine femur bone cancer (FBC) pain model.112 The medullary cavity of the distal femur of male C3H/HeN mice were injected with NCTC 2472 tumor cells, and tumor implantation induced a set of pain behaviors categorized as tactile allodynia, withdrawal threshold, guarding behavior, and limb-use abnormality. Administration of 7 (0.01 mg/kg, iv) or 8 (0.03, 0.1, and 0.3 mg/kg, iv) 11 days after tumor implantation ameliorated the aforementioned pain behaviors, and the effects were long-lasting (5−10 days postdose). An oral (po) dose of 7 (0.3 and 1 mg/kg) also provided robust, dose-dependent analgesic efficacy in the FBC model.112 Additionally, siRNA knockdown of spinal GlyT-2 improved pain-like behaviors in this model. Transfection with 2663



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activity (h5-HT2A IC50 = 1.3 μM).117 It has been argued that the dual GlyT-2 inhibitor/5-HT2A antagonist pharmacological profile of 16 provides a synergistic effect that yields robust preclinical in vivo analgesic efficacy despite moderate potency for each target. Indeed, the compound exhibited dose-dependent efficacy in the rat formalin test (12.5, 25, and 50 mg/kg, sc) with an ED50 = 20 mg/kg, and the efficacy observed for the 25 mg/kg sc dose was comparable to a 3 mg/kg (sc) dose of morphine.117 In addition, oral administration (50, 100, and 200 mg/kg) dose-dependently reduced mechanical allodynia in the rat spinal nerve ligation SNL (Chung) model, with an ED50 = 80 mg/kg. Good pharmacokinetic−pharmacodynamic (PK−PD) correlations were observed in both the formalin and SNL studies.117 Compound 16 is also the first GlyT-2 inhibitor to enter clinical trials. Single ascending dose (SAD) and multiple ascending dose (MAD) phase 1 clinical trials were conducted with healthy volunteers (NCT02333318, NCT01905410).118 For the SAD component of the trial, the drug was administered via iv infusion over 4 h period over a dose range of 0.25, 0.5, 1, 2, 4, 6, and 8 mg/kg. The MAD study involved low and high doses administered via iv infusion over a period of 4 h twice daily over 3 days. 16 was found to be safe and well tolerated in both SAD and MAD components of the phase 1 trial. A subsequent series of randomized, double-blind, and placebo-controlled phase 2a proof-of-concept trials to investigate the compound’s ability to treat postoperative pain have been initiated or were conducted with early gastric cancer patients following laparoscopic-assisted abdominal gastrectomy (NCT02522598), laparoscopic-assisted gastrectomy (NCT02844725), and laparoscopic colorectal surgery (NCT02489526, NCT02992041).118 Data from completed trials have yet to be published. Vivozon is also currently recruiting patients for a phase 1b/2a trial to evaluate the safety and efficacy of 16 with injections given to patients with lumbar radiculopathy (sciatica). 7.6. Mechanism-Based Safety Concerns for GlyT-2 Inhibitors: The GlyT-2−/− Knockout Mouse Phenotype and Human Hyperekplexia. Despite the promising preclinical proof-of-concept data for GlyT-2 inhibitors, clinical advancement within the field has largely been hampered due in part to perceived on-target liabilities associated with a lethal knockout mouse phenotype. Homozygous GlyT-2−/− knockout mice live only to the second postnatal week and present motor abnormalities including tremor, muscular spasticity, and impaired coordination.119 The animals exhibited significantly diminished glycinergic neurotransmission due to complete loss of GlyT-2, which prevents presynaptic vesicular reloading of glycine. Similarly, loss-of-function homozygous mice derived from a spontaneous GlyT-2 (SLC6A5) mutation express an identical hypoglycinergic phenotype.120 However, heterozygous GlyT-2± knockout and mutant mice are viable and devoid of adverse motor behaviors. In addition, PSNL mice that undergo siRNA GlyT-2 knockdown (75% GlyT-2 knockdown relative to wild-type controls) exhibit reduced mechanical allodynia over a time-course that correlates with reductions in GlyT-2 immunoreactivity, without adverse respiratory or motor effects.106 Taken collectively, these data suggest that partial loss of GlyT-2 activity is tolerated. These findings are recapitulated in humans as numerous missense, nonsense, and frameshift SLC6A5 mutations are associated with hyperekplexia, a rare paroxysmal neurological disorder caused by impaired glycinergic neurotransmission.121 The majority of these patients are homozygous

GlyT-2 siRNA was conducted 13 days after tumor implantation, and Western blot analysis confirmed that peak reduction of GlyT-2 occurred 3 days after siRNA transfection. Pain behaviors were attenuated by GlyT-2 knockdown in a manner similar in magnitude and duration observed for i.t. administration of 300 ng of 8.109 Time-dependent deteriorative and ameliorative effects for 8 and strychnine on pain behaviors were also noted, which could be due to initial changes in chloride gradients within the superficial dorsal horn that can occur in the early phases of peripheral nerve injury. Furthermore, administration of 8 (0.03, 0.1, and 0.3 mg/kg, iv or 0.3 and 1.0 mg/kg po) also exhibited synergistic effects with subtherapeutic doses of morphine (0.3 mg/kg, sc) and significantly improved pain-like behaviors.112 These findings are significant as bone cancer pain typically very difficult to treat as it often exhibits reduced responsiveness and becomes refractory to opioid treatment. GlyT-2 inhibition also provides supraspinal antinociception by restoring descending glycinergic inhibition. Intracerebroventricular (icv) administration of 7 (25, 50, and 100 μg) dosedependently suppressed phase II pain behaviors in the rat formalin test.113 Administration of 7 (25, 50, and 100 μg, icv) also attenuated mechanical hyperalgesia, cold hyperalgesia, and thermal in CCI rats. These analgesic effects observed in the CCI model could be antagonized by coadministration of icv strychnine.113 7.5. In Vivo Activity of Other GlyT-2 Inhibitors. The endogenous fatty acid 9 induces analgesic effects in part via inhibition of GlyT-2 (GlyT-2 IC50 = 3 μM).96 The highest concentrations of 9 are found in the spinal cord, and the compound has also been found to inhibit other pain targets that include COX-2, T-type Ca2+ ion channels, and fatty acid amide hydrolase (FAAH). Furthermore, 9 acts as a GlyR PAM, which may also contribute to its analgesic properties (vide infra). Bath application of 9 (60 μM) was shown to prolong the timecourse of glycine-evoked currents within the substansia gelatinosa of rat spinal cord slices.93 Superfusion of 9 (30 μM) also produced GlyR-mediated inward currents without effecting mIPSCs and increased the decay-time of GlyR-mediated eIPSCs.96 Furthermore, sc application (275 nmol) significantly reduced pain behaviors in the rat formalin test114 and ameliorated mechanical allodynia and thermal hyperalgesia in the rat partial nerve ligation (PNL) (700 nmol, i.t.)115 and CFA models (70−700 nmol, i.t.).116 Toray Industries Inc. reported the discovery of 10, which selectively inhibits GlyT-2-mediated [3H]-glycine uptake in stably transfected HEK-293 cells expressing hGlyT-2 with an IC50 = 105 nM.97 Potent antiallodynia effects were observed in the mouse PSNL neuropathic pain model upon single-dose iv administration (3 mg/kg). Statistically significant dose-dependent reductions in allodynia scores were also observed upon po (3, 10, and 30 mg/kg) and i.t. (1, 20, and 100 μg) administration. The highest doses for 10 administered (30 mg/kg, po and 100 μg, i.t., respectively) exhibited analgesic efficacy comparable to a 10 mg/kg (po) and 10 μg (i.t.) dose of gabapentin. Lastly, coadministration of strychnine abolishes the analgesic activity of 10, providing verification that the compound is ameliorating pain behaviors via augmentation of GlyR-mediated neurotransmission by elevating extracellular glycine concentrations.97 Vivozon Inc. has reported the discovery of 16 (VVZ-149) (structure not disclosed), a modestly potent GlyT-2 inhibitor (hGlyT-2 IC50 = 0.86 μM) with ancillary 5-HT2A antagonist 2664



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Figure 13. GlyT-1 inhibitors studied in preclinical pain models.

administration, which are outcomes consistent with the GlyT-2−/− knockout mouse phenotype. A PK−PD mismatch was also observed as the minimally efficacious dose (0.06 mg/kg) and toxic dose (20 mg/kg) were lower than expected as per estimated transporter occupancies based on brain drug exposures (6% and 82%, respectively).98 Conversely, structurally related analogue 11 presented apparent reversible dissociation binding kinetics and ip administration (3, 10, 25, and 100 mg/kg, ip) dose-dependently reduced pain behaviors without adverse effects and with good PK−PD correlation. Unlike 8, low doses of 11 with predicted transporter occupancies below 10% did not induce analgesia. The minimally effective dose of 25 mg/kg had an estimated transporter occupancy of 36% and reduced phase II paw licking behaviors by 33%. The 100 mg/kg dose produced a profound 80% reduction in pain behaviors with no convulsions or mortality, and the estimated transporter occupancy at this dose was 60%.98 Subsequent Irwin tests revealed that 7 has a narrow TI, induced tremors and stereotypies at analgesic doses (0.2 and 2 mg/kg), and presented an overall excitatory profile. Contrarily, 11 did not impair motor function at all doses tested (limited to 100 mg/kg as the highest dose due to poor solubility) and it exhibited a significantly improved TI relative to 8 with an overall sedative profile comparable to gabapentin.98 Drugs with reversible dissociation binding kinetics and short residence times have been shown to be advantageous for targets that pose a potential risk for mechanism-based toxicity. Multiple cases have been reported whereby the design of safe therapeutics for such targets was accomplished via utilization of fast-off binding kinetics to optimize the TI.122 Acknowledging the findings reported by Mingorance-Le Meur et al., it is possible that a potent GlyT-2 inhibitor with reversible dissociation binding kinetics could avoid mechanism-based adverse toxicity liabilities and provide a therapeutic with a desirable balance of efficacy and tolerability. 7.8. GlyT-1 Inhibitors. The selective GlyT-1 inhibitors 17 (ALX-5407) (hGlyT-1 IC50 = 3 nM),123 18 (Org-25935) (hGlyT-1 IC50 ∼ 100 nM),124 19 (sarcosine) (hGlyT-1 IC50 = 91 μM),125 and 20 (N-ethylglycine)126 have also exhibited analgesic efficacy in preclinical pain models (Figure 13). As glycine serves as an obligatory coagonist at the NMDA receptor, increasing glycine concentrations via GlyT-1 inhibition is generally thought to promote excitatory glutamatergic neurotransmission and could potentially be pro-algesic. However, glycine also primes the NMDA receptor for internalization127 and prolonged GlyT-1 inhibition has been shown to reduce NMDA receptor expression in the spinal cord.108 Chronic administration of 17 administered to CCI rats over 14 days via sc osmotic infusion pump (0.2, 2, 20, and 200 μg/kg/day) significantly reduced the nociceptive response to thermal and mechanical allodynia.108 Western blot analysis revealed that long-term administration of 17 significantly

or compound heterozygous recessive inheritance carriers. Heterozygous carriers are asymptomatic, providing evidence for required biallelic loss of GlyT-2 for disease expression. Rare dominant negative mutations involve mutants that impede wild-type trafficking or hetero-oligomer pairing.121 These data provide further evidence that submaximal pharmacological inhibition of GlyT-2 will be tolerated in humans. Studies with cultured spinal neurons show that prolonged exposure to very high concentrations of inhibitor 794,103 or 8103,104 show an initial increase followed by reduction in glycinergic neurotransmission, presumably resulting from unfavorable disruption of glycine resupply into presynaptic vesicles. These observations, in conjunction with the GlyT-2−/− knockout mouse phenotype, further suggest that complete loss of GlyT-2 transport activity would ultimately lead to diminished glycinergic activity and potentially induce undesirable effects. However, the aforementioned reported efficacy studies are largely devoid of observed undesirable effects that mimic the knockout mouse phenotype, which includes a 14-day chronic dosing study. This discrepancy may be because in vivo exposures attained for GlyT-2 inhibitors 7 or 8 in model studies where no adverse effects were observed were not sufficient for full transporter occupancy and complete shutdown of GlyT-2 mediated glycine uptake. The asymptomatic presentation of heterozygous GlyT-2± knockout and mutant mice, siRNA knockdown mice, and human heterozygous carriers of SLC6A5 mutations suggests that partial reduction of GlyT-2 expression permits sufficient reuptake of glycine, thereby maintaining normal levels of glycinergic neurotransmission. Additional data correlating transporter occupancy, analgesic efficacy, and safety for GlyT-2 inhibitors are required to allow a more detailed appraisal of the target. However, the completed phase 1 and ongoing phase 2 clinical trials with 16 does provide some support that GlyT-2 inhibition can be safe in humans at exposures that are predicted to be efficacious. 7.7. Reversible Dissociation Binding Kinetics for GlyT-2 Inhibition: Reducing the Potential Risk of Mechanism-Based Toxicity. Mingorance-Le Meur et al. report that apparent slow dissociation rate binding kinetics observed for 8 renders the inhibitor pharmacologically irreversible, and this characteristic may have led to observed acute mechanism-based motor effects and a narrow therapeutic index (TI) in a mouse formalin test.98 Inhibited glycine-evoked currents in Xenopus laevis oocytes expressing hGlyT-2 and incubated with 8 were unchanged after wash-out, indicating that the compound possesses a very slow off-rate. In vivo study in a formalin test with adult male mice revealed that intraperitoneal (ip) administration (0.02, 0.06, 0.2, 2, and 20 mg/kg) decreased phase II paw licking time in a dose-dependent manner.98 However, efficacy was seen at doses as low as 0.06 mg/kg, and the maximal tolerated dose (MTD) was 2 mg/kg. The 20 mg/kg dose induced convulsions and lethality within 30 min post 2665



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time-course for CSF glycine elevation correlated well with the antinociception timecourse observed in the mouse pain model.126 The measurement of preclinical and clinical CSF glycine concentrations has been widely used by multiple GlyT-1 inhibitor programs to verify in vivo drug−target engagement and to establish PK−PD correlations.128 The study with 20 suggests that CSF glycine may serve as a potential translational biomarker for GlyT-1 and GlyT-2 inhibitor pain programs by providing dose-dependent PK−PD data that can be correlated with analgesic efficacy.

reduced NMDA receptor NR1 subunit expression in the CCI rat ipsilateral spinal cord, which was not observed with chronic administration of GlyT-2 inhibitor 8.108 In addition to GlyT-2 inhibition, Morita and colleagues also investigated the potential analgesic effects of GlyT-1 inhibitors in a battery of murine pain models. Intravenous injection of 18 or 19 (0.3 mg/kg) presented a lag time of 1.5−2 h prior to providing statistically significant antiallodynia effects in PSNL mice, an effect that was not observed with administration of GlyT-2 inhibitors 7 and 8.106 This lag-time could be abolished by pretreatment with glycine B-site NMDA receptor antagonists L-701,324 and 5,7-dichlorokynurenic acid (5,7-DCK) (structures not shown). These findings suggest that increased extracellular glycine concentrations resulting from GlyT-1 inhibition initially activates NMDA receptor-mediated neurotransmission, which may delay the onset of antiallodynia effects. Spinal application of 18 (300 ng, i.t.) or 19 (20 ng, i.t.) also reduced mechanical allodynia in mice administered CFA. Interestingly, GlyT-1 inhibitor 18 (10 μg/kg, 0.1, 1, and 10 mg/kg, iv) exhibited inverted U-shaped dose-dependent effects in the murine STZ diabetic model, which was not observed for GlyT-2 inhibitor 8 throughout the same dose range.106 Although it is unclear as to why 18 produces an inverted-U dose−response relationship in this model, this dose−response phenomenon has been widely observed for other GlyT-1 inhibitors in both preclinical and clinical studies for various CNS indications. Compound 18 also produced analgesic effects the PSNL mouse model, which were attenuated via strychnine or siRNA knockdown of GlyRα3β. Furthermore, siRNA GlyT-1 knockdown induced analgesia and ameliorated mechanical allodynia in PSNL mice.106 Motoyama et al. reported that 18 also produced multiday improvements in allodynia scores in the murine FBC pain model.112 A single iv dose (0.3 mg/kg) significantly improved allodynia scores, increased withdrawal thresholds, attenuated guarding behaviors, and reduced limb-use abnormality. Reductions in allodynia scores were also achieved via siRNA spinal knockdown of GlyT-1.112 Wedehausen and co-workers reported that a metabolite of lidocaine, 20, may be contributing to the drug’s analgesic effects via inhibition of GlyT-1.126 20 selectively inhibits glycineinduced inward currents in Xenopus laevis oocytes transfected with GlyT-1 with no ancillary activity at GlyT-2, GlyRs, or NMDA receptors. The compound also ameliorated hyperalgesia and allodynia in models of inflammatory and neuropathic pain. Systemic administration significantly and dosedependently reduced inflammatory mechanical hyperalgesia (EC50 = 98 mg/kg) in mice injected with CFA. Application of 20 (200 mg/kg, sc) also significantly reduced mechanical allodynia and thermal hyperalgesia in CCI mice.126 Withdrawal thresholds were significantly increased within 1 h postdose, and the duration of these effects lasted for approximately 4−6 h. Furthermore, 20 (10, 30, 100, and 1000 μM, sc) dose-dependently reduced hyperexcitable dorsal horn WDR secondary order neuron action potentials in response to electrical, thermal, and mechanical stimulation in carrageenan treated mice with complete suppression of neuronal hyperexcitability achieved at 100 and 1000 μM. These findings demonstrate that 20 is imparting inhibitory control to inflammation-induced hyperexcitable dorsal horn neurons.126 The authors also report that exposure levels of 20 (200 mg/kg, sc) in rat plasma and CSF correlated with an increase in extracellular glycine concentrations in the CSF (25% versus basal controls). Moreover, the

8. GLYCINE RECEPTOR PAMS Selective potentiation of GlyR activity via positive allosteric modulation presents a highly attractive approach to augment spinal glycinergic neurotransmission and treat chronic pain.129 In general, PAMs present distinct pharmacological advantages over orthosteric agonists in that they may impart significant receptor subtype selectivity as allosteric binding sites may not be as evolutionarily conserved as orthosteric sites. Furthermore, PAMs also operate within the confines of endogenous agonist spatial and temporal release, thereby reducing the potential for receptor overstimulation, desensitization, and tachyphylaxis. Agents capable of selectively potentiating GlyR function would provide powerful tools to enable a better understanding of GlyR pharmacology as well as potentially lead to the discovery of novel therapeutics to treat chronic pain. Several structurally diverse endogenous messenger molecules and some known drugs have been found to bind at sites other than the orthosteric glycine-binding site and modulate GlyR activity (Figure 14). Such agents include divalent cations (zinc), neuroactive steroids (i.e., 21 (alphaxalone)130 and 22 (minaxalone)131), tropeines such as 23 (tropisetron),132 24 (glucose),133 alcohols such as 25 (ethanol)134), 26 (ivermectin),135 neurotransmitters other than 3 (i.e., 1), general anesthetics such as 27 (propofol),136 and 28 (isoflurane),137 and alkaloids such as 29 (gelsemine).138 Point mutation studies have revealed various molecular binding sites for many of the aforementioned modulators.139 Although these modulators are not GlyR subtype selective nor are they selective for GlyRs, some of them may provide excellent scaffolds for ligand or structure-based rational design efforts directed toward the discovery of drug like and developable subtype selective GlyR PAMs. Although GlyRs have yet to be fully exploited as drug targets and clinically available selective GlyR PAMs have yet to emerge, the approach has recently garnered significant attention.140 We will provide a brief summary of the current state of the art for the field. 8.1. Cannabinoid GlyR PAMs. Studies have shown that certain cannabinoids and endocannabinoids selectively potentiate GlyR-mediated neurotransmission and induce analgesia in models of acute, chronic inflammatory, and neuropathic pain. GlyRs were first reported as targets for endocannabinoid signaling in a study conducted with 30 (N-arachidonyl ethanol amide) (AEA) and 31 (2-arachidonyl-glycerol) (2-AG) (31) (Figure 15).141 These endocannabinoids were found to reduce the amplitude and alter the kinetics, desensitization, and rise time of glycine-activated currents (IGly) in neonatal rat hippocampal and cerebellar pyramidal and Purkinje neurons in a concentration-dependent manner. Cannabinoids 32 (Δ9-tetrahydrocannabinol) (Δ9-THC), the major psychoactive component of Cannabis sativa, and 30 induce concentration-dependent potentiation in glycine-activated (IGly) currents in acutely isolated ventral tegmental area (VTA) neurons and in Xenopus laevis oocytes cells expressing 2666



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Figure 14. Nonselective GlyR PAMs.

Figure 15. GlyR modulating cannabinoid ligands.

homomeric GlyRα1 and heteromeric GlyRα1β receptors.142 Agents 30 and 32 were also found to enhance glycine-activated currents in HEK-239 cells expressing the α1 and α3 subunits,143 and 30 increased IGly amplitudes in both rat cultured spinal neurons and in HEK-293 cells expressing human α1 and α3 GlyRs.143 Additional studies revealed that certain endocannabinoids exhibit distinct pharmacological profiles for GlyR subtypes. For example, 30-induced IGly potentiation is greater in HEK-239 cells expressing α1 relative to α2 and α3 subunits. Endocannabinoid 9 potentiates IGly in HEK-239 cells expressing α1 and inhibits IGly in HEK-239 cells expressing α2 and α3 subunits.144 Xiong and colleagues reported that 32 dose-dependently enhanced IGly in cultured spinal neurons and HEK-239 cells expressing recombinant homomeric GlyRα1 and heteromeric GlyRα1β receptors.145 Furthermore, coapplication of the selective CB1 receptor antagonist 7, the CB2 receptor inverse agonist 33 (SR144528) (Figure 16), and vanilloid receptor

in vivo analgesic effects of 32 in the mouse tail-flick reflex test could be antagonized by strychnine, suggesting that the observed antinociceptive activity was due to potentiation of GlyR-mediated neurotransmission. Analgesic effects were also observed in CB1R−/− knockout, CB1R−/−-CB2R−/− doubleknockout, and GlyRα2−/− knockout mice, providing corroborating evidence that the mechanism of action for pain reduction was via GlyRα1/GlyRα1β receptor activity.145 Importantly, analgesia in the tail-flick reflex test was absent in GlyRα3−/− but not in CB1R−/− or CB2R−/− knockout mice, providing evidence that 32 also ameliorates pain behaviors in part via potentiation of spinal GlyRα3β receptors. Importantly, these data suggest that the GlyR PAM activity of 32 contributes to cannabisinduced therapeutic analgesic effects.145 The nonpsychotropic cannabinoids 35 (cannabidiol) (CBD) and 36 (dehydroxyl-CBD) (DH-CBD) (Figure 17) potentiate

Figure 17. Nonpsychotropic cannabinoids 35 and 36.

IGly in HEK-239 cells expressing α1146 and α3147 subunits in a concentration-dependent manner. 36 also dose-dependently induced IGly in spinal lamina II neurons, and this activity was completely abolished by strychnine. Spinal application (10, 50, 100 μg, i.t.) significantly attenuated inflammatory thermal and mechanical pain hypersensitivity in adult male Sprague−Dawley rats subjected to intraplantar CFA injection.147 Intrathecal

Figure 16. CB2 receptor inverse agonist 33 and vanilloid receptor antagonist 34.

antagonist 34 (capsazepine) did not alter 32-mediated IGly in either the spinal neurons or the HEK-239 cells. In addition, 2667



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Additional optimization of the series led to the identification of 39 (AM-1488), a potent PAM for multiple GlyR subtypes that did not show off-target in vitro pharmacology in a comprehensive selectivity profile (Table 2). Compound 39 exhibits

administration also reduced mechanical allodynia in SNL rats and completely reversed the reduction of paw withdrawal latency induced by PEG2 administered to mice. Additional studies revealed that the analgesic effects of 36 were attenuated in Glyα3β−/− knockout mice.147 8.2. Aza-Naphthyridinone GlyR PAMs. Using a functional and heterologous cell-based FLIPR assay, researchers at Amgen recently reported HTS and subsequent hit-to-lead optimization efforts that led to the discovery of a novel class of orally bioavailable GlyR PAMs.148 The reported HTS and primary screening assay utilized a HEK-239 cell line stably transfected with human GlyRα3β, and changes in membrane potential resulting from GlyR-mediated ion flux were measured in the presence of a standard potential dye. Potentiated Cl− influx was assessed in the presence of low levels of endogenous glycine (EC10 or EC20), and Schild-shift assay plots recorded leftward shifts in glycine dose−response curves. The hits identified induced leftward shifts in glycine dose−response curves, indicating an increase in the potency of glycine for the receptor in the presence of the potentiator. None of the compounds screened increased glycine maximum efficacy. Compounds were rank-ordered based on the minimum tested concentration of potentiator required to shift the glycine dose−response curve at least 3-fold (mEC3).148 The HTS campaign yielded quinolone-based hit 37 (Figure 18), which was a modestly potent GlyRα3β potentiator

Table 2. In Vitro Potencies of 39 in HEK-293 Cells Overexpressing Human and Mouse GlyRs149 GlyR subtype hGlyRα1 hGlyRα1β hGlyRα3 hGlyRα3β mGlyRα1 mGlyRα3

EC50 (μM)a 0.327 0.171 0.454 0.330 0.268 0.490

± ± ± ± ± ±

0.027 0.094 0.067 0.085 0.173 0.141

EC50 max value (%)b 102.4 104.3 100 102.1 93.5 63.5

± ± ± ± ± ±

3.3 14 0 0.1 6.5 3.7

a EC50 values were obtained using a fluorescence readout based on a membrane-potential dye and in the presence of 10% maximal effective glycine concentration (EC10). bThe EC50 maximal (max) value is the top value of a nonlinear fit, in which 100% represents the maximal response to glycine alone.

good mouse PK characteristics with high oral bioavailability (%F > 100, 10 mg/kg po dose, 1 mg/kg iv dose), moderate half-life (t1/2 = 1.24 h), and suitable volume of distribution (Vss = 1.28 L/kg). Oral administration (20 mg/kg) induced a 94% reduction in tactile allodynia 60 min postdose in the mouse spared-nerve injury (SNI) model of neuropathic pain, and brain and plasma measurements taken immediately after 60 min measurement of tactile threshold showed favorable CNS permeability (Cu plasma = 2.31 ± 0.25 μM; Cu brain = 0.76 ± 0.18 μM).148 The effect of methylation of the pyrrolidine ring of 39 on mEC3 potency was then examined. Methylation on either side of the sulfonamide nitrogen for both enantiomers of 38 produced eight isomers (not shown). Of this set of compounds, the only active compound to emerge was 40 (AM-3607), which presented a methyl group projected into the concave face of the tricyclic ring system and was nearly an order of magnitude more potent than the des-methyl congener 38 (hGlyRα3β mEC3 = 0.025 μM).148 An X-ray cocrystal structure of 40 with the engineered construct hGlyRα3cryst in the presence of glycine was solved at a resolution of 2.6 Å. The crystallographic data showed glycine bound to all five canonical orthosteric binding sites within the ECD and 40 bound in an induced-pocket at each of the subunit interfaces of the ECD (Figure 19A).149 The location of the allosteric binding site, which is novel and had not been noted in the literature prior to this study, is adjacent to the glycine binding site with a distance of ∼10 Å between the benzodiazole of 40 and glycine. The crystallographic data also showed 40 adopting a conformation that was consistent with a calculated global minima conformation (0.05 kcal/mol difference) (Figure 19B). Molecular modeling studies had indicated that in addition to restricting free rotation of the sulfonamide, the α-methyl facilitated orientation of the 40 tricyclic framework into the putative chairlike bioactive conformation that presents excellent shape complementarity to the hGlyRα3cryst binding surface. This orientation was approximately 3.08 kcal above the global minima for the des-methyl congener 38 (PyMOL Molecular Graphics System, version 1.7.05, Schrö dinger, LLC.).148,149 The X-ray structure shows the sulfonamide of 40 engaged in an H-bond interaction with the main chain NH of neighboring Arg29. The pyyrolidine−naphthyridinone core is nestled within a hydrophobic pocket and engaged in

Figure 18. Amgen naphthyridinone GlyR PAMs.

(hGlyRα3β mEC3 = 3.1 μM). 37 exhibited high passive permeability and low efflux in the Madin−Darby canine kidney (MDCK) cell line assay, but the compound suffered from poor metabolic stability and high intrinsic clearance in mouse liver microsomes (MLM CLint = 901 μL/min/mg).148 Further structure−property relationship (SPR)-guided optimization efforts lead to the identification of proof-of-concept ex vivo tool compound 38 (hGlyRα3β mEC3 = 0.20 μM, MLM CLint = 151 μL/min/mg), which replaced the imidazole of hit 37 with a naphthyridinone motif. Bath application of 38 (0.5 μM) in adult mouse spinal cord slices produced a 4.9-fold increase in substantia gelatinosa eIPSC amplitudes in response to focal application of 20 μM glycine, and this effect was fully reversed in the presence of strychnine (10 μM).148 Interestingly, a stereopreference for potentiation was observed as the enantiomer of 38 did not enhance glycine-evoked currents. 2668



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Figure 19. (A) X-ray crystallographic data of 40 bound to engineered construct hGlyRα3cryst in the presence of glycine.149 Compound 40 binds to an allosteric site located at each of the subunit interfaces of the ECD, which is within 10 Å of the glycine binding site. (B) Orientation of 40 bound within the hGlyRα3cryst allosteric site. The naphthyridinone tricyclic core adopts a chairlike conformation. A sulfonamide oxygen is engaged in an H-bond interaction with Arg29 (H-bond shown as a pink dotted line). The water-mediated hydrogen bond interactions between the naphthyridinone carbonyl oxygen and Arg27 and Asp86, as well as resident hydrophobic residues are omitted for simplicity. The naphthyridinone pyridine nitrogen is directed toward solvent.149 Pictures for Figure 20A,B obtained from PDB 5TIN.

Figure 20. Pfizer confirmed GlyR PAM HTS hits.

automated electrophysiology assay using a microfluidic IonFlux HT assay to validate cherry picked identified hits. A conventional manual patch-clamp assay was then conducted on triaged hits to further validate HTS hits. Measurements for all three assays were conducted using a stably transfected CHO cell line expressing hGlyRα3 receptors. HTS screening of a 56558 compound library for PAM activity conducted in the presence of 20 μM glycine (EC20) yielded 214 actives (0.38% hit rate, Z′ = 0.71). Sequential screening and characterization through the IonFlux HT and manual patch-clamp assays identified seven genuine GlyRα3β PAMs, and the activities derived from both electrophysiological assays were in good agreement with one another (within 2-fold).150 Activity was quantified in the manual patch-clamp assay as the mean potentiation of GlyRmediated currents to 50 μM glycine (EC5−10) in the presence of 10 μM of compound. The mean potentiation for GlyRmediated currents for the seven PAMs (41−47) (Figure 20) ranged from 136% to 2710%, relative to control (Table 3).

hydrophobic van der Waals interactions. The naphthyridinone carbonyl oxygen is engaged in water-mediated hydrogen bond interactions with Arg27 and Asp86, while the pyridine nitrogen is directed toward solvent.148,149 The binding of 40 induces a change in hGlyRα3cryst conformation that stabilizes glycine binding at the orthosteric site, thereby increasing binding affinity. It should be noted that 40 potentiates hGlyRα3cryst with an EC50 similar to that of wildtype hGlyRα3. Surface plasmon resonance (SPR) measurements determined the KD of 40 at hGlyRα3cryst to be 0.011 ± 0.005 μM. Both SPR and isotheral titration calorimetry binding experiments determined that affinity for glycine increases by ∼200-fold upon binding of 40.148,149 8.3. Aryloxyethylsulfonamido GlyR PAMs. Pfizer has also recently disclosed efforts to identify selective GlyRα3β receptor PAMs using a three-tiered in vitro assay screening system.150 An initial high-throughput fluorescent membrane potential HTS screen was followed by a medium-throughput 2669



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Table 3. Activity Data for Pfizer GlyR PAM Hit Compounds147 fluorescence membrane potential HTS assay

ion flux HT assay

compd

% activity of GlyRα3β EC20 at 5.9 μMa

mean % potentiation of GlyRα3β EC20 at 10 μMa

41 42 43 44 45 46 47 38 (DH−CBD) 55 (DTBP)

67 65 52 52 28 161 23 94 134

182 182 49 59 64 44 98 95 116

manual patch clamp assay SEM

pEC50 (n = 2)b

mean % potentiation of GlyRα3β EC5−10 at 10 μMa

95% CIc

21 18 11 9 11 14 23 11 22

7.87 5.40 4.99 6.18 NDd ND ND ND ND

2061 2710 255 396 303 259 136 1351 309

1284:2838 1801:3618 107:404 76:717 98:508 −155:673 68:204 −121:2824 165:452

a Percent potentiation of EC20 glycine response at a single compound concentration. bpEC50 determined in the presence of EC20 glycine (two independent determinations, each containing four or five replicates per concentration). c95% CI = 95% confidence interval. dND = not determined.

Figure 21. Sesterterpene glycinyl-lactam GlyR PAMs 48 and 49.

8.5. Propofol-Derived Subtype-Selective GlyRα1β Receptor PAMs. Leuwer and co-workers have filed a patent application claiming the discovery of selective GlyRα1 PAMS derived from propofol, examples of which are shown in Figure 22 (50−54).153 GlyR PAM 50 (LT-01-25) exhibited selective potentiation of GlyRα1 versus GlyRα2, GlyRα3, and GABAA receptors. The compound also possesses suitable rat PK characteristics, and the inventors claim that oral administration induced statistically significant dose-dependent, robust, and long-lasting analgesic effects against mechanical hyperalgesia, cold sensitivity, and tactile allodynia in the partial ligation of the sciatic nerve (PLSN) model with rats.153 50 also dose-dependently increased paw withdrawal thresholds (PWT) in STZ-diabetic neuropathic rats. Importantly, no adverse motor or respiratory effects were observed in the any of the aforementioned pain model studies with 50.153 8.6. Phosphorylation State-Dependent Allosteric Modulation of GlyRα3β Receptors. Zeilhofer and co-workers report that the nonanesthetic propofol derivative 55 (2,6-ditert-butylphenol) (2,6-DTBP) (Figure 23) imparts selective potentiation of phosphorylated GlyRα3β receptors.83 55 induced a leftward shift in glycine concentration−response curves in HEK-293 cells expressing homomeric GlyRs, which were in agreement with an observed increase in channel-open probability in single-channel recording experiments. Application of 55 (100 μM) to VGAT::ChR2BAC transgenic mice lumbar spinal cord slices exhibited no significant effect on the amplitude or the decay time constants of blue-light elicited IPSCs in lamina II dorsal horn neurons. However, 55 significantly increased the decay time of glycinergic IPSCs after priming the slices with PGE2 (1 μM).83 Superfusion of PGE2 reduced the amplitude of glycine-mediated IPSCs by 35 ± 5% relative to controls without altering rise or decay time kinetics, consistent with inhibition of α3-containing GlyRs via EP2mediated phosphorylation. Subsequent application of 55

N-(2-(Quinolin-8-yloxy)ethyl)benzenesulfonamide 42 was the most potent analogue identified from this sample set (efficacy of potentiation = 2710%) and was subsequently studied for selectivity against GABAA receptors (α1/3β3γ2) and GlyRα1.150 Sulfonamide 42 (10 μM) did not affect GABAA receptor-mediated currents evoked by 1 μM GABA (EC10). However, the same concentration of 42 did potentiate a current evoked by 20 μM glycine (EC5−15) in a CHO cell line stably expressing GlyRα1 with a median potentiation of 3974%. Thus, 42 shows promise as an early pharmacological tool compound and is a suitable chemotype for the potential development of more potent and selective GlyR PAMs.150 8.4. Sesterpene Glycinyl-Lactam GlyR PAMs. Researchers in Queensland, Australia screened processed extracts for GlyR allosteric modulation activity from a library of more than 2500 marine invertebrate and algae samples collected from coastal and deep-sea locations across southern Australia and Antarctica.151,152 A yellow fluorescent protein (YFP)-based anion influx assay utilizing a HEK-293 cell lines stably transfected with either hGlyRα1 or hGlyRα3 was employed for the initial HTS of the marine extracts. Subsequent electrophysiological planar chip whole-cell patch-clamp recordings with the same cell lines was subsequently used to validate and confirm identified hits. These screens led to the discovery of sesterpene glycinyl-lactam GlyR modulators, which were isolated from Australian marine sponges of the family Irciniidae151 and of the genus Psammocinia.152 Figure 21 highlights stand-out GlyR PAMs 48 ((−)-irinianin lactam A) and 49 ((−)-oxoircinianin lactam A), which exhibited good activity among the hits identified from the screen.152 Application of 49 (100 μM) selectively potentiated GlyRα3-mediated inward current flux induced by glycine (EC20, 100 μM) by 260 ± 15% (GlyRα3 EC50 = 8.5 ± 2.1 μM). Lactam 49 (100 μM) selectively potentiated GlyRα1-mediated currents evoked by glycine (EC20, 100 μM) by 110 ± 8%.152 2670



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Figure 22. Propofol-derived GlyRα1 selective PAMs.

9. CONCLUSIONS Currently available therapeutics to treat chronic pain suffer from poor efficacy, undesirable side effects, or abuse liability potential. There is a tremendous need for analgesics that treat pathological pain through novel mechanisms of action that provide significantly improved efficacy, safety, and tolerability relative to current standard of care. Augmentation of spinal glycinergic neurotransmission may present such an opportunity. Several recent studies have emerged that elucidate the role of glycinergic signaling in the dorsal horn and its importance with regard to pain signaling. Potentiation of glycinergic inhibitory circuits within the dorsal horn could offer an innovative approach to treat chronic pain via direct modulation of a central gating mechanism controlling nociceptive signaling. The findings outlined in this Perspective suggest that diverse nociceptive and neuropathic pathological pain states present a disinhibition of excitatory drive within the dorsal horn. Thus, restoring or enhancing inhibitory spinal glycinergic neurotransmission has the potential to treat a variety of chronic pain states regardless of origin. In addition, the restricted distribution of glycinergic targets to caudal regions may allow for restoration of inhibitory pain control with limited CNS side effects typically associated with some current analgesic therapies. There may be evidence of clinical proof-of-concept for the approach. The GlyRα3β receptor PAM activity of some cannabinoids and endocannabinoids may be playing a role in the clinically observed therapeutic analgesic effects of cannabis. The active metabolite of lidocaine may be contributing to the drug’s analgesic effects via GlyT-1 inhibition. In addition, positive phase 2 data for the GlyT-2 inhibitor VVZ-149 may also provide clinical validation for the approach to treat pain. Augmentation of spinal glycinergic neurotransmission to treat chronic pain has recently begun to garner significant attention. In addition to the glycine transporter inhibition and GlyR PAM approaches described in this review, other methods to augment spinal glycinergic neurotransmission are also now under investigation. These include designing RNA aptamers as subtype selective GlyR PAMs,154 developing platelet-activating factor antagonists that may reduce pain in part by increasing

Figure 23. Propofol derivative 55.

(100 μM) significantly increased the IPSC decay time constant by 54 ± 11% without effecting amplitudes. These effects were diminished by preincubating the slices with PKA inhibitor H89 (5 μM). Furthermore, PGE2 application failed to render IPSC potentiation susceptible to 55 in slices obtained from GlyRa3−/− knockout mice. Potentiation of IPSCs by 55 measured from spinal cord slices of mice administered zymosan A into the left paw was also examined. Although 55 did not significantly alter IPSC amplitudes or rise times, it did significantly increase decay time kinetics (23.5 ± 7.5%), indicating that priming had occurred in response to zymosan A-induced peripheral inflammation.83 The modulatory effects of 55 also translated to in vivo analgesic activity in different inflammatory pain models.83 The compound significantly reversed mechanical and thermal hyperalgesia in mice 48 h after zymosan A injection, when central sensitization had reached its maximum. However, the compound did not produce analgesic effects in an acute pain ̈ mice, providing evidence that the study (pinprick) in naive analgesic activity of 55 requires GlyRα3β phosphorylation. Additional studies comparing the analgesic effects in CFA-treated wild-type versus GlyRa3−/− knockout mice were then conducted. 83 Compound 55 significantly reduced mechanical hyperalgesia in wild-type mice by 44.3 ± 5.2%, whereas the reduction in GlyRa3−/− knockout mice was significantly lower at 16.0 ± 6.2%. The effects against neuropathic hyperalgesia were also examined in CCI wild type and GlyRa3−/− knockout mice. Interestingly, 55 reduced hyperalgesia to a similar degree in both wild type and GlyRa3−/− knockout mice, suggesting that the compound is exerting analgesic effects against neuropathic pain via mechanisms in addition to restoration of phosphorylated GlyRα3β receptor activity.83 2671



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GlyRα3β activity,155 targeting inflammatory mediators such as IL-1β that facilitate GlyR-mediated LTP that diminish GABAergic signaling,78 and the utilization of gene therapy to increase GlyRα1β expression levels.156 As further investigation of spinal glycinergic neurotransmission and GlyR pharmacology continues and new findings emerge, the field will continue to gain momentum and bolster drug discovery efforts that may someday lead to innovative therapeutics that effectively target and suppress central nociceptive signaling and provide patients with safe and effective relief from chronic pain.

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receptor; 5-HT, serotonin; DLMT, dorsolateral pontomesencephalic tegmentum

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Phone: 518-694-7224. E-mail: christopher.cioffi@acphs.edu. ORCID

Christopher L. Cioffi: 0000-0003-0642-7905 Notes

The author declares no competing financial interest. Biography Christopher L. Cioffi is an Assistant Professor of Medicinal Chemistry in the Department of Basic and Clinical Sciences and the Department of Pharmaceutical Sciences at Albany College of Pharmacy and Health Sciences. Christopher received his doctorate in Organic Chemistry from Rensesselaer Polytechnic Institute in 2000 under the supervision of Professor Mark P. Wentland. He then subsequently conducted a 16-year industrial career as a medicinal chemist at Albany Molecular Research, Inc. (AMRI). While at AMRI, Christopher led numerous medicinal chemistry teams and made significant drug design contributions to programs that advanced drug candidates into preclinical development and clinical trials. He has worked in several therapeutic areas that include cardiovascular, gastrointestinal, CNS, and ophthalmic indications. Christopher has recently joined the faculty of Albany College of Pharmacy and Health Sciences in July of 2016.

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ABBREVIATIONS USED CNS, central nervous system; PNS, peripheral nervous system; NSAIDs, nonsteroidal anti-inflammatory drugs; GlyR, glycine receptor; NMDA receptor, N-methyl-D-aspartate receptor; DRG, dorsal root ganglia; GlyT-1, glycine transporter-1; GlyT-2, glycine transporter-2; iGluRs, ionotropic glutamate receptors; eIPSC, evoked inhibitory postsynaptic currents; mIPSC, miniature inhibitory postsynaptic currents; LTP, longterm potentiation; LTD, long-term depression; CSF, cerebral spinal fluid; SAR, structure−activity relationship; PK, pharmacokinetics; PD, pharmacodynamics; iv, intravenous; po, oral; i.t., intrathecal; sc, subcutaneous; icv, intracerebroventricular; NK1R, neurokinin 1 receptor; BDNF, brain-derived neurotrophic factor; KCC2, potassium-chloride cotransporter; ATP, adenosine triphosphate; SNI, spinal nerve injury; SNL, spinal nerve ligation; PSNL, partial sciatic nerve ligation; CCI, chronic constriction injury; PAF, platelet-activating factor; NO, nitric oxide; STZ, streptozotocin; CFA, complete Freund’s adjuvant; RVM, rostral ventromedial medulla; GABA, γ-aminobenzoic acid; AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; PE2, prostaglandin E2; IL-1β, interleukin-1β; IL-6, interleukin-6; NGF, nerve growth factor; TNF-α, tumor necrosis factor-alpha; CGRP, calcitonin gene related peptide; CB1R, cannabinoid 1 receptor; PKA, protein kinase A; PKC, protein kinase C; mGluR, metabotropic glutamate receptor; DRG, dorsal root ganglion; PAG, periaqueductal gray; PFC, prefrontal cortex; nAChR, nicotinic acetylcholine 2672



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