Torben R. Neelands

Involvement of the TTX-resistant sodium channel Nav 1.8 in inflammatory and neuropathic, but not post-operative, pain states

Abstract Antisense (AS) oligodeoxynucleotides (ODNs) targeting the Nav 1.8 sodium channel have been reported to decrease inflammatory hyperalgesia and L5/L6 spinal nerve ligation‐induced mechanical allodynia in rats. The present studies were conducted to further characterize Nav 1.8 AS antinociceptive profile in rats to better understand the role of Nav 1.8 in different pain states. Consistent with earlier reports, chronic intrathecal Nav 1.8 AS, but not mismatch (MM), ODN decreased TTX‐resistant sodium current density (by 60.5 ± 10.2% relative to MM; p < 0.05) in neurons from L4 to L5 dorsal root ganglia and significantly attenuated mechanical allodynia following intraplantar complete Freund’s adjuvant. In addition, 10 days following chronic constriction injury of the sciatic nerve, Nav 1.8 AS, but not MM, ODN also attenuated mechanical allodynia (54.3 ± 8.2% effect, p < 0.05 vs. MM) 2 days after initiation of ODN treatment. The anti‐allodynic effects remained for the duration of the AS treatment, and CCI rats returned to an allodynic state 4 days after discontinuing AS. In contrast, Nav 1.8 AS ODN failed to reduce mechanical allodynia in the vincristine chemotherapy‐induced neuropathic pain model or a skin‐incision model of post‐operative pain. Finally, Nav 1.8 AS, but not MM, ODN treatment produced a small but significant attenuation of acute noxious mechanical sensitivity in naïve animals (17.6 ± 6.2% effect, p < 0.05 vs. MM). These data demonstrate a greater involvement of Nav 1.8 in frank nerve injury and inflammatory pain as compared to acute, post‐operative or chemotherapy‐induced neuropathic pain states.

Repeated dosing of ABT-102, a potent and selective TRPV1 antagonist, enhances TRPV1-mediated analgesic activity in rodents, but attenuates antagonist-induced hyperthermia

ABSTRACT Transient receptor potential vanilloid type 1 (TRPV1) is a ligand‐gated ion channel that functions as an integrator of multiple pain stimuli including heat, acid, capsaicin and a variety of putative endogenous lipid ligands. TRPV1 antagonists have been shown to decrease inflammatory pain in animal models and to produce limited hyperthermia at analgesic doses. Here, we report that ABT‐102, which is a potent and selective TRPV1 antagonist, is effective in blocking nociception in rodent models of inflammatory, post‐operative, osteoarthritic, and bone cancer pain. ABT‐102 decreased both spontaneous pain behaviors and those evoked by thermal and mechanical stimuli in these models. Moreover, we have found that repeated administration of ABT‐102 for 5–12 days increased its analgesic activity in models of post‐operative, osteoarthritic, and bone cancer pain without an associated accumulation of ABT‐102 concentration in plasma or brain. Similar effects were also observed with a structurally distinct TRPV1 antagonist, A‐993610. Although a single dose of ABT‐102 produced a self‐limiting increase in core body temperature that remained in the normal range, the hyperthermic effects of ABT‐102 effectively tolerated following twice‐daily dosing for 2 days. Therefore, the present data demonstrate that, following repeated administration, the analgesic activity of TRPV1 receptor antagonists is enhanced, while the associated hyperthermic effects are attenuated. The analgesic efficacy of ABT‐102 supports its advancement into clinical studies.

Pharmacology of Modality-Specific Transient Receptor Potential Vanilloid-1 Antagonists That Do Not Alter Body Temperature

The transient receptor potential vanilloid-1 (TRPV1) channel is involved in the development and maintenance of pain and participates in the regulation of temperature. The channel is activated by diverse agents, including capsaicin, noxious heat (≥ 43°C), acidic pH (< 6), and endogenous lipids including N-arachidonoyl dopamine (NADA). Antagonists that block all modes of TRPV1 activation elicit hyperthermia. To identify efficacious TRPV1 antagonists that do not affect temperature antagonists representing multiple TRPV1 pharmacophores were evaluated at recombinant rat and human TRPV1 channels with Ca2+ flux assays, and two classes of antagonists were identified based on their differential ability to inhibit acid activation. Although both classes of antagonists completely blocked capsaicin- and NADA-induced activation of TRPV1, select compounds only partially inhibited activation of the channel by protons. Electrophysiology and calcitonin gene-related peptide release studies confirmed the differential pharmacology of these antagonists at native TRPV1 channels in the rat. Comparison of the in vitro pharmacological properties of these TRPV1 antagonists with their in vivo effects on core body temperature confirms and expands earlier observations that acid-sparing TRPV1 antagonists do not significantly increase core body temperature. Although both classes of compounds elicit equivalent analgesia in a rat model of knee joint pain, the acid-sparing antagonist tested is not effective in a mouse model of bone cancer pain.

TRPV1b overexpression negatively regulates TRPV1 responsiveness to capsaicin, heat and low pH in HEK293 cells

Transient receptor potential channel type V (TRPV) 1 is a non‐selective cation channel that can be activated by capsaicin, endogenous vanilloids, heat and protons. The human TRPV1 splice variant, TRPV1b, lacking exon 7, was cloned from human dorsal root ganglia (DRG) RNA. The expression profile and relative abundance of TRPV1b and TRPV1 in 35 different human tissues were determined by quantitative RT‐PCR using isoform‐specific probes. TRPV1b was most abundant in fetal brain, adult cerebellum and DRG. Functional studies using electrophysiological techniques showed that recombinant TRPV1b was not activated by capsaicin (1 µm), protons (pH 5.0) or heat (50°C). However, recombinant TRPV1b did form multimeric complexes and was detected on the plasma membrane of cells, demonstrating that the lack of channel function was not due to defects in complex formation or cell surface expression. These results demonstrate that exon 7, which encodes the third ankyrin domain and 44 amino acids thereafter, is required for normal channel function of human TRPV1. Moreover, when co‐expressed with TRPV1, TRPV1b formed complexes with TRPV1, and inhibited TRPV1 channel function in response to capsaicin, acidic pH, heat and endogenous vanilloids, dose‐dependently. Taken together, these data support the hypothesis that TRPV1b is a naturally existing inhibitory modulator of TRPV1.

Acidification of rat TRPV1 alters the kinetics of capsaicin responses

TRPV1 (vanilloid receptor 1) receptors are activated by a variety of ligands such as capsaicin, as well as by acidic conditions and temperatures above 42°C. These activators can enhance the potency of one another, shifting the activation curve for TRPV1 to the left. In this study, for example, we observed an approximately 10-fold shift in the capsaicin EC50 (640 nM to 45 nM) for rat TRPV1 receptors expressed in HEK-293 cells when the pH was lowered from 7.4 to 5.5. To investigate potential causes for this shift in capsaicin potency, the rates of current activation and deactivation of whole-cell currents were measured in individual cells exposed to treatments of pH 5.5, 1 μM capsaicin or in combination. Acidic pH was found to both increase the activation rate and decrease the deactivation rate of capsaicin-activated currents providing a possible mechanism for the enhanced potency of capsaicin under acidic conditions. Utilizing a paired-pulse protocol, acidic pH slowed the capsaicin deactivation rate and was readily reversible. Moreover, the effect could occur under modestly acidic conditions (pH 6.5) that did not directly activate TRPV1. When TRPV1 was maximally activated by capsaicin and acidic pH, the apparent affinity of the novel and selective capsaicin-site competitive TRPV1 antagonist, A-425619, was reduced ~35 fold. This shift was overcome by reducing the capsaicin concentration co-applied with acidic pH. Since inflammation is associated with tissue acidosis, these findings enhance understanding of TRPV1 receptor responses in inflammatory pain where tissue acidosis is prevalent.

Distribution and functional characterization of human Nav1.3 splice variants.

The focus of the present study is the molecular and functional characterization of four splice variants of the human Nav1.3 α subunit. These subtypes arise due to the use of alternative splice donor sites of exon 12, which encodes a region of the α subunit that resides in the intracellular loop between domains I and II. This region contains several important phosphorylation sites that modulate Na+ channel kinetics in related sodium channels, i.e. Nav1.2. While three of the four Nav1.3 isoforms, 12v1, 12v3 and 12v4 have been previously identified in human, 12v2 has only been reported in rat. Herein, we evaluate the distribution of these splice variants in human tissues and the functional characterization of each of these subtypes. We demonstrate by reverse transcriptase‐polymerase chain reaction (RT‐PCR) that each subtype is expressed in the spinal cord, thalamus, amygdala, cerebellum, adult and fetal whole brain and heart. To investigate the functional properties of these different splice variants, each α subunit isoform was cloned by RT‐PCR from human fetal brain and expressed in Xenopus oocytes. Each isoform exhibited functional voltage‐dependent Na+ channels with similar sensitivities to tetrodotoxin (TTX) and comparable current amplitudes. Subtle shifts in the V1/2 of activation and inactivation (2–3 mV) were observed among the four isoforms, although the functional significance of these differences remains unclear. This study has demonstrated that all four human splice variants of the Nav1.3 channel α subunit are widely expressed and generate functional TTX‐sensitive Na+ channels that likely modulate cellular excitability.

A-1048400 is a novel, orally active, state-dependent neuronal calcium channel blocker that produces dose-dependent antinociception without altering hemodynamic function in rats.

Blockade of voltage-gated Ca²⁺ channels on sensory nerves attenuates neurotransmitter release and membrane hyperexcitability associated with chronic pain states. Identification of small molecule Ca²⁺ channel blockers that produce significant antinociception in the absence of deleterious hemodynamic effects has been challenging. In this report, two novel structurally related compounds, A-686085 and A-1048400, were identified that potently block N-type (IC₅₀=0.8 μM and 1.4 μM, respectively) and T-type (IC₅₀=4.6 μM and 1.2 μM, respectively) Ca²⁺ channels in FLIPR based Ca²⁺ flux assays. A-686085 also potently blocked L-type Ca²⁺ channels (EC₅₀=0.6 μM), however, A-1048400 was much less active in blocking this channel (EC₅₀=28 μM). Both compounds dose-dependently reversed tactile allodynia in a model of capsaicin-induced secondary hypersensitivity with similar potencies (EC₅₀=300-365 ng/ml). However, A-686085 produced dose-related decreases in mean arterial pressure at antinociceptive plasma concentrations in the rat, while A-1048400 did not significantly alter hemodynamic function at supra-efficacious plasma concentrations. Electrophysiological studies demonstrated that A-1048400 blocks native N- and T-type Ca²⁺ currents in rat dorsal root ganglion neurons (IC₅₀=3.0 μM and 1.6 μM, respectively) in a voltage-dependent fashion. In other experimental pain models, A-1048400 dose-dependently attenuated nociceptive, neuropathic and inflammatory pain at doses that did not alter psychomotor or hemodynamic function. The identification of A-1048400 provides further evidence that voltage-dependent inhibition of neuronal Ca²⁺ channels coupled with pharmacological selectivity vs. L-type Ca²⁺ channels can provide robust antinociception in the absence of deleterious effects on hemodynamic or psychomotor function.

2', 3'-O-(2,4,6,trinitrophenyl)-ATP and A-317491 are competitive antagonists at a slowly desensitizing chimeric human P2X3 receptor.

Rapid desensitization of ligand‐gated ion channel receptors can alter the apparent activity of receptor modulators, as well as make detection of fast‐channel activation difficult. Investigation of the antagonist pharmacology of ATP‐sensitive homomeric P2X3 receptors is limited by agonist‐evoked fast‐desensitization kinetics. In the present studies, chimeric receptors were created using the coding sequence for the N‐terminus and the first transmembrane domain of either the nondesensitizing human P2X2a or fast‐desensitizing P2X3 receptor joined to the sequence encoding the extracellular loop, second transmembrane domain, and C‐terminus of the other receptor (designated P2X2–3 and P2X3–2, respectively). These clones were stably transfected into 1321N1 astrocytoma cells for biophysical and pharmacological experiments using both electrophysiological and calcium‐imaging methods. Chimeric P2X2–3 and P2X3–2 receptors were inwardly rectifying and agonist responses showed desensitization properties similar to the wild‐type human P2X2a and P2X3 receptors, respectively. The P2X2–3 chimera displayed an agonist pharmacological profile similar to the P2X3 wild‐type receptor being activated by low concentrations of both ATP and α,β‐meATP. In contrast, the P2X3–2 chimera had markedly reduced sensitivity to both agonists. The P2X3 receptor antagonists TNP‐ATP and A‐317491 were shown to be potent, competitive antagonists of the P2X2–3 chimera (Ki=2.2 and 52.1 nM, respectively), supporting the hypothesis that rapid receptor desensitization can mask the competitive antagonism of wild‐type homomeric P2X3 receptors.

[3H]A-778317 [1-((R)-5-tert-Butyl-indan-1-yl)-3-isoquinolin-5-yl-urea]: a Novel, Stereoselective, High-Affinity Antagonist Is a Useful Radioligand for the Human Transient Receptor Potential Vanilloid-1 (TRPV1) Receptor

1-((R)-5-tert-Butyl-indan-1-yl)-3-isoquinolin-5-yl-urea (A-778317) is a novel, stereoselective, competitive antagonist that potently blocks transient receptor potential vanilloid-1 (TRPV1) receptor-mediated changes in intracellular calcium concentrations (pIC50 = 8.31 ± 0.13). The (S)-stereoisomer, 1-((S)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea (A-778316), is 6.8-fold less potent (pIC50 = 7.47 ± 0.07). A-778317 also potently blocks capsaicin and acid activation of native rat TRPV1 receptors in dorsal root ganglion neurons. A-778317 was tritiated ([3H]A-778317; 29.3 Ci/mmol) and used to study recombinant human TRPV1 (hTRPV1) receptors expressed in Chinese ovary cells (CHO) cells. [3H]A-778317 labeled a single class of binding sites in hTRPV1-expressing CHO cell membranes with high affinity (KD = 3.4 nM; Bmax = 4.0 pmol/mg protein). Specific binding of 2 nM [3H]A-778317 to hTRPV1-expressing CHO cell membranes was reversible. The rank-order potency of TRPV1 receptor antagonists to inhibit binding of 2 nM [3H]A-778317 correlated well with their functional potencies in blocking TRPV1 receptor activation. The present data demonstrate that A-778317 blocks polymodal activation of the TRPV1 receptor by binding to a single high-affinity binding site and that [3H]A-778317 possesses favorable binding properties to facilitate further studies of hTRPV1 receptor pharmacology.