DESCRIPTION (provided by applicant): Pain afflicts over 116 million Americans annually and costs an estimated $635 billion dollars in medical treatments and lost productivity. The treatment of pain (both acute/post-surgical and chronic) often involves opioid analgesics. We have shown that peripheral inflammatory pain (PIP) promotes BBB dysfunction characterized by changes in expression and localization of efflux transporter and tight junction (TJ) proteins as well as increased paracellular permeability (i.e. leak). The clinical relevance of these changes i altered brain uptake of opioid analgesics. In this renewal we propose to test the novel hypothesis that BBB dysfunction during PIP which alters paracellular permeability and CNS delivery of opioid analgesics can be modulated by therapeutic targeting of pathology-induced changes in trafficking of P- glycoprotein (Pgp) and of TJ proteins occludin and claudins that are critical to the formation and integrity of TJ oligomeric protein assemblies. Two specific aims willtest this hypothesis. Aim 1: To determine how PIP-induced changes in Pgp trafficking result in increased Pgp drug efflux activity. In this aim we will identify components of Pgp storage compartment(s) isolated from cerebral microvessels (Aim 1A). We will also correlate changes in Pgp opioid efflux activity with changes in Pgp trafficking over a 1-72 hr time course (one-hit or rechallenge) (Aim 1B). Since production of reactive oxygen species (ROS) is a critical component of pain/inflammation we will evaluate the effect of administration of TEMPOL (i.e. 4-hydroxy-2266-tetramethylpiperidine-N- oxyl) a ROS scavenger on Pgp trafficking as well as brain uptake and analgesic efficacy of opioids over a 1-72 hr time course of PIP (one-hit or rechallenge) (Aim 1C). Aim 2: To examine how PIP-induced changes in TJ protein trafficking modulate paracellular BBB permeability. In this aim we will identify proteins associated with TJs isolated from cerebral microvessels (Aim 2A). We will also correlate changes in paracellular BBB permeability with changes in protein-protein interactions that cause changes in intracellular trafficking of occludin and claudins over a 1-72 hr time course of PIP (one-hit or rechallenge) (Aim 2B). We will then evaluate the effect of TEMPOL administration on occludin/claudin trafficking on BBB paracellular permeability to codeine and codeine efficacy over a 1-72 hr time course of PIP (one-hit or rechallenge). (Aim 2C). Our goal in this renewal is to discover novel therapeutic targets for treating both pain and pain-induced BBB dysfunction by defining the role of protein trafficking in promoting changes in BBB structure/function that lead to altered CNS drug delivery during PIP.