Cultured CD4T cells and primary human lymphocytes express hOATPs: intracellular accumulation of saquinavir and lopinavir

Abstract

Background and purpose: Drug efflux tranporters (P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP)) limit the cellular uptake of human immunodeficiency virus protease inhibitors but the contribution of influx transporters in cells that (over)express P-gp or MRP is less clear. Here, we studied the expression of one influx transporter system, human organic anion-transporting polypeptide (hOATP), in some T-cell lines (CEM, CEM(VBL), CEM(E1000)) and in peripheral blood mononuclear cells (PBMCs) and examined the effects of manipulation of influx/efflux transporters on the uptake of saquinavir and lopinavir.

Experimental approach: The expression of hOATPs was studied by PCR. We used hOATP substrate or inhibitor (estrone-3-sulphate (E-3-S) or montelukast, respectively) and inhibitors of P-gp (XR9576) and MRP (MK571 and frusemide) to study functional interactions between influx and efflux transporters in the uptake of saquinavir and lopinavir. Lipophilicity of the drugs was measured by octanol/saline partition coefficient.

Key results: CEM cells, their variants and PBMCs express various hOATP isoforms, with OATP3A1 detected in all of the cells. MK571, XR9576 and frusemide increased the uptake of saquinavir and lopinavir. E-3-S and montelukast reduced the uptake of saquinavir and lopinavir in some, but not all, of the cells. Pretreatment of the cells with MK571, XR9576 or frusemide, followed by E-3-S co-incubation reduced the cellular accumulation of saquinavir and lopinavir. Lopinavir is much more lipophilic than saquinavir.

Conclusions and implications: Human OATPs, MRP, P-gp and lipophilicity determine the cellular uptake and retention of saquinavir and lopinavir. These data may have important implications for drug-drug interactions, drug safety and efficacy.

Figure 1

Polymerase chain reaction products to show that CEM, its variant cells and peripheral blood mononuclear cells (PBMCs) express various organic anion-transporting polypeptide (OATP) isoforms. PCR products were separated on 2% agarose gels (1 × TBE) and stained with ethidium bromide (see Materials and methods section for details). Lane A, negative control (water); lane B, positive control (human liver); lane C, PBMCs; lane D, CEM; lane E, CEM_VBL; and lane F, CEM_E1000. Arrows indicate base pair (bp) sizes for each OATP.

Figure 2

Effects of the organic anion-transporting polypeptide (OATP) substrate, estrone-3-sulphate (E-3-S) and OATP inhibitor montelukast on the accumulation (CAR) of saquinavir (SQV) and lopinavir (LPV) in CEM, CEM_VBL, CEM_E1000 cells and PBMCs. Concentration-dependent effects of (a) E-3-S on the CAR of saquinavir, (b) E-3-S on the CAR of lopinavir, (c) montelukast on the CAR of saquinavir and (d) montelukast on the CAR of lopinavir in CEM and its variant cells. Concentration-dependent effects of E-3-S and montelukast (at 50 μM) on the CAR of (e) saquinavir and lopinavir in PBMCs. The data on montelukast (at 50 μM) were generated from four other PBMC samples different from those used to assay the effects of E-3-S in the same figure. Note that the volume of each CEM cell is assumed as 1 pL. CAR, cellular accumulation ratio, is the amount of radiolabelled drug associated with the cell pellets relative to the same volume of supernatant. Each bar represents the mean±s.d. (n=4). *P<0.05, **P<0.01, ***P<0.0001 compared with controls. E-3-S, estrone-3-sulphate; PBMC, peripheral blood mononuclear cells; CAR, cellular accumulation ratio.

Figure 3

The impact of E-3-S in reversing the effects of efflux modulators (XR9576, MK571 and frusemide) on the cellular accumulation of saquinavir (SQV) and lopinavir (LPV) in CEM, CEM_VBL, CEM_E1000 cell lines and in PBMCs. Untreated cells are presented for comparative purposes only. (a) Effects of E-3-S (at 30 and 100 μM) co-incubation on the CAR of saquinavir after pretreatment of CEM and its variant cells with 50 μM MK571. (b) Effects of E-3-S (at 30 and 100 μM) co-incubation on the CAR of saquinavir after pretreatment of CEM and its variant cells with 0.1 μM XR9576 and (c) effects of pretreatment of the cells with 50 μM MK571 followed by E-3-S (at 30 and 100 μM) on the CAR of lopinavir in CEM and its variant cells. (d) Effects of E-3-S co-incubation after pretreatment of the cells with 0.1 μM XR9576 in CEM and its variant cells. (e) Effects of E-3-S (at 30 and 100 μM) co-incubation on the uptake of saquinavir and lopinavir in PBMCs pretreated with 50 μM frusemide. Significance between cells incubated with inhibitor and cells coincubated with E-3-S after pretreatment with inhibitor was tested by Shapiro–Wilk followed by Kruskal–Wallis tests. Each bar represents the mean±s.d. (n=3–4, with four incubations per treatment). *P<0.05, ^**P<0.01, ***P<0.0001 compared with inhibitor-treated controls. E-3-S, estrone-3-sulphate; PBMC, peripheral blood mononuclear cells; CAR, cellular accumulation ratio.