ABSTRACT
BACKGROUND: The CCR2/CCL2 system has been identified as a regulator in the pathogenesis of neuropathy-induced pain. However, CCR2 target validation in analgesia and the mechanism underlying antinociception produced by CCR2 antagonists remains poorly understood. In this study, in vitro and in vivo pharmacological approaches using a novel CCR2 antagonist, AZ889, strengthened the hypothesis of a CCR2 contribution to neuropathic pain and provided confidence over the possibilities to treat neuropathic pain with CCR2 antagonists. RESULTS: We provided evidence that dorsal root ganglia (DRG) cells harvested from CCI animals responded to stimulation by CCL2 with a concentration-dependent calcium rise involving PLC-dependent internal stores. This response was associated with an increase in evoked neuronal action potentials suggesting these cells were sensitive to CCR2 signalling. Importantly, treatment with AZ889 abolished CCL2-evoked excitation confirming that this activity is CCR2-mediated. Neuronal and non-neuronal cells in the spinal cord were also excited by CCL2 applications indicating an important role of spinal CCR2 in neuropathic pain. We next showed that in vivo spinal intrathecal injection of AZ889 produced dose-dependent analgesia in CCI rats. Additionally, application of AZ889 to the exposed spinal cord inhibited evoked neuronal activity and confirmed that CCR2-mediated analgesia involved predominantly the spinal cord. Furthermore, AZ889 abolished NMDA-dependent wind-up of spinal withdrawal reflex pathway in neuropathic animals giving insight into the spinal mechanism underlying the analgesic properties of AZ889. CONCLUSIONS: Overall, this study strengthens the important role of CCR2 in neuropathic pain and highlights feasibility that interfering on this mechanism at the spinal level with a selective antagonist can provide new analgesia opportunities.
Subject(s)
Hyperalgesia/drug therapy , Neuralgia/drug therapy , Piperazines/therapeutic use , Receptors, CCR2/antagonists & inhibitors , Spinal Cord/pathology , Animals , Calcium Signaling , Drug Delivery Systems , Ganglia, Spinal/pathology , Piperazines/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, CCR2/physiology , Signal TransductionABSTRACT
1-isoquinolinylguanidines were previously disclosed as potent and selective inhibitors of urokinase-type plasminogen activator (uPA). Further investigation of this template has revealed that incorporation of a 7-sulfonamide group furnishes a new series of potent and highly selective uPA inhibitors. Potency and selectivity can be achieved with sulfonamides derived from a variety of amines and is further enhanced by the incorporation of sulfonamides derived from amino acids. The binding mode of these 1-isoquinolinylguanidines has been investigated by X-ray cocrystallization studies. uPA inhibitor 26 was selected for further evaluation based on its excellent enzyme potency (Ki 10 nM) and selectivity profile (4000-fold versus tPA and 2700-fold versus plasmin). In vitro, compound 26 is able to inhibit exogenous uPA in human chronic wound fluid (IC50=0.89 microM). In vivo, in a porcine acute excisional wound model, following topical delivery, compound 26 is able to penetrate into pig wounds and inhibit exogenous uPA activity with no adverse effect on wound healing parameters. On the basis of this profile, compound 26 (UK-371,804) was selected as a candidate for further preclinical evaluation for the treatment of chronic dermal ulcers.
Subject(s)
Anti-Ulcer Agents/chemical synthesis , Guanidines/chemical synthesis , Quinolines/chemical synthesis , Sulfonamides/chemical synthesis , Urokinase-Type Plasminogen Activator/antagonists & inhibitors , Acute Disease , Animals , Anti-Ulcer Agents/chemistry , Anti-Ulcer Agents/pharmacology , Binding Sites , Chronic Disease , Crystallography, X-Ray , Guanidines/chemistry , Guanidines/pharmacology , Humans , In Vitro Techniques , Models, Molecular , Protein Binding , Quinolines/chemistry , Quinolines/pharmacology , Skin/injuries , Skin Diseases/enzymology , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacology , Swine , Ulcer/enzymology , Urokinase-Type Plasminogen Activator/chemistry , Wound Healing/drug effectsABSTRACT
BACKGROUND: Understanding the relationship between dose, lung exposure, and drug efficacy continues to be a challenging aspect of inhaled drug development. An experimental inhalation platform was developed using mometasone furoate to link rodent lung exposure to its in vivo pharmacodynamic (PD) effects. METHODS: We assessed the effect of mometasone delivered directly to the lung in two different rodent PD models of lung inflammation. The data obtained were used to develop and evaluate a mathematical model to estimate drug dissolution, transport, distribution, and efficacy, following inhaled delivery in rodents and humans. RESULTS: Mometasone directly delivered to the lung, in both LPS and Alternaria alternata rat models, resulted in dose dependent inhibition of BALf cellular inflammation. The parameters for our mathematical model were calibrated to describe the observed lung and systemic exposure profiles of mometasone in humans and in animal models. We found that physicochemical properties, such as lung fluid solubility and lipophilicity, strongly influenced compound distribution and lung retention. CONCLUSIONS: Presently, we report on a novel and sophisticated mathematical model leading to improvements in a current inhaled drug development practices by providing a quantitative understanding of the relationship between PD effects and drug concentration in lungs.
Subject(s)
Alternariosis/drug therapy , Anti-Inflammatory Agents/administration & dosage , Drug Dosage Calculations , Lung Diseases, Fungal/drug therapy , Lung/drug effects , Models, Biological , Mometasone Furoate/administration & dosage , Pneumonia/drug therapy , Administration, Inhalation , Aerosols , Alternaria , Alternariosis/metabolism , Alternariosis/microbiology , Alternariosis/physiopathology , Animals , Anti-Inflammatory Agents/pharmacokinetics , Disease Models, Animal , Humans , Lipopolysaccharides , Lung/metabolism , Lung/physiopathology , Lung Diseases, Fungal/metabolism , Lung Diseases, Fungal/microbiology , Lung Diseases, Fungal/physiopathology , Male , Mometasone Furoate/pharmacokinetics , Pneumonia/chemically induced , Pneumonia/metabolism , Pneumonia/physiopathology , Rats, Inbred BN , Rats, Sprague-Dawley , Species Specificity , Tissue DistributionABSTRACT
The role of muscarinic receptor subtype-1 (M1) in chronic pain is unclear. In an attempt to gain an understanding of its role, we have tested xanomeline, an M1/M4-preferring agonist, together with nonselective (scopolamine and pirenzepine), and selective (MT-7 and MT-3) muscarinic receptor (M1 and M4, respectively) antagonists in a number of inflammatory and neuropathic pain models. Xanomeline potently and effectively reversed tactile allodynia and heat hyperalgesia associated with established neuropathic and inflammatory pain in both rat and mouse models. Scopolamine and pirenzepine completely blocked the analgesic response to xanomeline, confirming that the analgesic effect is mediated by the muscarinic system. The highly selective M1 receptor toxin, MT-7, almost completely abolished the analgesic response to xanomeline when administered supraspinally. However, the highly selective M4 receptor toxin, MT-3, only marginally reversed the analgesia when given supraspinally, and had no effect when given spinally. In conclusion, the data presented show that the nonselective muscarinic agonist xanomeline is analgesic in models of persistent pain and suggest that the activation of supraspinal M1 receptors, and to a lesser extent supraspinal M4 receptors, contributes to that analgesia.