Integrative And Molecular Studies Of Pain And Pain Control

Clinical Center

Investigators

Linked publications, trials & patents

Abstract

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 axonal projections peripherally to skin and deep tissues and make connections centrally to neurons within the dorsal spinal cord. This is the first CNS site of synaptic information processing for pain. Mechanisms of nociception are investigated through models of pathophysiological tissue 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 treating severe pain. Studies with the TRPV1 agonist resiniferatoxin (RTX) have resulted in a Phase I clinical trial in patients with intractable pain from advanced cancer. In this cycle we have begun a clinical trial of RTX to treat neuropathic pain from a nerve injury. Agonist activation of TRPV1 causes an influx of sodium and calcium ions through the pore of TRPV1. The unique property of RTX for therapeutic purposes is its pseudo-irreversible binding to TRPV1 which props open the ion channel causing an intracellular calcium overload and local, cell specific 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 when applied near the neuronal perikarya. At the same time non-TRPV1 expressing neurons are not affected by RTX, thereby sparing most forms of somatosensation and motor function. We have shown in previous studies that RTX produces very effective pain control in a wide variety of pre-clinical models. Now, in the human clinical trial, we administer RTX into the lumbar CSF (intrathecal). To date, we have treated 19 patients with pain from advanced cancer. This Phase I study is provisionally complete although we plan to examine a higher dose tier. The results are being readied for publication. Earlier we published studies of RTX injections around or directly into sensory ganglia. These preclinical experiments formed the basis of our clinical trial protocol to treat localized chronic pain by periganglionic RTX injection. As a proof-of-concept study, we published a case report using intraganglionic lidocaine in a patient with unilateral phantom limb pain of 30 yrs duration. This treatment blocked the patients pain completely for 2 hrs as expected and shows that blocking nociceptive inputs to the CNS is an extraordinarily effective analgesic manipulation. Our goal is to replace lidocaine with RTX and obtain long-term pain control in regional cancer and other types of localized pain problems. This can be achieved with a single injection. Other peripheral routes of RTX administration include injection into skin, joints, nerve bundles, or topically. Preclinical studies show that analgesia by these routes is long-lasting but reversible since the peripheral nerve endings regrow. Human protocols for post-operative incision and Mortons neuroma pain indications were generated in collaboration with the Thoracic and Oncologic Surgery Branch, NCI, and local podiatrists, respectively. The initial post-operative study evaluates preemptive treatment with RTX injected subcutaneously and preemptively along the track of the incision. This protocol passed CC scientific review and was evaluated by the FDA which asked for additional experiments related to wound healing (pending). The protocol for treating Mortons neuroma will be by perineural infiltration around the nerve just proximal to the neuroma. This protocol passed CC scientific review, passed review of the IND at the FDA and the clinical protocol was approved by the NIH IRB. We are poised to treat the first patient. Early Translational Investigations: In this cycle we extended our systems approach for integrated RNA-Seq and lipidomics to humans to a human intraoperative tissue biopsy protocol. We obtained samples from surgical wound margins over time. This longitudinal tissue procurement was completed, and we are presently analyzing the samples transcriptomically, anatomically, and lipidomically. This is nearing completion as we finish the anatomical evaluations of incised tissue with in situ hybridization and immunofluorescence staining. During this cycle we have made a strong effort to incorporate direct in human studies of nociceptive molecular biology. This was prompted by our repeated observations of species differences in gene expression in dorsal root ganglion (DRG) and spinal cord. Most of the human skin, nerve, DRG and spinal cord tissues are recovered from organ donors or obtained by ourselves at autopsy of NIH patients. These tissues are being analyzed intensively by whole tissue sequencing, single nucleus sequencing, in situ hybridization and immunofluorescence staining for proteins. In collaboration with the Dr. Ashok Kulkarnis lab, we have examined DRG from patients with painful diabetic peripheral neuropathy (DPN). The first paper describing these results was published in the previous cycle. We observed a loss of a select subpopulation of nociceptors suggesting a dynamic interplay between hyperexcitability and neurotoxicity. We emphasize the importance of enhancing the research focus on human nociceptive tissues in a recent commentary (PMID: 35504570). Basic Pain Mechanisms: Underlying the translational and clinical studies are investigations of neuronal function, behavior, and molecular biological 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 to obtain a comprehensive, quantitative foundational molecular understanding of nociceptive processes related to inflammation, surgical incision, nerve injury, and most recently burn injury via collaboration. One of the main methods used is RNA-Seq where we sequence all of the mRNAs in a given tissue or cell population. Our work now integrates RNA-Seq into most of our investigations. Two other main methods are anatomical: multiplex fluorescence in situ and immunofluorescence of tissues in the nociceptive circuit. By combining techniques, we now investigate humans with genetic variations that affect pain sensitivity. At present we are investigating a group of rare individuals with a copy number variant (CNV) involving a specific gene locus. Individuals with three copies of this region exhibit profound decreases pain sensitivity. Using RNA-Seq we identified one gene in the CNV which contains 25 genes, that is an excellent candidate for mediating analgesic actions when overexpressed. The results are both compelling and informative and define a previously unidentified genetic mechanism for governing pain sensitivity. These findings have been submitted for publication. These approaches are especially useful for evaluating new candidate molecules for development of non-opioid analgesic agents.