Near infrared readouts offer sensitive and rapid assessments of intestinal permeability and disease severity in inflammatory bowel disease models.

Intestinal permeability and neutrophil activity are closely linked to inflammatory bowel disease (IBD) pathophysiology. Here we discuss two techniques for assessing permeability and neutrophil activity in mouse IBD models using near infrared (NIR) detection. To address the limitation of visible light readouts-namely high background-IRDye 800CW was used to enable rapid, non-terminal measurements of intestinal permeability. The increased sensitivity of NIR readouts for colon permeability is shown using dextran sulfate sodium (DSS) and anti-CD40 murine colitis models in response to interleukin-22 immunoglobulin Fc (IL22Fc) fusion protein and anti-p40 monoclonal antibody treatments, respectively. In addition to enhanced permeability, elevated levels of neutrophil elastase (NE) have been reported in inflamed colonic mucosal tissue. Activatable NIR fluorescent probes have been extensively used for disease activity evaluation in oncologic animal models, and we demonstrate their translatability using a NE-activatable reagent to evaluate inflammation in DSS mice. Confocal laser endomicroscopy (CLE) and tissue imaging allow visualization of spatial NE activity throughout diseased colon as well as changes in disease severity from IL22Fc treatment. Our findings with the 800CW dye and the NE probe highlight the ease of their implementation in preclinical IBD research.

Design of Aminobenzothiazole Inhibitors of Rho Kinases 1 and 2 by Using Protein Kinase†A as a Structure Surrogate.

We describe the design, synthesis, and structure-activity relationships (SARs) of a series of 2-aminobenzothiazole inhibitors of Rho kinases (ROCKs) 1 and 2, which were optimized to low nanomolar potencies by use of protein kinase A (PKA) as a structure surrogate to guide compound design. A subset of these molecules also showed robust activity in a cell-based myosin phosphatase assay and in a mechanical hyperalgesia in vivo pain model.

Selective blockade of TRPA1 channel attenuates pathological pain without altering noxious cold sensation or body temperature regulation.

Despite the increasing interest in TRPA1 channel as a pain target, its role in cold sensation and body temperature regulation is not clear; the efficacy and particularly side effects resulting from channel blockade remain poorly understood. Here we use a potent, selective, and bioavailable antagonist to address these issues. A-967079 potently blocks human (IC(50): 51 nmol/L, electrophysiology, 67 nmol/L, Ca(2+) assay) and rat TRPA1 (IC(50): 101 nmol/L, electrophysiology, 289 nmol/L, Ca(2+) assay). It is >1000-fold selective over other TRP channels, and is >150-fold selective over 75 other ion channels, enzymes, and G-protein-coupled receptors. Oral dosing of A-967079 produces robust drug exposure in rodents, and exhibits analgesic efficacy in allyl isothiocyanate-induced nocifensive response and osteoarthritic pain in rats (ED(50): 23.2 mg/kg, p.o.). A-967079 attenuates cold allodynia produced by nerve injury but does not alter noxious cold sensation in naive animals, suggesting distinct roles of TRPA1 in physiological and pathological states. Unlike TRPV1 antagonists, A-967079 does not alter body temperature. It also does not produce locomotor or cardiovascular side effects. Collectively, these data provide novel insights into TRPA1 function and suggest that the selective TRPA1 blockade may present a viable strategy for alleviating pain without untoward side effects.

Discovery and biological evaluation of potent, selective, orally bioavailable, pyrazine-based blockers of the Na(v)1.8 sodium channel with efficacy in a model of neuropathic pain.

Na(v)1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons. It has been implicated in the pathophysiology of inflammatory and neuropathic pain, and we envisioned that selective blockade of Na(v)1.8 would be analgesic, while reducing adverse events typically associated with non-selective VGSC blocking therapeutic agents. Herein, we describe the preparation and characterization of a series of 6-aryl-2-pyrazinecarboxamides, which are potent blockers of the human Na(v)1.8 channel and also block TTx-r sodium currents in rat dorsal root ganglia (DRG) neurons. Selected derivatives display selectivity versus human Na(v)1.2. We further demonstrate that an example from this series is orally bioavailable and produces antinociceptive activity in vivo in a rodent model of neuropathic pain following oral administration.

Characterization of Fasudil in preclinical models of pain.

UNLABELLED: Activation of Rho kinase (ROCK) has been shown to play a role in neuronal regeneration and development of posttraumatic neuropathic pain. The ROCK inhibitor Fasudil, used clinically for the treatment of vasospasm, was used to investigate the analgesic profile of a ROCK inhibitor. Fasudil was evaluated in different preclinical models of neuropathic, osteoarthritic (OA), and inflammatory pain as well as capsaicin-induced acute pain and secondary mechanical hypersensitivity. In addition, Fasudil was tested in in vivo electrophysiology to determine the mechanism by which Fasudil produces analgesia. Fasudil at the highest dose tested (30 mg/kg) significantly attenuated mechanical allodynia in spinal-nerve ligation (SNL; 77%), chronic constriction injury (CCI; 53%), capsaicin-induced secondary mechanical hypersensitivity (63%), sodium iodoacetate-induced OA pain (88%), and capsaicin-induced acute flinching behaviors (56%). However, Fasudil (at 30 mg/kg) failed to attenuate or had only modest effects on inflammatory thermal hyperalgesia following carrageenan injection and mechanical allodynia following Complete Freund's Adjuvant (CFA) injection. Fasudil produced ED(50) of 10.8 mg/kg in the SNL, and 5.7 mg/kg in the OA pain models. The ED(50) and 95% CI could not be obtained in the other models. Furthermore, administration of Fasudil (10 mg/kg, iv) significantly reduced both spontaneous and evoked firing of wide dynamic range (WDR) neurons in SNL, but not sham rats. Finally, Fasudil significantly decreased exploratory behaviors at 30 mg/kg. These results suggest that the acute administration of a ROCK inhibitor produces efficacy in both neuropathic and nociceptive pain states at doses devoid of locomotor side effects, with specific effects on WDR neurons. PERSPECTIVE: In this article, the potential analgesic effects of Fasudil in a range of preclinical pain models were assessed. Fasudil was shown to have efficacy in neuropathic and nociceptive pain models. These findings may help identify new therapeutic treatments for pain in the clinic.

Comparison of mechanical allodynia and the affective component of inflammatory pain in rats.

Most animal models of pain cannot separate the sensory and affective components of pain. One model that has been used to assess affective pain is the place escape avoidance paradigm (PEAP). The aim of the current study is two-fold. First, validate PEAP with Complete Freund's Adjuvant (CFA)-induced inflammation for the assessment of the affective component of pain using the reference analgesics celecoxib, diclofenac and duloxetine; fluoxetine and scopolamine were tested as negative controls. Secondly, determine if there is a difference in efficacy in PEAP in comparison to the effects of the same compounds on von Frey-evoked mechanical allodynia in CFA animals. All compounds were tested in mechanical allodynia, place escape/avoidance, and for potentially confounding side effects in locomotor activity. Results show that celecoxib, diclofenac, and duloxetine significantly increased the time spent on the side associated with stimulation of the injured paw, whereas fluoxetine and scopolamine had no effect. Higher doses of celecoxib, diclofenac, duloxetine, and fluoxetine were required to attenuate von Frey-evoked mechanical allodynia. In the side effect assays, only fluoxetine decreased locomotor activity at doses used in PEAP. These results show that in inflammatory pain induced by CFA injection, PEAP is more sensitive to the effects of pain relieving compounds than mechanical allodynia. Fluoxetine showed efficacy in the mechanical allodynia test, but not PEAP, whereas duloxetine showed efficacy in mechanical allodynia and PEAP. These studies show that methods other than reflex based measures of pain such as affective pain models could be more predictive of efficacy/potency in the clinic.