The data here comply with this knowledge somewhat, suggesting that nifurtimox appears to be a substrate of functional OATP systems, but OATs and MRP1 appear not

to be involved. This was demonstrated by use of the broad spectrum MRP, OAT and OATP inhibitor, probenecid; the MRP1 inhibitor, indomethacin, the OATP competitive inhibitor; TCA and the OAT competitive inhibitor, PAH. OATPs are bidirectional drug transporters ( Nozawa et al., 2005) and the increases in accumulation with the addition of TCA could provide some evidence for their role in nifurtimox transport. However, although indomethacin is commonly used as a MRP1 inhibitor, it has also been shown to be a substrate of OATPs and OATs, albeit at different concentration ranges than used here ( Parepally et al., 2006). Notably the changes in [3H]nifurtimox Transferase inhibitor accumulation were much smaller than those seen with the BCRP specific drugs and accumulation following ATP depletion, suggesting only minor roles for OATPs in comparison to the role played by BCRP. It is also important to point out that

some groups have found that probenecid is a BCRP substrate in vitro, and this is also a possible explanation for the increase in [3H]nifurtimox accumulation using this drug ( Merino et al., 2006). However, other groups have found no such evidence of BCRP/probenecid interaction ( Pan et al., 2007). It has also been see more reported that neither TCA ( Suzuki et al., 2003) nor indomethacin ( Elahian et al., 2010) interacts with BCRP. The concentrations of drugs used in this study were carefully chosen to follow those used in previous in vivo studies by our group, to be in line with the clinically relevant doses used in the field (with the anti-HAT Verteporfin concentration drugs) and to be in line with those reported in publications such as Matsson et al. 2009. These data and findings by other groups highlight that drugs

affecting transport activity must be used at specific concentrations to affect the targeted transport proteins. With combination therapy becoming the most promising method of clinical S2 HAT treatment, we also investigated whether other unlabelled anti-HAT drugs could modulate the accumulation of [3H]nifurtimox in human brain endothelium. In line with previous work by our group, an increase in [3H]nifurtimox accumulation was seen with the addition of 10 μM pentamidine. Pentamidine, a S1 acting drug, has been previously shown by our group to be a substrate for P-gp and is also transported by other transport proteins including MRP. This present study with P-gp inhibitors suggests that perhaps nifurtimox also interacts with other membrane transporters. BCRP and members of the OATPs could be candidates as indicated by their interactions with PhA, ko143, probenecid and PAH.