ADX71943 and ADX71441, novel positive allosteric modulators of the GABAB receptor with distinct central/peripheral profiles, show efficacy in the monosodium iodoacetate model of chronic osteoarthritis pain in the rat.

We tested novel positive allosteric modulators (PAMs) of the γ-aminobutyric acid receptor B (GABAB), ADX71943 and ADX71441in the monosodium iodoacetate model of chronic osteoarthritis pain in rats with the objective to delineate the role of peripheral versus central GABAB receptor populations in modulation of chronic pain. Anesthetized Sprague-Dawley rats received an injection of monosodium iodoacetate into the knee and were tested for hyperalgesia starting post-MIA day 14. Effects of compounds on ipsilateral joint compression threshold were evaluated on post-MIA day 14 (after acute treatment), as well as after repeated, daily treatment on days 21 and 28 (ADX71943 only) and were compared to those of celecoxib (30mg/kg, p.o.). The PAMs were also tested in the rat rotarod test for potential muscle-relaxant effects. Acutely, ADX71943 (1-30mg/kg, p.o.), the peripherally restricted PAM, resulted in similar increases in pain threshold across the doses on day 14, while showing reduced efficacy on day 21 and no efficacy on day 28. A clear reduction in the efficacy of celecoxib across testing was also noted in this experiment. Acutely ADX71441 (0.3-15mg/kg, p.o.), the central-peripheral PAM, resulted in over 2-fold increases in pain threshold at 15mg/kg (but not at lower doses) on day 14, while causing more modest effects on day 21. Celecoxib increased pain threshold after both acute and daily treatment, showing overall similar efficacy. Thus, early, presumably more inflammatory phase of osteoarthritis pain in more sensitive to GABAB PAMs with peripherally restricted profile, while later, presumably more neuropathic phase is more sensitive to PAMs with central-peripheral profile.

Preclinical Pharmacological Approaches in Drug Discovery for Chronic Pain.

In recent years, animal behavioral models, particularly those used in pain research, have been increasingly scrutinized and criticized for their role in the poor translation of novel pharmacotherapies for chronic pain. This chapter addresses the use of animal models of pain used in drug discovery research. It highlights how, when, and why animal models of pain are used as one of the many experimental tools used to gain better understanding of target mechanisms and rank-order compounds in the iterative process of establishing structure-activity relationship. Together, these models help create an "analgesic signature" for a compound and inform the indications most likely to yield success in clinical trials. In addition, the authors discuss some often underappreciated aspects of currently used (traditional) animal models of pain, including simply applying basic pharmacological principles to study design and data interpretation as well as consideration of efficacy alongside side effect measures as part of the overall conclusion of efficacy. This is provided to add perspective regarding current efforts to develop new models and endpoints both in rodents and in larger animal species as well as assess cognitive and/or affective aspects of pain. Finally, the authors suggest ways in which efficacy evaluation in animal models of pain, whether traditional or new, might better align with clinical standards of analysis, citing examples where applying effect size and number needed to treat estimations to animal model data suggest that the efficacy bar often may be set too low preclinically to allow successful translation to the clinical setting.

An industry perspective on the role and utility of animal models of pain in drug discovery.

In recent years, animal behavioral models, particularly those used in pain research, have been increasingly scrutinized and criticized for their role in the poor translation of novel pharmacotherapies for chronic pain. This article addresses the use of animal models of pain from the perspective of industrial drug discovery research. It highlights how, when, and why animal models of pain are used as one of the many experimental tools used to gain better understanding of target mechanisms and rank-order compounds in the iterative process of establishing structure-activity relationships (SAR). Together, these models help create an 'analgesic signature' for a compound and inform the indications most likely to yield success in clinical trials. In addition, the authors discuss some often under-appreciated aspects of currently used (traditional) animal models of pain, including how industry balances efficacy with side effect measures as part of the overall conclusion of efficacy. This is provided to add perspective regarding current efforts to develop new models and endpoints both in rodents and larger animal species as well as assess cognitive and/or affective aspects of pain. Finally, the authors suggest ways in which efficacy evaluation in animal models of pain, whether traditional or new, might better align with clinical standards of analysis, citing examples where applying effect size and NNT estimations to animal model data suggest that the efficacy bar often may be set too low preclinically to allow successful translation to the clinical setting.

The monosodium iodoacetate model of osteoarthritis pain in the rat.

Osteoarthritis (OA) is one form of degenerative joint disease characterized by progressive loss of articular cartilage, decreased function and is frequently accompanied by chronic pain. Given the success of arthroplasty as a treatment for late-stage OA, there is considerable interest in developing therapies pertaining to the management of pain associated with OA as well as therapies designed to slow or reverse the progression of the disease. To this end, establishment of relevant animal models that are amenable to testing novel therapies is of considerable value to the scientific community. Here, we describe a model of OA-related pain in which progressive joint destruction is induced by injection of monosodium iodoacetate into the articular space of the knee of the rat. Further, we describe three different methods to measure pain-related behaviors in this model: hind limb weight bearing, primary mechanical hyperalgesia, and hind limb grip strength.

Complement activation in the peripheral nervous system following the spinal nerve ligation model of neuropathic pain.

Neuroinflammatory and neuroimmune mechanisms, as exemplified by infiltrating immune cells and activation of resident endothelial/glial cells, respectively, are known to be involved in the establishment and maintenance of chronic pain. An immune system pathway that may be involved in the activation of both immune and glial cells is complement. The complement pathway is made up of a large number of distinct plasma proteins which react with one another to opsonize pathogens and induce a series of inflammatory responses to help fight infection. Cleaved products and complexes produced by complement activation are responsible for a range of effects including mediation of immune infiltration, activation of phagocytes, opsonization/lysis of pathogens and injured cells, and production of vasoactive amines such as histamine and serotonin. Gene-expression microarray-analysis performed on the rat spinal nerve ligation (SNL) model of neuropathic pain revealed that multiple complement components including the C1 inhibitor, C1q alpha, beta, and gamma, C1r, C1s, C2, C3, C4, C7, and factors B, D, H, and P, were up-regulated while DAF was down-regulated. Regulation of C3 and DAF was confirmed by real-time RT-PCR and in situ hybridization. To test the hypothesis that complement plays a role in neuropathic pain, SNL rats were treated with cobra venom factor (CVF) to deplete plasma of complement component C3. Pain behavior was significantly attenuated in SNL rats treated with CVF as was complement activity at the ipsilateral dorsal root ganglia. Our results suggest the complement pathway might be a novel target for the development of neuropathic pain therapeutics.

Pharmacology of 2-[4-(4-chloro-2-fluorophenoxy)phenyl]-pyrimidine-4-carboxamide: a potent, broad-spectrum state-dependent sodium channel blocker for treating pain states.

Voltage-gated Na(+) channels may play important roles in establishing pathological neuronal hyperexcitability associated with chronic pain in humans. Na(+) channel blockers, such as carbamazepine (CBZ) and lamotrigine (LTG), are efficacious in treating neuropathic pain; however, their therapeutic utility is compromised by central nervous system side effects. We reasoned that it may be possible to gain superior control over pain states and, in particular, a better therapeutic index, by designing broad-spectrum Na(+) channel blockers with higher potency, faster onset kinetics, and greater levels of state dependence than existing drugs. 2-[4-(4-Chloro-2-fluorophenoxy)phenyl]-pyrimidine-4-carboxamide (PPPA) is a novel structural analog of the state-dependent Na(+) channel blocker V102862 [4-(4-fluorophenoxy)benzaldehyde semicarbazone]. Tested on recombinant rat Na(v)1.2 channels and native Na(+) currents in cultured rat dorsal root ganglion neurons, PPPA was approximately 1000 times more potent, had 2000-fold faster binding kinetics, and > or =10-fold higher levels of state dependence than CBZ and LTG. Tested in rat pain models against mechanical endpoints, PPPA had minimal effective doses of 1 to 3 mg/kg p.o. in partial sciatic nerve ligation, Freund's complete adjuvant, and postincisional pain. In all cases, efficacy was similar to clinically relevant comparators. Importantly, PPPA did not produce motor deficits in the accelerating Rotarod assay of ataxia at doses up to 30 mg/kg p.o., indicating a therapeutic index >10, which was superior to CBZ and LTG. Our experiments suggest that high-potency, broad-spectrum, state-dependent Na(+) channel blockers will have clinical utility for treating neuropathic, inflammatory, and postsurgical pain. Optimizing the biophysical parameters of broad-spectrum voltage-gated Na(+) channel blockers may lead to improved pain therapeutics.

Development and pharmacological characterization of a rat model of osteoarthritis pain.

Osteoarthritis (OA) is an age-related joint disease characterized by degeneration of articular cartilage and is associated with chronic pain. Although several experimental models of OA have been employed to investigate the underlying etiologies of the disease, there has been relatively little investigation into development of animal models of OA to study the pain associated with the condition. In the present study, we investigated OA induced by injection of either iodoacetate or papain into the knee joint of rats, and assessed the joint degeneration with radiographic analyses and measured pain behavior using hind limb weight bearing. We found that injection of iodoacetate, but not papain, resulted in a chronic joint degeneration as measured by decreased bone mineral content and bone mineral density, necrosis of articular cartilage and osteophyte formation. These pathological changes were associated with pain that manifested as time- and concentration-dependent alterations in hind limb weight bearing. These alterations in hind limb weight bearing were reversed with morphine, but were not significantly affected by acute administration of either indomethacin or celecoxib. However, administration of 30 mg/kg celecoxib twice daily for 10 days resulted in a significant restoration of hind limb weight bearing. We conclude that the iodoacetate model of OA is a relevant animal model to study pain associated with OA, and can be used to test potential therapeutic agents.

Intra-amygdalar injection of DAMGO: effects on c-Fos levels in brain sites associated with feeding behavior.

It is well known that the mu opioid agonist, Tyr-D-Ala-Gly-(me) Phe-Gly-ol (DAMGO), increases food intake in rats when injected into a variety of brain sites including the central nucleus of the amygdala (CeA). Immunohistochemical studies measuring c-Fos immunoreactivity (IR) suggest that the CeA contributes to opioid-related feeding. In the current study, we injected 2 nmol of DAMGO and measured food intake, c-Fos IR levels in various brain sites involved in feeding behavior, and mu opioid receptor internalization. We also studied the effect of CeA-injected DAMGO on LiCl-induced increases in c-Fos IR in the amygdala. As was expected, intra-CeA injection of DAMGO increased food intake of rats over a 4-h period. DAMGO injection into the CeA also resulted in mu opioid receptor internalization in the CeA, indicating activation of mu opioid receptor expressing neurons in this site. Administration of DAMGO into the CeA increased c-Fos IR levels in the shell of the nucleus accumbens (NAcc), but not in 17 other brain sites that were studied. We also found that intra-CeA injection of DAMGO, prior to LiCl injection, decreased c-Fos IR levels in the CeA compared to vehicle-injected rats. Thus, intra-CeA administration of DAMGO may increase feeding, in part, by activating neurons in the shell of the nucleus accumbens and by inhibiting activity of selected neurons in the CeA.

DiPOA ([8-(3,3-diphenyl-propyl)-4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]dec-3-yl]-acetic acid), a novel, systemically available, and peripherally restricted Mu opioid agonist with antihyperalgesic activity: II. In vivo pharmacological characterization in the rat.

Mu opioid receptors are expressed throughout the central and peripheral nervous systems. Peripheral inflammation leads to an increase in mu receptor present on the peripheral terminals of primary sensory neurons. Activation of peripheral mu receptors produces potent antihyperalgesic effects in both humans and animals. Here, we describe the in vivo pharmacological properties of the structurally novel, highly potent, systemically available yet peripherally restricted mu opioid agonist, [8-(3,3-diphenyl-propyl)-4-oxo-1-phenyl-1,3,8-triaza-spiro[4.5]dec-3-yl]-acetic acid (DiPOA). DiPOA administered i.p. produced naltrexone-sensitive, dose-dependent reversal of Freund's complete adjuvant-induced inflammatory mechanical hyperalgesia (1-10 mg/kg). Maximum percent reversal (67%) was seen 1 h postadministration at 10 mg/kg (the highest dose studied). DiPOA also proved antihyperalgesic in a model of postsurgical pain with a maximum percent reversal of 85% 1 h postadministration at 30 mg/kg i.p. (the highest dose studied). DiPOA administered i.p. had no effect in the tail flick assay of acute pain (0.1-10 mg/kg), produced no ataxia as measured by latency to fall from an accelerating rotarod (3-30 mg/kg), and was not antihyperalgesic in the Seltzer model of neuropathic pain (1-10 mg/kg). This is the first report of a peripherally restricted, small-molecule mu opioid agonist that is nonsedating, antihyperalgesic, and effective against inflammatory and postsurgical pain when administered systemically.

Proteomic analysis of the reactive phenotype of astrocytes following endothelin-1 exposure.

Reactive gliosis is an invariant feature of the pathology of central nervous system (CNS) injury and a major determinant of neuronal survival and regeneration. To begin to understand the alterations in astrocyte protein expression that drive glial changes that occur following injury, we used an established model system (endothelin-1 stimulation of hypertrophy) and proteomic analysis to define a discrete set of differentially expressed proteins and post-translational modifications that occur as the astrocytes change from a quiescent to a reactive state. This orchestrated set of changes included proteins involved in cytoskeletal reorganization (caldesmon, calponin, alpha B-crystallin, stathmin, collapsing response mediator protein-2), cell adhesion (vinculin, galectin-1), signal transduction (RACK-1) and astrocyte differentiation (glutamine synthetase). Using proteomic analysis to understand what drives astrocyte expression of these functionally divergent molecules may offer insight into the mechanisms by which astrocytes can exhibit both pro-regenerative and anti-regenerative activities following CNS injury.

N-(4-Tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine -1(2H)-carbox-amide (BCTC), a novel, orally effective vanilloid receptor 1 antagonist with analgesic properties: II. in vivo characterization in rat models of inflammatory and neuropathic pain.

The vanilloid receptor 1 (VR1) is a cation channel expressed predominantly by nociceptive sensory neurons and is activated by a wide array of pain-producing stimuli, including capsaicin, noxious heat, and low pH. Although the behavioral effects of injected capsaicin and the VR1 antagonist capsazepine have indicated a potential role for VR1 in the generation and maintenance of persistent pain states, species differences in the molecular pharmacology of VR1 and a limited number of selective ligands have made VR1 difficult to study in vivo. N-(4-Tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropryazine-1(2H)-carbox-amide (BCTC) is a recently described inhibitor of capsaicin- and acid-mediated currents at rat VR1. Here, we report the effects of BCTC on acute, inflammatory, and neuropathic pain in rats. Administration of BCTC (30 mg/kg p.o.) significantly reduced both mechanical and thermal hyperalgesia induced by intraplantar injection of 30 micro g of capsaicin. In rats with Freund's complete adjuvantinduced inflammation, BCTC significantly reduced the accompanying thermal and mechanical hyperalgesia (3 mg/kg and 10 mg/kg p.o., respectively). BCTC also reduced mechanical hyperalgesia and tactile allodynia 2 weeks after partial sciatic nerve injury (10 and 30 mg/kg p.o.). BCTC did not affect motor performance on the rotarod after administration of doses up to 50 mg/kg p.o. These data suggest a role for VR1 in persistent and chronic pain arising from inflammation or nerve injury.

Endothelin receptor expression in the normal and injured spinal cord: potential involvement in injury-induced ischemia and gliosis.

The endothelins (ETs) are a family of peptides that exert their biological effects via two distinct receptors, the endothelin A receptor (ET(A)R) and the endothelin B receptor (ET(B)R). To more clearly define the potential actions of ETs following spinal cord injury, we used immunohistochemistry and confocal microscopy to examine the protein expression of ET(A)R and ET(B)R in the normal and injured rat spinal cord. In the normal spinal cord, ET(A)R immunoreactivity (IR) is expressed by vascular smooth muscle cells and a subpopulation of primary afferent nerve fibers. ET(B)R-IR is expressed primarily by radial glia, a small population of gray and white matter astrocytes, ependymal cells, vascular endothelial cells, and to a lesser extent in smooth muscle cells. Fourteen days following compression injury to the spinal cord, there was a significant upregulation in both the immunoexpression and number of astrocytes expressing the ET(B)R in both gray and white matter and a near disappearance of ET(B)R-IR in ependymal cells and ET(A)R-IR in primary afferent fibers. Conversely, the vascular expression of ET(A)R and ET(B)R did not appear to change. As spinal cord injury has been shown to induce an immediate increase in plasma ET levels and a sustained increase in tissue ET levels, ETs would be expected to induce an initial marked vasoconstriction via activation of vascular ET(A)R/ET(B)R and then days later a glial hypertrophy via activation of the ET(B)R expressed by astrocytes. Strategies aimed at blocking vascular ET(A)R/ET(B)R and astrocyte ET(B)Rs following spinal cord injury may reduce the resulting ischemia and astrogliosis and in doing so increase neuronal survival, regeneration, and function.

Endothelin B receptors are expressed by astrocytes and regulate astrocyte hypertrophy in the normal and injured CNS.

The ability of mammalian central nervous system (CNS) neurons to survive and/or regenerate following injury is influenced by surrounding glial cells. To identify the factors that control glial cell function following CNS injury, we have focused on the endothelin B receptor (ET(B)R), which we show is expressed by the majority of astrocytes that are immunoreactive for glial acid fibrillary protein (GFAP) in both the normal and crushed rabbit optic nerve. Optic nerve crush induces a marked increase in ET(B)R and GFAP immunoreactivity (IR) without inducing a significant increase in the number of GFAP-IR astrocytes, suggesting that the crush-induced astrogliosis is due primarily to astrocyte hypertrophy. To define the role that endothelins play in driving this astrogliosis, artificial cerebrospinal fluid (CSF), ET-1 (an ET(A)R and ET(B)R agonist), or Bosentan (a mixed ET(A)R and ET(B)R antagonist) were infused via osmotic minipumps into noninjured and crushed optic nerves for 14 days. Infusion of ET-1 induced a hypertrophy of ET(B)R/GFAP-IR astrocytes in the normal optic nerve, with no additional hypertrophy in the crushed nerve, whereas infusion of Bosentan induced a significant decrease in the hypertrophy of ET(B)R/GFAP-IR astrocytes in the crushed but not in the normal optic nerve. These data suggest that pharmacological blockade of astrocyte ET(B)R receptors following CNS injury modulates glial scar formation and may provide a more permissive substrate for neuronal survival and regeneration.

Efficacy of systemic morphine suggests a fundamental difference in the mechanisms that generate bone cancer vs inflammatory pain.

Pain is the cancer related event that is most disruptive to the cancer patient's quality of life. Although bone cancer pain is one of the most severe and common of the chronic pains that accompany breast, prostate and lung cancers, relatively little is known about the mechanisms that generate and maintain this pain. Recently, we developed a mouse model of bone cancer pain and 16 days following tumor implantation into the intramedullary space of the femur, significant bone destruction and bone cancer pain-related behaviors were observed. A critical question is how closely this model mirrors human bone cancer pain. In the present study we show that, as in humans, pain-related behaviors are diminished by systemic morphine administration in a dose dependent fashion that is naloxone-reversible. Humans suffering from bone cancer pain generally require significantly higher doses of morphine as compared to individuals with inflammatory pain and in the mouse model, the doses of morphine required to block bone cancer pain-related behaviors were ten times that required to block peak inflammatory pain behaviors of comparable magnitude induced by hindpaw injection of complete Freund's adjuvant (CFA) (1-3mg/kg). As these animals were treated acutely, there was not time for morphine tolerance to develop and the rightward shift in analgesic efficacy observed in bone cancer pain vs. inflammatory pain suggests a fundamental difference in the underlying mechanisms that generate bone cancer vs. inflammatory pain. These results indicate that this model may be useful in defining drug therapies that are targeted for complex bone cancer pain syndromes.