br Membrane transporters as a novel
Membrane transporters as a novel therapeutic target in pediatric TBI The concept of targeting membrane transporters began with identifying a neuroprotective drug and a corresponding transporter inhibitor. Somewhat serendipitously, we discovered that N-acetylcysteine (NAC), a cysteine donor for GSH synthesis and antioxidant, was a transporter substrate for the probenecid-inhibitable transporters SLC22A6 (OAT1) and SLC22A8 (OAT3) (Hagos et al., 2017). Using juvenile (PND 17) Sprague-Dawley rats, we showed that administration of NAC in the presence of probenecid increased the plasma and membrane transport area under the concentration curve by 1.65- and 2.41-fold, respectively, vs. NAC administered alone. Additionally, using HEK-293 cells that overexpress SLC22A6 and SLC22A8 transporters, we showed that NAC exhibits time- and concentration-dependent uptake vs. mock-transfected cells, inhibitable by probenecid. These data provided insight regarding how NAC bidirectionally crosses biological barriers and identified a novel therapeutic strategy to increase NAC exposure (and possibly other drugs) in the brain by inhibiting membrane transporters (Fig. 1). Since this study examined membrane transporter function in the developing brain, similar studies in the mature adult brain are warranted. The combination strategy of co-administering the transporter inhibitor, probenecid, and its recently discovered substrate, NAC, had several theoretical advantages. First, by inhibiting OATs, probenecid could increase the exposure of NAC in plasma and importantly in the brain. Second, NAC, chemically a thiol, can act as an antioxidant by reacting with and scavenging a number of radicals including OH, NO2, CO3, and thiyl radicals (Samuni et al., 2013). Third, by inhibiting ABCC transporters such as ABCC1 which are involved in the efflux of GSH and its conjugates from the brain, probenecid could increase the overall GSH pool available for neurons and other brain cells. Fourth, both NAC and probenecid are FDA approved drugs with favorable safety profiles, hence providing an opportunity for rapid translation to the clinic. Fifth, the combination therapy targets multiple processes vis-à-vis TBI and oxidative stress. We verified brain exposure of systemically administered NAC and probenecid in a double-blind, placebo controlled Phase I study in children with severe TBI (Pro-NAC Trial) (Clark et al., 2017). In the Pro-NAC trial, fourteen patients (n = 7/group) received probenecid (25 mg/kg load, then 10 mg/kg/dose q6h × 11 doses) and NAC (140 mg/kg load, then 70 mg/kg/dose q4h × 17 doses), or placebos via naso/orogastric tube. Serum and CSF samples were collected for 96 h after injury. Treatment resulted in detectable CSF concentrations of NAC and probenecid throughout the treatment period and was not associated with undesirable effects after TBI. This provided important proof-of-principle for transporter modulation as a novel therapeutic strategy for pediatric TBI. In a follow-up study, our group utilized metabolomics, a powerful tool for comprehensively profiling metabolites and altered biochemical processes, to evaluate the CSF metabolome in children with severe TBI treated with probenecid and NAC (Hagos et al., 2018). The goal was to determine whether the combination modulated glutathione metabolism and related pathways after TBI (the purported mechanism of action) to evaluate pharmacodynamic target engagement. The CSF metabolome was analyzed at 24 h post-injury in seven Pro-NAC and five placebo treated patients as well as five control subjects. A combination of metabolomics and pathway/network analyses showed that seven glutathione-centered pathways and two glutathione-centered networks were enriched in the CSF of Pro-NAC treated vs. placebo treated TBI patients. Several pathways/networks consisting of components that are known substrates of probenecid-inhibitable membrane transporters such as prostaglandins, kynurenate and urate were also enriched, providing additional mechanistic validation. This first of its kind “neuropharmacometabolomics” assessment revealed alterations in known and previously unidentified metabolic derangements after TBI, and supported target engagement of the combination of probenecid and NAC in the treatment of severe TBI in children. The study also underscored the value of metabolomics as a tool to understand the range of substrates and functions of transporters, and in identifying markers of efficacy and toxicity for drugs that target transporters in TBI.