Summary: Overview: This research program addresses basic molecular and physiological processes of nociceptive (pain-sensing) transmission in the central and peripheral nervous systems 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 innervate the skin and deep tissues and their connections in the dorsal spinal cord, which is the first site of synaptic information processing for pain. Our research has identified the DRG and spinal cord as loci of neuronal plasticity and altered gene expression in persistent pain states. The mechanisms of transduction of physical pain stimuli are investigated through examination of events and molecules in damaged or inflamed peripheral tissue, in primary cultures or using reductionist preparations such as heterologous expression systems in cultured cells. Our goals are (1) to understand the molecular and cell biological mechanisms of acute and chronic pain at the initial steps in the pain pathway, (2) to investigate mechanisms underlying human chronic pain disorders, and (3) to use this knowledge to devise new treatments for pain. New Treatments for Pain: We address the new treatment goal by a translational research and human clinical trials program aimed at developing new analgesics and interventions for severe pain. The current approach, based on our studies of pain transduction through the vanilloid transient receptor potential ion channel (TRPV1), has resulted in a Phase I clinical trial of the TRPV1 agonist resiniferatoxin (RTX) as a new treatment for advanced cancer pain. RTX activates TRPV1, which is an inflammation and heat-sensitive calcium/sodium ion channel that normally converts painful heat or low pH into nerve action potentials by opening the pore of the ion channel. The influx of ions depolarizes pain-sensing nerve endings and triggers electrical impulses that are conducted to the spinal cord. RTX is a potent capsaicin analog that props open the TRPV1 ion channel, causing calcium cytotoxicity and death of TRPV1 pain-sensing neurons or their axonal projections or endings. RTX treatments produced very effective pain control in preclinical models by several routes of administration: into the cerebrospinal fluid around the spinal cord (intrathecal) or directly into the sensory dorsal root ganglion (DRG) (permanent effect) or into peripheral sites to expose nerve endings in skin, joints, cornea or axons in a peripheral nerve (temporary effect since the peripheral endings regenerate). To date, following approval of the clinical protocol and the Investigational New Drug application, we have treated 5 patients with severe pain from advanced cancer and went through one dose escalation. The aim of the Phase I study is to determine the safety of RTX upon administration into the spinal CSF (intrathecal). Our plan is to complete the Phase I study, generate new protocols for postherpetic neuralgia an other acute and chronic pain conditions such as osteoarthritis, head and neck cancer, post-amputation and neuropathic pain. Basic Pain Mechanisms: Underlying the translational studies are investigations of molecular regulation of gene expression, neuronal function, behavior, and mechanisms of pain transduction. We are systematically investigating the first three steps in the pain pathway beginning with injured peripheral tissue, the dorsal root ganglion and the dorsal (sensory) spinal cord. The goal of this approach is to obtain a comprehensive molecular understanding of nociceptive process. Our studies reveal a complex, dynamic modulation of gene expression at all three steps. We have identified prominent roles for new, key molecules with distinct combinatorial patterns of expression among the three tissues. We are now examining human DRG and spinal cord to determine if the same expression and enrichment occurs in human pain circuits. We established collaboration with the Neuropathology Section, NIMH and have collected over 100 human trigeminal ganglia and cervical spinal cord samples. These tissues are being used for transcriptome and protein analysis of human nociceptive circuits. We are in the process of establishing collaboration with Rush University to obtain postmortem DRG and spinal cord of patients with defined pain disorders;thus both baseline and pain-state-dependent changes can be investigated. Through this research we hope to obtain a fundamental understanding of the relationships between tissue damage, inflammation and pain sensation. In a broader framework, these studies explore the fundamental molecular basis of synaptic plasticity. We hypothesize modularity in neuronal responses when a new level of synaptic or pharmacological input occurs that will be relevant to pain, and to neurological disorders like epilepsy or drug abuse whereby "generic" alterations are combined with circuit-specific genes to meet the demands of new stimulation or activity. Understanding the molecular repertoire and its dynamic interactions will lead to a deeper understanding of mechanisms that trigger and sustain chronic pain and other disorders of the nervous system. In terms of behavioral studies, we have begun to dissect out the role of the lightly myelinated, rapidly conducting A-delta neuron using an infrared diode laser stimulator. These studies show that A-delta neurons are sensitized in inflammatory pain, similar to C-fibers, and have led to the hypothesis that they are mediators or triggers for breakthrough pain in osteoarthritis or cancer. We also have determined that the A-delta fibers are the most sensitive to RTX axotomy, possibly because the lesion occurs at multiple nodes of Ranvier that the cell cannot repair easily. We shall be examining this issue in more detail. Becauese the stimulus is so short this allows for detailed studies of sensory motor integration in the first few milliseconds after stimulation,before compensatory pain control processes can intervene. These types of fast transient stimuli are often the stimuli that trigger episodes of breakthrough pain. Early Translational Investigations: A final set of studies concerns the identification of small chemicals that act as positive allosteric modulators (PAMs) of TRPV1 activation by pH or vanilloid agonists. Our human cancer study showed that TRPV1 is one of the most important transducers of painful stimuli and understanding how the ion channel can be blocked, activated or sensitized is fundamental to understanding pain. We have identified a new action on TRPV1 via screening of small molecule libraries. We discovered compounds that enhance the activation of TRPV1 upon agonist binding to the orthosteric site or by elevated H+ ions. These studies suggest allosteric modulation of the TRPV1 ion channel open state that may be accessed for pain transmission as well as the existence of a new class of pharmacological agents for pain modulation and pain control. Over the past year we screened the 300,000 compound Molecular Probes library and identified over 100 direct agonists and over 900 PAMs. Clearly not all of these single-point determinations are correct and we are in the process of rescreening for validation. Secondary screens for specificity will use other TRP and ligand activated ion channels coupled with electrophysiological recordings and Ca-45 uptake. We hope to obtain new probes for TRPV1 functional studies and new leads for PAMs that can be used for pain control. These studies have progressed to the point where we are synthesizing analogs of the lead PAMs for evaluation.