There is a sense of urgency to move forward with pharmacological therapies that improve outcome after brain injury. Notably, traumatic brain injury (TBI) remains the leading cause of death and disability amongst children and young adults. To date, all clinical trials exploring single agents or therapies for TBI have failed. Unique impediments to effective treatment for TBI are ATP-binding cassette transporters and solute carriers on the blood-brain (BBB) and blood-cerebrospinal fluid (CSF) barriers which limit bioavailability of drugs to normal and injured brain by active and rapid re-uptake and export of drug back into blood. These barriers are often cited as a major explanation for the failure of clinical drug trials for CNS injury, and clearly limit the therapeutic indications for many drugs otherwise effective in non-CNS diseases. Membrane transporters include the multidrug resistance proteins, multidrug resistance-associated proteins, and organic anion transporters. Importantly, pharmacological inhibitors of these transporters have existed for decades, used to enhance bioavailability of drugs that are membrane transporter substrates. A prototype membrane transporter inhibitor is probenecid. Probenecid is currently in clinical use to treat uric acidemia, and was developed during World War II to increase the bioavailability of penicillin to wounded soldiers. The combination of probenecid or any membrane transporter inhibitor with a potentially neuroprotective substrate has never been evaluated for the treatment of TBI, although probenecid alone has been used (safely) to evaluate brain-CSF concentrations of organic acids after TBI. Since these inhibitors utilize reduced glutathione (GSH) to co-export substances out of cells, probenecid also maintains intracellular stores of GSH, a prominent endogenous antioxidant. As such, probenecid itself may be neuroprotective by maintaining endogenous antioxidant reserves (AOR), complimenting its capacity to improve brain bioavailability of exogenous treatments by reducing efflux across membrane barriers. The PIs have provocative preliminary data showing that the combination of probenecid and the FDA-approved antioxidant N-acetylcysteine (NAC), whose CNS use is limited by poor brain bioavailability, synergistically restore total AOR in injured brain after TBI in mice. This coupled with the PIs' previous report showing that total AOR in CSF from patients are reduced by > 50% after TBI, provides compelling translational data in support of this combinational strategy. The PIs' hypothesis is that combinational strategies that include therapies that overcome membrane transport barriers will synergistically improve bioavailability and efficacy of both clinically used and novel therapies after TBI. Specific aims are to define the capacity of the combination of probenecid and NAC to synergistically reduce oxidative stress and improve neurological outcome in neurons after stretch-induced trauma in vitro and in mice after TBI in vivo; and to define the capacity of the combination of probenecid and NAC to safely and synergistically reduce oxidative stress in children with severe TBI. PUBLIC HEALTH RELEVANCE: Traumatic brain injury is a major cause of morbidity and mortality in adults, and is the leading cause of death in children. This proposal will evaluate the promising and clinically available combination of a membrane transport inhibitor (probenecid) and an antioxidant (N-acetylcysteine) for the capacity to synergistically improve outcome after TBI. This proposal includes contemporary in vitro and in vivo models of brain injury and a Phase I clinical safety/pharmacokinetics trial.