Community Research and Development Information Service - CORDIS

Periodic Report Summary 3 - GLORIA (Understanding chronic pain and new druggable targets: Focus on glial-opioid receptor interface)

Project Context and Objectives:
Chronic pain is prevalent; it decreases the quality of life of the patient and causes significant costs to the society. Currently available analgesics are either not effective enough or patients cannot take them due to adverse effects. Analgesic efficacy can be improved by targeting pain mechanisms that are specific for different conditions: fibromyalgia (FM), osteoarthritis (OA), rheumatoid arthritis (RA), and neuropathic pain (NP), and by improving the safety of the current analgesics, such as opioids.

GLORIA focuses on the role of glial activation by tissue and nerve injury, inflammation, and high doses of opioids in both large cohorts of chronic pain patients and in respective experimental disease models. We develop a biomarker profile for the clinical conditions by assessing inflammatory markers in cerebrospinal fluid and glial activation in the brain with positron emission tomography (PET), by performing well-validated experimental pain tests, using conditioned pain modulation as a biomarker of endogenous analgesia, functional magnetic resonance imaging (fMRI) to study pain-related networks in the brain, and genetics. New bioinformatics tools are developed for data analysis.

Individual differences in glial activation and pain perception are used to design different research lines to develop personalized analgesics. We will use well-validated in vivo models relevant for RA, OA, FM, and NP. Glial activation in these models will be studied using novel methodology to better understand the pathophysiology of these conditions. GLORIA will also focus on the design and synthesis of two lines of new analgesic molecules, those that target the activation of glia or are GDNF family ligand (GFL) mimetics.

Main lines of research in GLORIA:

Mapping of pheno- and genotypes in chronic pain
The main goal is to identify biomarkers for chronic pain and those related to the glia-TLR4-opioid systems. We will also perform systematic pathway analyses to tag new potential genes via systems biology bioinformatics methods.

Mapping of phenotypes in chronic pain using advanced proteomics
We use advanced proteomics to identify biomarkers for pain patterns and for different phenotypes in chronic pain. We will establish a proteomic platform, perform epidemiological characterization of pain patterns and geno-phenotypes in large patient cohorts and identify biomarkers for pain phenotypes in RA, OA, FM and NP.

In vivo studies of glial cell activation and pain regulation by imaging
We study the role of neuroinflammation in chronic pain. We will assess endogenous pain modulation and resting state networks with fMRI in chronic pain patients; establish patterns of neuroinflammation by analysing cerebrospinal fluid and develop methods to assess glia cell activation using PET in humans and in vivo models, and develop cellular models for quantitative characterization of opioid receptor-mediated signalling dynamics by functional fluorescence microscopy imaging (fFMI).

Glial activation in animal models of chronic pain
The aim of this work is to examine the role of glia cells and opioid receptors expressed in them in in vivo models representing inflammatory, nociceptive, neuropathic, and fibromyalgia-type pain.

Role of opioid receptors on glial cells in chronic pain: conditional knockouts
We will establish genetic in vivo models to study the role of mu and delta opioid mechanisms in glial cells in NP. We aim to identify key opioid receptor populations expressed by glial cells in the development and persistence of chronic pain and opioid tolerance/hyperalgesia.

Novel compounds for managing chronic pain
We search for novel, more potent compounds to target pain-related receptors using computational methods; and optimize existing drugs for improved safety by combining computational methods with synthesis of derivative molecules and in vitro/vivo testing of the compounds. The focus is on two lines of molecules: those that target opioid receptors/glia and GFL mimetics.
Project Results:
We have established a panel of human pain-associated genes, a next generation sequencing (NGS) workflow to study opioid and toll-like receptor (TLR) genes, and bioinformatics methods and tools for the data analysis. We have screened a cohort of pain patients who need unusually high opioid doses, for genetic variants in the opioid receptor genes. We have invented an innovative data-science based biomarker that uses contemporary artificial intelligence techniques. We have used machine-learning to analyse clinical parameters from 1000 post-mastectomy patients and established different pain and mood-related phenotypes to be associated with underlying complex NGS-derived genotypes. Human genetic associations with persistent pain suggest the immune system as a target for potential future drugs for persistent pain.

We have defined and validated clinical pain phenotypes (remaining pain and widespread pain) in early RA with epidemiological methods. Remaining pain is common in early RA, and pain may persist despite good clinical response to anti-rheumatic treatment. We have established a platform for mass spectrometry-based mapping of candidate peptides in clinical patient cohorts, and formed a target list for further linking of biomarker data with clinical pain data.

We have analysed pro- and anti-inflammatory cytokines/chemokines in the cerebrospinal fluid of RA, FM and OA patients and found increased levels of IL-1b in RA, and of IL-8 in FM and OA patients, suggesting that IL-1b is involved in inflammatory pain, and IL-8 in disturbances in autonomic nervous system and dysfunctional pain modulation. Moreover, MCP1 levels were elevated in OA patients, and the data suggest that MCP1 has a special role in neuro-immune signalling in women.

We have tested RA patients for pain sensitivity with quantitative sensory tests, and for brain activity during resting state vs. evoked pain using fMRI. Our findings suggest changes in resting state activity and cerebral processing of painful stimuli from the inflamed joints. By assessing glial activation with PET, we discovered two different patterns: one in patients suffering from inflammatory pain (RA) and another in FM patients, suggesting specific patterns of glia activation in different pain conditions, with potential relevance for future treatment strategies.

Furthermore, using fMRI we could tie a genetic mechanism, associated with glia activation, to altered cerebral pain processing in FM. In addition, we have established a dual plasmid system for simultaneous expression of the mu-opioid and serotonin receptor and an assay to visualize the heterodimers by fFMI.

We have established and optimized a microglial enrichment and flow cytometric quantification method for the characterization of microglia in the naïve state and in different pain models. We have also established a quantitative 3D method to calculate minor morphological changes of microglia.

In vivo models representing RA, OA and NP have been established and pain-like behaviours in these systems have been evaluated. We detected increased spinal microglia and astrocyte reactivity in the models of RA and NP, but not in the OA model. The glia inhibitor minocycline delayed or reversed mechanical hypersensitivity in the RA and NP models.

We have successfully produced and analysed genetic models for selective mu or delta opioid receptor deletion in astrocytes and microglia.

We have developed and selected candidates for TLR4 inhibitors, opioid receptor antagonists, and GFL mimetics from public and commercial databases and by performing similarity screening by chemical structure. Three GFL mimetics have been tested in NP models and were shown to be useful starting compounds for the development of innovative analgesics for NP. We have also devised routes of chemical synthesis for several opioid derivatives. The pharmacokinetic and toxicological properties have been evaluated for the best compounds which will be further tested in in vivo pain models.
Potential Impact:
Chronic pain is an important cause of decreased quality of life, function, and work capacity. GLORIA can be expected to have a significant impact on future pain research by providing tools for both basic research (e.g. experimental disease-specific pain models, new genetic models, biomarkers, and targets for analgesic drug development) and clinical research (biomarkers, pain phenotypes and specific pain-related gene panels and the combinations of these). GLORIA will increase our understanding of the mechanisms (molecular, systems biological, genetic, behavioural) of pain in inflammatory (RA, OA), neuropathic (nerve injury, diabetes) and dysfunctional (FM) pain. GLORIA will also elucidate the changes that take place, particularly in the central nervous system, with time due to tissue or nerve injury and inflammation. All this information will also facilitate future analgesic drug development.

Most importantly, GLORIA will provide improvement in clinical diagnosis, early intervention, and more personalized therapy for persistent pain. This information can be applied into patient care very early on. Our results already suggest that in RA, such knowledge may be important for the early identification of patients at risk for the development of treatment refractory widespread pain. This would enable early introduction of pain relieving therapy and non-pharmacological interventions that can be used efficiently to prevent the development of widespread pain. Our first results revealed differential neuroinflammatory profiles in patients suffering from autoimmune inflammatory pain (RA) and patients suffering from a chronic pain syndrome associated with dysfunctional pain modulation (FM). In addition we have found that a genetic mechanism affecting the receptor binding by activated glia affects the severity of FM symptoms. These findings indicate a rational for the development of new therapeutic strategies targeting these systems for intractable chronic pain conditions with dysfunctional pain modulation.

One important goal of GLORIA is involving patients in the research. Several meetings have been held with the Swedish patient organization Reumatikerförbundet. We will continue involving patient organizations in the project in order to enhance the role of patients as partners e.g. in developing meaningful outcomes in clinical research. We have also organised lectures and provided patients with feedback from the studies in which they have contributed.

Another important aim of GLORIA is training of young scientists. At the moment, 38 undergraduate and graduate students and postdoctoral researchers are being trained within GLORIA at the five partner institutes. We have organized courses for postgraduate level students at the University of Helsinki and Strasbourg. We hope to inspire school children to study natural sciences that are currently considered too demanding by most pupils, and to this end have participated at various school events in Stockholm and Helsinki, and have presented pain research. Hopefully they will be inspired to choose to study natural sciences and medicine.
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