New Analgesic Targets

• purinergic P2X and P2Y receptors;

• ionotropic and metabotropic glutamate receptors; and

• tetrodotoxin-resistant voltage-gated sodium channels (Nay) and some voltage-gated calcium and potassium channels.

The putative roles of most of these molecular entities have been examined by either knocking out (genetically null mice), knocking down (antisense oligonucleotide or siRNA strategies), or blocking (antagonists) the receptors to reveal altered behaviors to application of noxious stimuli. Transient Receptor Potential (TRP) Ion Channels

Members of the transient receptor potential (TRP) family are nonselective cation channels that are involved in several physiological conditions, including pain.1'2 TRPV1 is of particular interest because it responds to thermal stimuli in the normal noxious range for skin (42-52 °C), to protons and a reduction in tissue pH (as commonly occurs during inflammation), and to chemicals, notably capsaicin, but also a variety of endogenous lipids produced when tissue is insulted. Other members of the TRP family of receptors respond principally to very high (> 52 °C) or very low (<18 °C) temperatures in the tissue-damaging range, and are thus noxious.

TRPV1 is a serpentine protein that has six transmembrane (TM) segments flanked by specific ankyrin domains in the cystolic N-terminal portion.3 The channel pore (P-loop) is located between TM5 and TM6. It is believed to exist in oligomeric form, and heterogeneous assembly of TRPV1 with other channels has been speculated to account for observed discrepancies in cellular assays of vanilloid activity in native and cloned receptors.3 TRPV1 is the prototypical member of the TRPV subfamily which comprises six members, TRPV1-TRPV6.4 Among TRPV channels, TRPV1 is unique in its interaction with two potent natural products, capsaicin and resiniferatoxin, which appear to gate the channel by decreasing the threshold of temperature activation.2

Three different classes of lipids, derived from the metabolism of arachidonic acid, have been characterized as activators of TRPV1. These are the endocannabinoid anandamide, several lipoxygenase products of arachidonic acid, and N-arachidonyldopamine.5,6 Characteristically, these endogenous lipids have the ability to modulate TRPV1, functionally increasing or enhancing its activation. TRPV1 is expressed in neuronal and non-neuronal tissues, including nociceptors and central nervous system neurons. Wherever located, activation of TRPV1 results in rapid increase in intracellular Ca2 + and consequent activation of intracellular mechanisms.6

Studies have shown that capsaicin-sensitive afferent neurons participate in the regulation of normal urinary bladder function, gastrointestinal circulation, secretion, mucosal homeostasis, motility, and both somatic and visceral nociception.4,7,8 For example, TRPV1 null mice exhibit reduced hyperalgesia in inflammatory9 and neuropathic8 pain models when compared to wild-type mice, reduced bladder function,7 and colon mechanosensitivity.10 These (and other) reports emphasize why the TRPV1 receptor is an attractive target for the development of novel molecules for the treatment of both inflammatory somatic and visceral pain. Acid-Sensing Ion Channels

ASICs are members of the NaC/DEG superfamily. This family includes epithelial Na+ channels (ENaC), degenerins (DEG), and a peptide-gated Na + channel, Phe-Met-Arg-Phe-NH2 (FaNaC).11 The major structural features of ASICs, based on the cloning of ASIC1a, are two TM domains (TM1 and TM2), a large extracellular loop, and the C- and N-terminus are intracellular.12 Evidence suggests that the ASIC1a assembles as a tetramer. ASICs are mainly expressed in nervous system tissue, but not in glia, and in some non-neuronal sites. In the periphery, ASICs are present in all sensory ganglia and are regulated by the autonomic nervous system in different organs.

At present, few pharmacological approaches are effective or selective for ASICs. These channels are blocked by amiloride, a nonselective antagonist; a tarantula psalmotoxin specifically blocks the ASIC1a subunit. Phenylalanine amide-related peptides (FRMF amides) are a family of peptides that slow down desensitization of ASIC3 homomers and heteromers. High concentrations of nonsteroidal anti-inflammatory drugs (NSAIDs) reportedly inhibit ASICs and their inflammation-induced expression,13 suggesting that ASICs may be a new target for the development of novel NSAIDs.14 P2 Receptors

Neurons in the central nervous system are endowed with adenosine triphosphate (ATP)-sensitive receptors belonging to the P2X and P2Y types.15 P2X receptors are multimeric ligand-gated ion channels whereas P2Y receptors are

G protein-coupled receptors. ATP is ubiquitous and is the principal agonist for P2X receptors. ATP itself, when administered exogenously, has long been appreciated as 'algogenic' (pain-producing). When released from epithelium in the skin or internal organs (e.g., bladder urothelium), ATP activates P2X receptors on nerve terminals, including nociceptors, and is believed to be an important contributor to pain.

P2X receptors are purine nucleotide-gated cation channels that mediate fast cell-cell signaling in excitable tissues.16 Seven P2X receptors have been idenitifed and are designated P2X^. Their molecular structure consists of two membrane-spanning regions, an intracellular N- and C-terminal, and a cysteine-rich extracellular loop.16 P2X receptors are oligomeric and composed of more than one subunit. A functional receptor appears to be a trimer. These receptors form cation-selective channels with almost equal permeability to Na+ and K+. P2X receptor antagonists selective for P2X1/3 (TNP-ATP) and P2X2/3 (A-317491) reveal a role for P2X channels in inflammatory and other pain states,17'18 which is supported by results obtained in P2X3 and P2X2/3 null mutant mice.19'20

P2Y receptors are G protein-coupled and are also activated by ATP, but adenosine diphosphate and related analogs have greater potency.15 There are 10 cloned and functionally defined P2Ysubtypes. Eight receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) are present in human tissue and several (P2Y1, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) occur in the central nervous system. At present less information is available on the potential role of P2Y receptors in nociceptive processes compared to P2X receptors. However, P2Y1 and P2Y2 receptors have been implicated in the ATP-mediated sensitization of TRPV1 receptors.16 In addition, there is additional evidence that P2Y receptors may be involved in chronic pain.16 Excitatory Amino Acid Receptors

The excitatory amino acid glutamate plays a major role in nociceptive processing.21 Glutamate released from sensory neurons, including nociceptors, exerts its actions on two major types of receptors: ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). iGluRs are ligand-gated ion channels that include N-methyl-D-aspartate (NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate receptors. mGluRs are G protein-coupled receptors that modulate the actions of glutamate through various second-messenger systems.

The metabotropic glutamate receptor family consists of at least eight members (mGluR1_mGluR8) which can be divided into three main groups based on sequence similarity.21 Group 1 consists of mGluR1 and mGluR5, which couple most commonly to phospholipase C. Group 2 includes mGluR2 and mGluR3, whereas group 3 includes mGluR4, mGluR6, mGluR7, and mGluR8. Groups 2 and 3 inhibit adenyl cyclase and modulate G protein-coupled inwardly rectifying potassium channels (GIRKs) or voltage-gated calcium channels.

NMDA receptors have been studied in great detail as potential targets for the development of novel analgesics. While efficacious, NMDA receptor antagonists suffer from side effects such as fatigue, dizziness, pyschosis, and at higher concentrations, amnesia and neurotoxicity.22,23 Thus, there has been movement away from the development of nonselective NMDA receptor antagonists24 to modulatory site (e.g., glycineB site) and NMDA receptor subunit site (e.g., NR2B) antagonists, which have shown potential for reduced side effects while maintaining efficacy. Agents selective for the various subtypes of mGluRs are also a target for development. However, validation of mGluRs as a drug target is incomplete at present. Voltage-Sensitive (NaV) Channels

The voltage-sensitive Na+ channel is a TM protein which generates action potentials in excitable cells. A variety of different isoforms of this channel have now been identified.25 Mammalian Na+ channels consist of an a subunit (around 260 kDa) and a b subunit (30-40 kDa).25 Different isoforms of the a subunit have been identified. Nine isoforms have been grouped into a single family, NaV1, and some of these isoforms have a role in nociceptive processing.

Genetic and pharmacological studies indicate that NaV 1.8 (SNS/PN3), a tetrodotoxin (TTX)-resistant channel, is likely important in nociception.24,26 Intrathecal administration of specific antisense oligodeoxynucleotides (ODNs) to knockdown this TTX-resistant sodium channel reversed tactile allodynia and thermal hyperalgesia in models of neuropathic pain in rats.27,28 In addition, NaV 1.8-/_ mice showed blunted responses to intracolonic capsaicin, but normal nociceptive behavior to acute noxious stimuli.29 Furthermore, nonselective NaV 1.8 channel blockers have been shown to produce antinociceptive effects in rodent models of neuropathic pain.30 Currently, there are no selective NaV 1.8 TTX-resistant channel blockers. Voltage-Gated Calcium Channels (VGCCs)

Voltage-gated calcium channels (VGCCs) have also been shown to play an important role in the modulation of nociceptive processing.31 Calcium ions are a universal second messenger for intracellular signaling in many cell types.

Several individual subtypes of calcium channels are under active investigation as targets for the treatment of chronic pain. VGCCs are found in the plasma membrane of all excitable cells, including peripheral and central neurons. They are localized throughout the neuron and trigger the release of neurotransmitters.31 Ten VGCCs have been identified, of which nine are expressed in the nervous system.32 L-type channels were originally classified as channels with a large single channel conductance and long open time. T-type channels were channels with a tiny single channel conductance and transient open time. Further work identified N-type (found in neurons) and P-type (found in cerebellar Purkinje neurons) channels.31 A numerical numbering system has been adopted to classifiy VGCCs into three families (CaV1-CaV3) according to the sequence homology of their a1 subunits.33'34

Among all VGCCs, N-type calcium channels encoded by the CaV2.2 gene have attracted the most attention as a target for the treatment of chronic pain.31 N-type channel blockers stop the release of neuropeptides such as substance P.35'36 Studies have shown that N-type channel null mice are less sensitive to neuropathic and inflammatory pain when compared to wild-type mice.37'38 Furthermore, ziconotide, a potent N-type channel blocker, has been approved for the treatment of neuropathic pain.32

Ziconotide is a synthetic form of the peptide o-conotoxin MVILA, originally isolated from the venom of the cone snail Conus magnus.39 Toxins from other cone snails have also been identified as potent VGCC inhibitors.40 Currently, ziconotide is only available as an intrathecal infusion41 and there is an effort to identify other small-molecule N-type calcium-channel blockers.

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