Integrative And Molecular Studies Of Pain And Pain Control

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Overview: This research program addresses basic molecular and physiological processes of nociceptive (pain-sensing) transmission in the peripheral and central nervous systems (CNS) and new ways to effectively control pain. The molecular research is performed using animal and in vitro cell-based models. We concentrate on primary afferent pain-sensing neurons located in dorsal root ganglion (DRG) that send nerve fibers to skin and deep tissues and make connections within dorsal spinal cord, which is the first CNS site of synaptic information processing for pain. The mechanisms of transduction of pain stimuli are investigated through models of pathophysiological damage or using reductionist preparations such as primary DRG cultures or heterologous expression systems of ion channels or receptors. Our goals are (a) to understand the molecular and cell biological mechanisms of acute and chronic pain at the initial steps in the pain pathway, (b) to investigate mechanisms underlying human chronic pain disorders, (c) to explore neuronal plasticity and altered gene expression in persistent pain states, and (d) to use this knowledge to devise new treatments for pain. New Treatments for Pain: We address the new treatment goal through translational research coupled with human clinical trials to develop and introduce new molecular interventions for severe pain. Studies with the TRPV1 agonist resiniferatoxin (RTX) have resulted in a Phase I clinical trial for in patients with intractable pain from advanced cancer. RTX activates an influx of sodium and calcium ions and once bound to TRPV1, RTX props open the ion channel causing an intracellular calcium cytotoxicity. Depending on the route of administration RTX disables TRPV1 pain-sensing nerve endings or axons (i.e., the nerve fiber) or deletes the neuron entirely. RTX produces very effective pain control in pre-clinical models. The central route involves administration into the cerebrospinal fluid around the spinal cord (intrathecal). We have treated 16 patients with pain from advanced cancer. This study is complete unless another, higher, dose tier is investigated and being readied for publication. Earlier we published studies of RTX injections around or directly into sensory ganglia. These formed the basis of our protocol to localized chronic pain by periganglionic RTX injection. Peripheral routes of RTX administration also include injection into skin, joints, nerve bundles, or topically. Analgesia by these routes is long-lasting but reversible, since peripheral nerve endings regrow. Peripheral administration formed the basis of three reports on treating (a) experimental burn pain, (b) surgical incision pain, and (c) a third in which we successfully treated clinical osteoarthritis (OA) pain by intraarticular injection in client owned dogs. The post-operative incision and OA pain indications are being translated to humans. We are conducting the postoperative pain study in the Clinical Center in collaboration with the Thoracic and Oncologic Surgery Branch, NCI. The initial study will evaluate preemptive treatment with local RTX injected into wound sites. This protocol has passed scientific review and will proceed to the IRB and IND review stages. The protocol for treating trigeminal neuralgia by injection into the trigeminal ganglion also is in the process of review. Early Translational Investigations: In collaboration, we have also examined the pharmacological activity of polyunsaturated ethanolamines and linoleic acid metabolites. In the previous cycle, we evaluated tissue biosynthetic pathways for new endogenous lipids and published our discovery of two previously unknown lipids related to nociception and itch in both animal and human studies. In this cycle we have applied a systems-based approach to characterize oxylipin precursor fatty acids, and the expression of genes coding for proteins involved in biosynthesis, transport, signaling and inactivation of pro- and anti-nociceptive oxylipins in rodent pain circuit tissues. We also measured basal and stimulated levels of predicted oxylipins, throughout the time course of an intraplantar carrageenan injection. These findings have been submitted for publication and advance our understanding of the molecular pathways linking oxylipins and their precursor fatty acids to nociceptive signaling pathways in rats. To extend the systems approach to humans we have established a new human protocol to obtain intraoperative tissue samples from surgical wound margins over time. This longitudinal study is currently underway. We have biopsied 5 subjects so far out of 12 and samples will be used to perform analyses of lipids and transcriptomic profiles over time. During the previous cycle we published a report on a protein therapeutic agent that is a conjugate between Substance P and a bioengineered Pseudomonas exotoxin (SP-PE35). This agent is endocytosed by the substance P receptor expressing second order spinal cord dorsal horn neurons and the exotoxin moiety stops protein synthesis thereby killing the neuron and interrupting the pain pathway to the brain. This is a potent analgesic agent. We are currently working with our NCI collaborators to generate high-expressing constructs for SP-PE35 to obtain large amounts of the active pharmaceutical ingredient for further testing in cancer pain and spinal cord injury pain. Basic Pain Mechanisms: Underlying the translational and clinical studies are investigations of molecular biology, neuronal function, behavior, and mechanisms of pain transduction and wound healing. We systematically investigate molecular alterations at the first three steps in the pain pathway beginning with injured peripheral tissue, the dorsal root ganglion and the dorsal (sensory) spinal cord in order to obtain a comprehensive quantitative foundational molecular understanding of nociceptive processes related to inflammation, surgical incision, and nerve injury. We use a method called RNA-Seq to sequence all of the mRNAs in a given tissue or cell population. Our work now integrates RNA-seq as a component in most of our investigations. We also investigate humans with genetic variations that affect pain sensitivity. At present we are investigating two groups of patients with copy number variants that decrease pain sensitivity. The results are both compelling and informative and define previously unidentified genetic substrates that can govern pain sensitivity. We also use RNA-seq to define genes involved in inherited peripheral neuropathies and other disorders. These investigations provide new quantitative assessments of neuronal and glial genes as well as immune process related to the nociceptive circuit. Through this basic research we aim to obtain a deeper understanding of mechanisms that trigger acute pain and sustain chronic pain and to identify molecular components to control pain.