Abstract
The SLC22A1 influx transporter is expressed on the basolateral membrane of hepatocytes and is involved in the excretion of numerous cations. Inhibition of SLC22A1 by several antiretrovirals, such as the protease inhibitor darunavir, has not previously been determined. In order to better understand and predict drug-SLC22A1 interactions, a range of antiretrovirals were screened for SLC22A1-associated inhibition and transport. Stable SLC22A1-expressing KCL22 cells were produced previously by nucleofection. Control KCL22 cells were transfected with the empty vector pcDNA3.1. Accumulation of tetraethylammonium (5.5 μM, 30 min) was determined in SLC22A1-expressing and mock-transfected cells with and without 50 μM of SLC22A1 inhibitor prazosin, or 50 μM of each antiretroviral drug. SLC22A1 IC50 values for efavirenz, darunavir, and prazosin were determined. Cellular accumulation of efavirenz and darunavir was also assessed in SLC22A1-expressing KCL22 cells and reversibility of this accumulation was assessed using prazosin. Tetraethylammonium accumulation was higher in SLC22A1-expressing cells compared to mock-transfected cells (10.6 ± 0.8 μM vs. 0.3 ± 0.004 μM, p = 0.009) and was significantly reduced in SLC22A1-expressing cells when co-incubated with all antiretrovirals tested except atazanavir, lamivudine, tenofovir, zidovudine, and raltegravir. Particularly noticeable was the predominance of SLC22A1 inhibitors in the protease inhibitor and non-nucleoside reverse transcriptase inhibitor classes. Absolute SLC22A1 IC50 values for efavirenz, darunavir, and prazosin were 21.8, 46.2, and 2.8 μM, respectively. Efavirenz accumulation was higher in SLC22A1-expressing cells compared to mock-transfected cells (17% higher, p = 0.009) which was reversed using prazosin, whereas no difference was observed for darunavir (p = 0.86). These data inform the mechanistic basis for disposition, drug-drug interactions and pharmacogenetic candidate gene selection for antiretroviral drugs.
Highlights
Eukaryotic drug transporting proteins play an important role in the absorption, distribution, and elimination of numerous antiretrovirals used in Human Immunodeficiency Virus (HIV) therapy
Results are given as tetraethylammonium concentration in cells after 30 min incubation ± SD
SLC22A1 inhibition was achieved using numerous antiretrovirals: all protease inhibitors apart from atazanavir were able to reduce tetraethylammonium accumulation. These findings agree with previous data, where saquinavir, indinavir, ritonavir, and nelfinavir were able to reduce tetraethylammonium accumulation in SLC22A1-overexpressing Hela cells (Zhang et al, 2000) and atazanavir was found to have no SLC22A1-inhibiting capability in vitro (Jung et al, 2008)
Summary
Eukaryotic drug transporting proteins play an important role in the absorption, distribution, and elimination of numerous antiretrovirals used in Human Immunodeficiency Virus (HIV) therapy. The rationale of this study was to determine the inhibitory potential across antiretroviral classes for SLC22A1-mediated tetraethylammonium transport This was used to confirm past data and to investigate the effects of previously un-assessed antiretrovirals such as darunavir, lopinavir, and etravirine. The SLC22A1-mediated transport of darunavir and efavirenz, which both showed moderate inhibition of SLC22A1 activity in initial screens, has not previously been investigated and this was assessed. This information will aid in our understanding of factors responsible for antiretroviral disposition, and may provide explanations and possible solutions for drug interactions involving antiretrovirals and co-administered substrates of SLC22A1
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