Mark O. Urban

Reversal of acid-induced and inflammatory pain by the selective ASIC3 inhibitor, APETx2

BACKGROUND AND PURPOSE Inflammatory pain is triggered by activation of pathways leading to the release of mediators such as bradykinin, prostaglandins, interleukins, ATP, growth factors and protons that sensitize peripheral nociceptors. The activation of acid‐sensitive ion channels (ASICs) may have particular relevance in the development and maintenance of inflammatory pain. ASIC3 is of particular interest due to its restricted tissue distribution in the nociceptive primary afferent fibres and its high sensitivity to protons.

BOLD and blood volume-weighted fMRI of rat lumbar spinal cord during non-noxious and noxious electrical hindpaw stimulation.

Spinal cord fMRI is a useful tool for studying spinal mechanisms of pain, hence for analgesic drug development. Its technical feasibility in both humans and rats has been demonstrated. This study investigates the reproducibility, robustness, and spatial accuracy of fMRI of lumbar spinal cord activation due to transcutaneous noxious and non-noxious electrical stimulation of the hindpaw in alpha-chloralose-anesthetized rats. Blood oxygenation level-dependent (BOLD) and blood volume-weighted fMRI data were acquired without and with intravenous injection of ultra small superparamagnetic iron oxide particles (USPIO), respectively, using a gradient echo (GE) echo planar imaging (EPI) technique at 4.7 T. Neuronal activation in the spinal cord induced by noxious stimulation to the hindpaw (2 ms wide, 5 mA amplitude, known to activate C-fibers) can be robustly detected by both fMRI techniques with excellent reproducibility and peaked at the stimulus frequency of 40 Hz. However, both fMRI techniques were not sensitive to neuronal activation in spinal cord induced by non-noxious stimulation (0.3 ms, 1.5 mA, known only to activate A-fibers). Spatially, the fMRI signal extended approximately 5 mm in the longitudinal direction, covering L(3)-L(5) segments. In the cross-sectional direction, the highest signal change of blood volume-weighted fMRI was in the middle of the ipsilateral dorsal horn, which roughly corresponds to laminae V and VI, while the highest signal change of BOLD fMRI was in the ipsilateral dorsal surface. This study demonstrates that spinal cord fMRI can be performed in anesthetized rats reliably and reproducibly offering it as a potential tool for analgesic drug discovery.

Amiloride derived inhibitors of acid-sensing ion channel-3 (ASIC3).

A series of amiloride derivatives modified at the 5-position of the pyrazine ring were evaluated as inhibitors of acid-sensing ion channel-3 (ASIC3), a novel target for the treatment of chronic pain.

Imidazopyridine CB2 agonists: Optimization of CB2/CB1 selectivity and implications for in vivo analgesic efficacy

A new series of imidazopyridine CB2 agonists is described. Structural optimization improved CB2/CB1 selectivity in this series and conferred physical properties that facilitated high in vivo exposure, both centrally and peripherally. Administration of a highly selective CB2 agonist in a rat model of analgesia was ineffective despite substantial CNS exposure, while administration of a moderately selective CB2/CB1 agonist exhibited significant analgesic effects.

fMRI of pain processing in the brain: A within-animal comparative study of BOLD vs. CBV and noxious electrical vs. noxious mechanical stimulation in rat

This study aims to identify fMRI signatures of nociceptive processing in whole brain of anesthetized rats during noxious electrical stimulation (NES) and noxious mechanical stimulation (NMS) of paw. Activation patterns for NES were mapped with blood oxygen level dependent (BOLD) and cerebral blood volume (CBV) fMRI, respectively, to investigate the spatially-dependent hemodynamic responses during nociception processing. A systematic evaluation of fMRI responses to varying frequencies of electrical stimulus was carried out to optimize the NES protocol. Both BOLD and CBV fMRI showed widespread activations, but with different spatial characteristics. While BOLD and CBV showed well-localized activations in ipsilateral dorsal column nucleus, contralateral primary somatosensory cortex (S1), and bilateral caudate putamen (CPu), CBV fMRI showed additional bilateral activations in the regions of pons, midbrain and thalamus compared to BOLD fMRI. CBV fMRI that offers higher sensitivity compared to BOLD was then used to compare the nociception processing during NES and NMS in the same animal. The activations in most regions were similar. In the medulla, however, NES induced a robust activation in the ipsilateral dorsal column nucleus while NMS showed no activation. This study demonstrates that (1) the hemodynamic response to nociception is spatial-dependent; (2) the widespread activations during nociception in CBV fMRI are similar to what have been observed in (14)C-2-deoxyglucose (2DG) autoradiography and PET; (3) the bilateral activations in the brain originate from the divergence of neural responses at supraspinal level; and (4) the similarity of activation patterns suggests that nociceptive processing in rats is similar during NES and NMS.

Antinociceptive effects of the non-selective cannabinoid receptor agonist CP 55,940 are absent in CB1-/- and not CB2-/- mice in models of acute and persistent pain

Previous studies have suggested a role for both CB1 and CB2 cannabinoid receptors in modulation of nociception. To further examine the role of CB1 and CB2 receptors in antinociception, we evaluated the efficacy of the non-selective cannabinoid receptor agonist, CP 55,940, in models of acute, inflammatory, and neuropathic pain in control mice, CB1 receptor knockout mice, and CB2 receptor knockout mice. In control C57BL/6 mice, administration of CP 55,940 (0.03-0.3 mg/kg, i.p.) reversed complete Freunds adjuvant-induced tactile allodynia, reversed tactile allodynia in the spinal nerve ligation model and inhibited the noxious heat-evoked tail withdrawal response. In addition to its antinociceptive effects, CP 55,940 produced an impairment of motor coordination in the rotarod test. The antinociceptive effects produced by CP 55,940 and associated motor deficits were found to be completely abolished in CB1 receptor knockout mice. In contrast, the antinociceptive effects of CP 55,940 in all pain models were fully retained in CB2 receptor knockout mice, along with the associated motor deficits. The results suggest that the antinociceptive effects of CP 55,940 in models of acute and persistent pain, along with the associated motor deficits, are mediated by CB1 receptors, and likely not CB2 receptors.

Decahydroquinoline amides as highly selective CB2 agonists: Role of selectivity on in vivo efficacy in a rodent model of analgesia

A novel series of decahydroquinoline CB2 agonists is described. Optimization of the amide substituent led to improvements in CB2/CB1 selectivity as well as physical properties. Two key compounds were examined in the rat CFA model of acute inflammatory pain. A moderately selective CB2 agonist was active in this model. A CB2 agonist lacking functional CB1 activity was inactive in this model despite high in vivo exposure both peripherally and centrally.

Synthesis, structure-activity relationship, and pharmacological profile of analogs of the ASIC-3 inhibitor A-317567.

The synthesis, structure-activity relationship (SAR), and pharmacological evaluation of analogs of the acid-sensing ion channel (ASIC) inhibitor A-317567 are reported. It was found that the compound with an acetylenic linkage was the most potent ASIC-3 channel blocker. This compound reversed mechanical hypersensitivity in the rat iodoacetate model of osteoarthritis pain, although sedation was noted. Sedation was also observed in ASIC-3 knockout mice, questioning whether sedation and antinociception are mediated via a non-ASIC-3 specific mechanism.

Identification of non-amidine inhibitors of acid-sensing ion channel-3 (ASIC3)

A series of benzothiophene methyl amines were examined in an effort to identify non-amidine chemotypes with reduced polypharmacology from existing leads with the goal of finding potent ASIC3 channel blockers to advance the therapeutic evaluation of ASIC3 inhibition.

The Effect of Amitriptyline on Ectopic Discharge of Primary Afferent Fibers in the L5 Dorsal Root in a Rat Model of Neuropathic Pain

BACKGROUND: The sodium channel blocker amitriptyline has been shown to inhibit ectopic discharge in injured nerves. In the present study, we characterized ectopic discharges of afferent fibers following L5/L6 spinal nerve ligation (SNL) by their electrophysiological properties and sensitivities to inhibition by amitriptyline in the decentralized L5 dorsal root in SNL rats. METHODS: Rats exhibiting withdrawal thresholds <4.0 g after SNL were selected for the present study. After laminectomy in pentobarbital-anesthetized rats, the L5 dorsal root was decentralized close to its entry to the spinal cord, and the spontaneous activities of single units were recorded peripherally before and after IV administration of amitriptyline. The mean frequency of afferent fiber activity and instantaneous frequency were measured. RESULTS: The spontaneous activities of afferent fibers in naïve rats had high frequency (35.23 ± 6.63 Hz) and pattern discharge based on their instantaneous frequencies and interspike interval distributions. In rats that had received SNL, afferent fibers exhibited spontaneous discharge (mean of 11.05 ± 3.66 Hz) with an irregular discharge pattern or short bursting activity in some cases. Only 5/13 (38%) afferent fibers from naïve rats showed reduced spontaneous activities after amitriptyline (2 mg/kg, IV), whereas amitriptyline significantly inhibited ectopic discharge in 13/18 (72%) afferent fibers from SNL rats (ID50 = 1.66 ± 0.17 mg/kg). Furthermore, the greatest inhibitory effect of amitriptyline was consistently observed on those afferent fibers exhibiting low frequency (<20 Hz) and/or bursting discharge. CONCLUSION: These results provide direct evidence that amitriptyline, which is used clinically for the treatment of neuropathic pain, selectively inhibits ectopic discharge of low frequency and bursting discharge in the rat neuropathic pain model.