Gin C. Hsieh

A-740003 [N-(1-{[(Cyanoimino)(5-quinolinylamino) methyl]amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide], a Novel and Selective P2X7 Receptor Antagonist, Dose-Dependently Reduces Neuropathic Pain in the Rat

ATP-sensitive P2X7 receptors are localized on cells of immunological origin including glial cells in the central nervous system. Activation of P2X7 receptors leads to rapid changes in intracellular calcium concentrations, release of the proinflammatory cytokine interleukin-1β (IL-1β), and following prolonged agonist exposure, cytolytic plasma membrane pore formation. P2X7 knockout mice show reduced inflammation as well as decreased nociceptive sensitivity following peripheral nerve injury. A-740003 (N-(1-{[(cyanoimino)(5-quinolinylamino) methyl] amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide) is a novel competitive antagonist of P2X7 receptors (IC50 values = 40 nM for human and 18 nM for rat) as measured by agonist-stimulated changes in intracellular calcium concentrations. A-740003 showed weak or no activity (IC50 > 10 μM) at other P2 receptors and an array of other neurotransmitter and peptide receptors, ion channels, reuptake sites, and enzymes. A-740003 potently blocked agonist-evoked IL-1β release (IC50 = 156 nM) and pore formation (IC50 = 92 nM) in differentiated human THP-1 cells. Systemic administration of A-740003 produced dose-dependent antinociception in a spinal nerve ligation model (ED50 = 19 mg/kg i.p.) in the rat. A-740003 also attenuated tactile allodynia in two other models of neuropathic pain, chronic constriction injury of the sciatic nerve and vincristine-induced neuropathy. In addition, A-740003 effectively reduced thermal hyperalgesia observed following intraplantar administration of carrageenan or complete Freunds adjuvant (ED50 = 38–54 mg/kg i.p.). A-740003 was ineffective in attenuating acute thermal nociception in normal rats and did not alter motor performance at analgesic doses. These data demonstrate that selective blockade of P2X7 receptors in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain.

Localization of histamine H4 receptors in the central nervous system of human and rat

Existing data on the expression of H(4) histamine receptor in the CNS are conflicting and inconclusive. In this report, we present the results of experiments that were conducted in order to elucidate H(4) receptor expression and localization in the brain, spinal cord, and dorsal root ganglia (DRG). Here we show that transcripts of H(4) receptor are present in all analyzed regions of the human CNS, including spinal cord, hippocampus, cortex, thalamus and amygdala, with the highest levels of H(4) mRNA detected in the spinal cord. In rat, H(4) mRNA was detected in cortex, cerebellum, brainstem, amygdala, thalamus and striatum. Very low levels of H(4) mRNA were detected in hypothalamus, and no H(4) signal was detected in the rat hippocampus. Fairly low levels of H(4) mRNA were detected in examined peripheral tissues including spleen and liver. Interestingly, strong expression of H(4) mRNA was detected in the rat DRG and spinal cord. Immunohistochemical analysis revealed expression of H(4) receptors on neurons in the rat lumbar DRG and in the lumbar spinal cord. Our observations provide evidence of the H(4) presence in both human and rodent CNS and offer some insight into possible role of H(4) in itch and pain.

In vitro and in vivo characterization of A-796260: a selective cannabinoid CB2 receptor agonist exhibiting analgesic activity in rodent pain models

Selective cannabinoid CB2 receptor agonists have demonstrated analgesic activity across multiple preclinical pain models. AM1241 is an indole derivative that exhibits high affinity and selectivity for the CB2 binding site and broad spectrum analgesic activity in rodent models, but is not an antagonist of CB2 in vitro functional assays. Additionally, its analgesic effects are μ‐opioid receptor‐dependent. Herein, we describe the in vitro and in vivo pharmacological properties of A‐796260, a novel CB2 agonist.

Neointimal formation after balloon-induced vascular injury in Yucatan minipigs is reduced by oral rapamycin.

Rapamycin, a macrolide antibiotic known to prevent allograft rejection, is a potent inhibitor of cell proliferation. Therefore we studied the effects of orally administered rapamycin in a pig model of balloon injury in an attempt to reduce the cellular proliferation and neointimal formation thought to play a role in restenosis. Twenty Yucatan minipigs, divided into groups of 10 animals each, were subjected to balloon inflation of the carotid arteries. One group received the methylcellulose vehicle for rapamycin, whereas the second group was treated for a total of 31 days with 2.0 mg/kg of rapamycin administered daily by oral gavage. This dose and treatment regimen produced significant (p < 0.05) reductions in neointimal area (59%) and in the maximal thickness of the neointima (59%) when comparisons were made with vehicle-treated animals. These effects were accompanied by a significant increase in the lumen area in animals that received rapamycin (33%). Medial area was decreased by 18% in these animals. Blood samples from rapamycin-treated pigs indicated peak concentrations of 1.87 +/- 0.45 and 1.70 +/- 0.24 ng/ml at 2 and 4 weeks after balloon angioplasty, respectively. Significant increases in blood pressure of 21 mm Hg and decreases in heart rate of 25 beats/min also were observed in rapamycin-treated animals relative to those that received vehicle. These results indicate that the antiproliferative effect of rapamycin can be demonstrated after oral dosing in a pig vascular injury model, suggesting a possible therapeutic utility for rapamycin or its analogs in patients undergoing balloon angioplasty.

Indol-3-ylcycloalkyl Ketones: Effects of N1 Substituted Indole Side Chain Variations on CB2 Cannabinoid Receptor Activity

Several 3-acylindoles with high affinity for the CB(2) cannabinoid receptor and selectivity over the CB(1) receptor have been prepared. A variety of 3-acyl substituents were investigated, and the tetramethylcyclopropyl group was found to lead to high affinity CB(2) agonists (5, 16). Substitution at the N1-indole position was then examined. A series of aminoalkylindoles was prepared and several substituted aminoethyl derivatives were active (23-27, 5) at the CB(2) receptor. A study of N1 nonaromatic side chain variants provided potent agonists at the CB(2) receptor (16, 35-41, 44-47, 49-54, and 57-58). Several polar side chains (alcohols, oxazolidinone) were well-tolerated for CB(2) receptor activity (41, 50), while others (amide, acid) led to weaker or inactive compounds (55 and 56). N1 aromatic side chains also afforded several high affinity CB(2) receptor agonists (61, 63, 65, and 69) but were generally less potent in an in vitro CB(2) functional assay than were nonaromatic side chain analogues.

Pharmacological modulation of movement-evoked pain in a rat model of osteoarthritis.

This study was conducted to characterize movement-induced pain in a rat model of knee joint osteoarthritis and validate this behavioral assessment by evaluating the effects of clinically used analgesic compounds. Unilateral intra-articular administration of a chondrocyte glycolytic inhibitor monoiodoacetate, was used to induce knee joint osteoarthritis in Sprague-Dawley rats. In this osteoarthritis model, histologically erosive disintegration of the articular surfaces of the ipsilateral joint are observed which closely mimic the clinical picture of osteoarthritis. Movement-induced pain behavior was measured using hind limb compressive grip force evaluation. The animals exhibited pain behaviors epitomized by a long-lasting decrement in bilateral compressive hind limb grip force following unilateral knee injury. The effects of clinically used reference analgesics were evaluated 20 days following i.a. injection of monoiodoacetate. Full analgesic activity was observed for tramadol, celecoxib and diclofenac; moderate effects for indomethacin, duloxetine and gabapentin but weak or no effects for acetaminophen, ibuprofen and lamotrigine. As morphine reduced grip force in naïve rats, its analgesic effects could not be accurately evaluated in this model. Finally, the effects of celecoxib were maintained following chronic dosing. The results indicate that this in vivo model utilizing a movement-induced pain behavior spawned by knee joint osteoarthritis may provide a valuable tool in examining the role of potential analgesic targets in osteoarthritic pain. As the model is clinically relevant, it will further enhance the mechanistic understanding of chronic arthritic joint pain and help in developing newer and better therapeutic strategies to manage osteoarthritis pain.

Differential effects of cannabinoid receptor agonists on regional brain activity using pharmacological MRI

Activation of cannabinoid CB1 and/or CB2 receptors mediates analgesic effects across a broad spectrum of preclinical pain models. Selective activation of CB2 receptors may produce analgesia without the undesirable psychotropic side effects associated with modulation of CB1 receptors. To address selectivity in vivo, we describe non‐invasive, non‐ionizing, functional data that distinguish CB1 from CB2 receptor neural activity using pharmacological MRI (phMRI) in awake rats.

Rotationally constrained 2,4-diamino-5,6-disubstituted pyrimidines: a new class of histamine H4 receptor antagonists with improved druglikeness and in vivo efficacy in pain and inflammation models.

A new structural class of histamine H 4 receptor antagonists (6-14) was designed based on rotationally restricted 2,4-diaminopyrimidines. Series compounds showed potent and selective in vitro H 4 antagonism across multiple species, good CNS penetration, improved PK properties compared to reference H 4 antagonists, functional H 4 antagonism in cellular and in vivo pharmacological assays, and in vivo anti-inflammatory and antinociceptive efficacy. One compound, 10 (A-943931), combined the best features of the series in a single molecule and is an excellent tool compound to probe H 4 pharmacology. It is a potent H 4 antagonist in functional assays across species (FLIPR Ca (2+) flux, K b < 5.7 nM), has high (>190x) selectivity for H 4, and combines good PK in rats and mice (t 1/2 of 2.6 and 1.6 h, oral bioavailability of 37% and 90%) with anti-inflammatory activity (ED 50 = 37 micromol/kg, mouse) and efficacy in pain models (thermal hyperalgesia, ED 50 = 72 micromol/kg, rat).

Central and peripheral sites of action for CB2 receptor mediated analgesic activity in chronic inflammatory and neuropathic pain models in rats

BACKGROUND AND PURPOSE Cannabinoid CB2 receptor activation by selective agonists has been shown to produce analgesic effects in preclinical models of inflammatory and neuropathic pain. However, mechanisms underlying CB2‐mediated analgesic effects remain largely unknown. The present study was conducted to elucidate the CB2 receptor expression in ‘pain relevant’ tissues and the potential sites of action of CB2 agonism in rats.

H4 receptor antagonism exhibits anti-nociceptive effects in inflammatory and neuropathic pain models in rats

The histamine H(4) receptor (H(4)R) is expressed primarily on cells involved in inflammation and immune responses. To determine the potential role of H(4)R in pain transmission, the effects of JNJ7777120, a potent and selective H(4) antagonist, were characterized in preclinical pain models. Administration of JNJ7777120 fully blocked neutrophil influx observed in a mouse zymosan-induced peritonitis model (ED(50)=17 mg/kg s.c., 95% CI=8.5-26) in a mast cell-dependent manner. JNJ7777120 potently reversed thermal hyperalgesia observed following intraplantar carrageenan injection of acute inflammatory pain (ED(50)=22 mg/kg i.p., 95% CI=10-35) in rats and significantly decreased the myeloperoxide activity in the carrageenan-injected paw. In contrast, no effects were produced by either H(1)R antagonist diphenhydramine, H(2)R antagonists ranitidine, or H(3)R antagonist ABT-239. JNJ7777120 also exhibited robust anti-nociceptive activity in persistent inflammatory (CFA) pain with an ED(50) of 29 mg/kg i.p. (95% CI=19-40) and effectively reversed monoiodoacetate (MIA)-induced osteoarthritic joint pain. This compound also produced dose-dependent anti-allodynic effects in the spinal nerve ligation (ED(50)=60 mg/kg) and sciatic nerve constriction injury (ED(50)=88 mg/kg) models of chronic neuropathic pain, as well as in a skin-incision model of acute post-operative pain (ED(50)=68 mg/kg). In addition, the analgesic effects of JNJ7777120 were maintained following repeated administration and were evident at the doses that did not cause neurologic deficits in rotarod test. Our results demonstrate that selective blockade of H(4) receptors in vivo produces significant anti-nociception in animal models of inflammatory and neuropathic pain.