Raltegravir permeability across blood-tissue barriers and the potential role of drug efflux transporters

Abstract

The objectives of this study were to investigate raltegravir transport across several blood-tissue barrier models and the potential interactions with drug efflux transporters. Raltegravir uptake, accumulation, and permeability were evaluated in vitro in (i) P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance-associated protein 1 (MRP1), or MRP4-overexpressing MDA-MDR1 (P-gp), HEK-ABCG2, HeLa-MRP1, or HEK-MRP4 cells, respectively; (ii) cell culture systems of the human blood-brain (hCMEC/D3), mouse blood-testicular (TM4), and human blood-intestinal (Caco-2) barriers; and (iii) rat jejunum and ileum segments using an in situ single-pass intestinal perfusion model. [(3)H]Raltegravir accumulation by MDA-MDR1 (P-gp) and HEK-ABCG2-overexpressing cells was significantly enhanced in the presence of PSC833 {6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-L-valine-cyclosporine}, a P-gp inhibitor, or Ko143 [(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester], a BCRP inhibitor, suggesting the inhibition of a P-gp- or BCRP-mediated efflux process, respectively. Furthermore, [(3)H]raltegravir accumulation by human cerebral microvessel endothelial hCMEC/D3 and mouse Sertoli TM4 cells was significantly increased by PSC833 and Ko143. In human intestinal Caco-2 cells grown on Transwell filters, PSC833, but not Ko143, significantly decreased the [(3)H]raltegravir efflux ratios. In rat intestinal segments, [(3)H]raltegravir in situ permeability was significantly enhanced by the concurrent administration of PSC833 and Ko143. In contrast, in the transporter inhibition assays, raltegravir (10 to 500 μM) did not increase the accumulation of substrate for P-gp (rhodamine-6G), BCRP ([(3)H]mitoxantrone), or MRP1 [2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)] by MDA-MDR1 (P-gp)-, HEK-ABCG2-, or HeLa-MRP1-overexpressing cells, respectively. Our data suggest that raltegravir is a substrate but not an inhibitor of the drug efflux transporters P-gp and BCRP. These transporters might play a role in the restriction of raltegravir permeability across the blood-brain, blood-testicular, and blood-intestinal barriers, potentially contributing to its low tissue concentrations and/or low oral bioavailability observed in the clinic setting.

FIG 1

Raltegravir interactions with ABC transporters. (A) Effect of raltegravir on R-6G accumulation by MDA-MDR1 (P-gp) monolayer cells. The cellular accumulation of R-6G (1 μM) was determined at 60 min by exposing confluent MDA-MDR1 (P-gp) cells to R-6G in assay buffer with or without the P-gp inhibitor CsA (25 μM) or raltegravir (10 to 500 μM). (B) Effect of raltegravir on [³H]mitoxantrone accumulation by HEK-ABCG2-overexpressing cells. The accumulation of [³H]mitoxantrone (20 nM) at 120 min was measured in the absence (control) or presence of BCRP inhibitor (5 μM Ko143) or raltegravir (10 to 100 μM), all in the presence of 2 μM PSC833. (C) Effect of raltegravir on BCECF accumulation by HeLa-MRP1 monolayer cells. The cellular accumulation (at 120 min) of BCECF was determined by exposing confluent HeLa-MRP1 cells to 5 μM BCECF-AM, with or without the MRP inhibitor MK571 (10 μM) or raltegravir (10 to 500 μM), all in the presence of 2 μM PSC833. The results are expressed as the mean ± standard error of the mean (SEM) from at least three independent experiments, with each data point in an individual experiment representing triplicate measurements. Statistical significance was assessed by applying the unpaired two-tailed Student's t test analysis. *, P < 0.05.

FIG 2

Interactions of raltegravir with ABC transporters in overexpressing cell lines. Shown is the accumulation of [³H]raltegravir by MDA-WT/MDA-MDR1 (P-gp) cells (A), HEK-WT/HEK-ABCG2 cells (B), and HeLa-WT/HeLa-MRP1 cells (C). The accumulation of 1 μM [³H]raltegravir (at 120 min) was measured in cell monolayers in the absence or presence of the inhibitors for P-gp (5 μM PSC833 and 25 μM CsA), BCRP (5 μM Ko143 and 10 μM FTC), or MRP1 (5 μM MK571). The results are expressed as mean ± SEM from at least three independent experiments, with each data point in an individual experiment representing triplicate measurements. Statistical significance was assessed applying one-way analysis of variance (ANOVA), followed by Bonferroni's correction for a test of repeated measures. *, P < 0.05.

FIG 3

(A) Time profile of [³H]raltegravir in hCMEC/D3 cells. The uptake of 1 μM [³H]raltegravir by the hCMEC/D3 confluent monolayer cells was measured over time at 37°C. The inset shows the linearity of initial uptake up to 3 min. (B) Accumulation of 1 μM [³H]raltegravir (for 120 min) by hCMEC/D3 cells in the absence or presence of inhibitors of P-gp (5 μM PSC833), BCRP (5 μM Ko143), or MRPs (5 μM MK571). The results are expressed as mean ± SEM from at least three independent experiments, with each data point in an individual experiment representing triplicate measurements. *, P < 0.05.

FIG 4

(A) Time profile of [³H]raltegravir in TM4 cells. The uptake of 1 μM [³H]raltegravir by the TM4 confluent monolayer cells was measured over time at 37°C. The inset shows the linearity of initial uptake up to 3 min. (B) Accumulation of 1 μM [³H]raltegravir (120 min) by TM4 cells in the absence or presence of inhibitors of P-gp (5 μM PSC833), Bcrp (5 μM Ko143) or Mrps (5 μM MK571). The results are expressed as mean ± SEM from at least three independent experiments, with each data point in an individual experiment representing triplicate measurements. *, P < 0.05.

FIG 5

Raltegravir accumulation and permeability in Caco-2 cells: interactions with ABC transporters. (A) Time profile of [³H]raltegravir in Caco-2 cells. The uptake of 1 μM [³H]raltegravir by the Caco-2 confluent monolayer cells was measured over time at 37°C. The inset shows the linearity of initial uptake up to 2 min. (B) Accumulation of 1 μM [³H]raltegravir (120 min) by Caco-2 cells in the absence or presence of inhibitors of P-gp (5 μM PSC833), BCRP (5 μM Ko143), or MRPs (5 μM MK571). (C) Raltegravir intestinal permeability across Caco-2 monolayer cells in the apical-to-basolateral and basolateral-to-apical directions. The flux of raltegravir was determined by introducing 1 μM [³H]raltegravir into the donor compartment (apical or basolateral) and measuring its appearance over time in the receiver compartment (basolateral or apical, respectively) in the absence or presence of 5 μM PSC833 or 5 μM Ko143 to calculate the apparent permeability coefficients (P_app). The results are expressed as the mean ± SEM from at least three independent experiments, with each data point in an individual experiment representing triplicate measurements. Statistical significance was assessed applying the one-way ANOVA, followed by Bonferroni's correction for a test of repeated measures. *, P < 0.05.

FIG 6

Raltegravir in situ intestinal permeability and interactions with ABC transporters. Effect of P-gp inhibitor (PSC833) (A), Bcrp inhibitor (Ko143) (B), or both P-gp and Bcrp inhibitors (PSC833 plus Ko143) (C) on raltegravir intestinal permeability, as measured by in situ single-pass perfusion of Sprague-Dawley rat intestinal segments, was quantified. The effective permeability (P_eff) of raltegravir was measured in the jejunum or ileum segment of rat intestine in each animal by in situ single-pass perfusion. The intestinal segment was perfused with 10 μM [³H]raltegravir at pH 6.5, first without inhibitor (first hour, control) and then in the presence of 5 μM PSC833, 5 μM Ko143, or both (second hour) to determine the effect of these inhibitors on raltegravir permeability. The raltegravir P_eff values measured in the presence of the P-gp inhibitor PSC833, the Bcrp inhibitor Ko143, or both the P-gp and the Bcrp inhibitors, PSC833 + Ko143, were compared to those of the control period in the same animal by paired two-tailed Student's t test analysis using five Sprague-Dawley rats per group.