Prasant Chandran

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.

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.

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.

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.

Development of opioid tolerance with repeated transcutaneous electrical nerve stimulation administration.

&NA; The analgesia produced by low and high frequency transcutaneous electrical nerve stimulation (TENS) is mediated by the release of &mgr;‐ or &dgr;‐opioids, respectively in the central nervous system. Repeated administration of either &mgr;‐ or &dgr;‐opioid agonists induce opioid analgesic tolerance. Thus, we tested if repeated administration of TENS (either low or high frequency) in rats leads to a development of tolerance to its antihyperalgesic effects with a corresponding cross‐tolerance to &mgr;‐ and &dgr;‐opioid agonists. Unilateral knee joint inflammation (3% carrageenan) was induced in adult Sprague–Dawley rats. Either low (4 Hz) or high frequency (100 Hz) TENS was administered for 6 days (20 min daily) to the inflamed knee joint under halothane anesthesia. The no TENS controls were administered anesthesia only for the same period. Withdrawal threshold to mechanical stimuli was measured before and after administration of TENS on each day and also on the sixth day. A separate group of animals was tested for tolerance to either the &mgr;‐opioid agonist, morphine (1.32, 3.95, 13.2 nmol/10 ml, intrathecal (i.t.)) or the &dgr;‐opioid agonist, SNC‐80 (6, 20, 60, 120 nmol/10 ml, i.t.) 30 min after i.t. administration. The reduced mechanical withdrawal threshold following the induction of inflammation was reversed by the application of TENS. However, repeatedly administering either low or high frequency TENS for 6 days, lead to a diminution in its effectiveness in reversing the ipsilateral secondary mechanical hyperalgesia by the fourth day. The effects of morphine in the low and SNC‐80 in the high frequency TENS groups were significantly less than the group that did not receive TENS. On the other hand, morphine and SNC‐80 were similar to the no TENS control in the high and low frequency TENS groups, respectively. Thus, repeated administration of low and high frequency TENS leads to a development of opioid tolerance with a corresponding cross‐tolerance to i.t. administered &mgr;‐ and &dgr;‐opioid agonists, respectively. Clinically, it can be inferred that a treatment schedule of repeated daily TENS administration should be avoided to possibly obviate the induction of tolerance.

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.

Comparison of antinociceptive actions of standard analgesics in attenuating capsaicin and nerve-injury-induced mechanical hypersensitivity

Intradermal capsaicin injection produces immediate spontaneous pain behaviors, and a secondary mechanical hypersensitivity (SMH) that is employed in the clinic as a model potentially predictive of human neuropathic pain. Presently, we have characterized capsaicin-induced SMH in rats, and compared pharmacological actions of standard analgesics in this and two nerve injury models, the L5/L6 spinal nerve ligation (SNL) and sciatic nerve chronic constriction injury (CCI) models. Intraplantar capsaicin produced dose-related SMH (enhanced paw withdrawal response to von Frey monofilament stimulation at an area away from injection site) that lasted for over 4 h. While pretreatment with a potent selective transient receptor potential vanilloid receptor-1 (TRPV1) antagonist A-425619 (1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea) prevented development of acute nocifensive (flinching) behavior immediately following capsaicin injection (ED(50)=4.9 mg/kg), the compound failed to attenuate the SMH when administered 2 h following capsaicin (10 microg/10 microl). Additional standard analgesics were also tested 3 h following intraplantar capsaicin in the SMH model. Comparison of their potencies in attenuating mechanical hypersensitivity in capsaicin, SNL and CCI models revealed similar ED(50)s for morphine (2.3 mg/kg, 1.6 mg/kg and 3.2 mg/kg, respectively), gabapentin (33.1 mg/kg, 33.9 mg/kg and 26.3 mg/kg, respectively) and lamotrigine (9.1 mg/kg, 8.9 mg/kg and 15.5 mg/kg, respectively). Duloxetine produced 50-65% effect at the highest tested dose (50 mg/kg), whereas the highest tested doses of morphine (10 mg/kg), gabapentin (85.5 mg/kg) and lamotrigine (30 mg/kg) all produced >70% efficacy in capsaicin SMH, SNL and CCI models. In contrast, celecoxib and ibuprofen showed weak effects in all three models. All standard analgesics generally had weak efficacy in attenuating capsaicin-induced immediate acute flinching behavior when administered before capsaicin. These results provide further support to the suggestions that distinct pharmacological mechanisms underlie capsaicin-induced acute nocifensive and SMH behaviors, and certain neuronal mechanisms underlying neuropathic pain states are also contributory to capsaicin-induced SMH.

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.

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.