Impact of drug transporters on cellular resistance towards saquinavir and darunavir

The membrane-associated drug transporter P-glycoprotein (P-gp) plays an essential role in drug efflux from the brain. Induction of this protein at the blood-brain barrier (BBB) could further affect the ability of a drug to enter the brain. At present, P-gp induction mediated by antiretroviral drugs at the BBB has not been fully investigated. Since P-gp expression is regulated by ligand-activated nuclear receptors, i.e., human pregnane X receptor (hPXR) and human constitutive androstane receptor (hCAR), these receptors could represent potential pathways involved in P-gp induction by antiretroviral drugs. The aims of this study were (i) to determine whether antiretroviral drugs currently used in HIV pharmacotherapy are ligands for hPXR or hCAR and (ii) to examine P-gp function and expression in human brain microvessel endothelial cells treated with antiretroviral drugs identified as ligands of hPXR and/or hCAR. Luciferase reporter gene assays were performed to examine the activation of hPXR and hCAR by antiretroviral drugs. The hCMEC/D3 cell line, which is known to display several morphological and biochemical properties of the BBB in humans, was used to examine P-gp induction following 72 h of exposure to these agents. Amprenavir, atazanavir, darunavir, efavirenz, ritonavir, and lopinavir were found to activate hPXR, whereas abacavir, efavirenz, and nevirapine were found to activate hCAR. P-gp expression and function were significantly induced in hCMEC/D3 cells treated with these drugs at clinical concentrations in plasma. Together, our data suggest that P-gp induction could occur at the BBB during chronic treatment with antiretroviral drugs identified as ligands of hPXR and/or hCAR.

Section: Discussionsupporting

confidence: 90%

Induction of P-Glycoprotein by Antiretroviral Drugs in Human Brain Microvessel Endothelial Cells

Chan

Patel

Cummins

et al. 2013

“…P-gp/ABCB1 and BCRP/ABCG2 inhibition was quantified by calcein and pheophorbide A efflux assays as described previously (23, 26). We used the growth inhibition assay in MDCKII cells overexpressing human P-gp/ABCB1 (7), BCRP/ABCG2 (17), and MRP1-3/ABCC1-3 (8) as a surrogate for substrate characteristics of etravirine as it has been described for other antiretroviral drugs (3,13,26). The induction assay, quantification of mRNA expression by real-time reverse transcriptase PCR (RT-PCR), and data evaluation by calibrator-normalized relative quantification with efficiency correction were also performed as published earlier (26).…”

mentioning

confidence: 99%

“…11609) from Tibotec, Inc. Other materials were used as described previously (13). Etravirine was tested for cytotoxic effects prior to P-gp/ABCB1 and BCRP/ABCG2 inhibition assays with a cytotoxicity detection kit (Roche Applied Science, Mannheim, Germany) according to manufacturer's instructions.…”

mentioning

confidence: 99%

Interaction Potential of Etravirine with Drug Transporters Assessed In Vitro

Zembruski

Haefeli

Weiß

2011

Etravirine is a novel nonnucleoside reverse transcriptase inhibitor (NNRTI) for the treatment of HIV-1 infections. ABC transporters potentially mediate clinically relevant drug-drug interactions. We assessed substrate characteristics and the inhibitory and inductive potential of etravirine on ABC transporters. Etravirine did not inhibit P-gp/ABCB1 and was not transported by the tested ABC transporters but was a potent inhibitor of BCRP/ABCG2. Etravirine induced several ABC transporters, especially BCRP/ABCG2. These data demonstrate that etravirine has the potential for drug-drug interactions by modulation of expression and function of several ABC transporters.Etravirine is a novel nonnucleoside reverse transcriptase inhibitor (NNRTI) for the treatment of HIV-1 infections. It is active against wild-type and some NNRTI-resistant HIV strains (1, 2) and offers a new treatment option for treatmentexperienced patients. Pharmacokinetic drug-drug interactions considerably influence efficacy and safety of antiretroviral therapy. Interactions might lower concentrations of antiretrovirals below therapeutic concentrations (5,12,14) and cause treatment failure and viral resistance. Interactions may also increase drug exposure and augment toxicity.The main mechanisms of interaction in antiretroviral combination therapy involve the drug-metabolizing cytochrome P450 enzymes (CYPs) as well as efflux and uptake transporters. Crucial efflux transporters are several ATP-binding cassette (ABC) transporters that have been identified as important interaction sites of antiretroviral drugs (9-11). Relevant uptake transporters are the organic anion-transporting polypeptides (OATPs/SLCOs) (6,16,20). Information on interactions of etravirine is sparse. We therefore investigated whether etravirine is a substrate of P-gp/ABCB1, BCRP/ABCG2, MRP1/ ABCC1, MRP2/ABCC2, or MRP3/ABCC3 and whether it inhibits P-gp/ABCB1 and BCRP/ABCG2. Furthermore, we investigated etravirine's potency to induce ABC transporters, important OATPs/SLCOs, CYPs, and the transcription factor pregnane X receptor.Etravirine was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, as etravirine TMC125 (catalog no. 11609) from Tibotec, Inc. Other materials were used as described previously (13). Etravirine was tested for cytotoxic effects prior to P-gp/ABCB1 and BCRP/ABCG2 inhibition assays with a cytotoxicity detection kit (Roche Applied Science, Mannheim, Germany) according to manufacturer's instructions. Cytotoxic concentrations were excluded in the respective assays. P-gp/ABCB1 and BCRP/ABCG2 inhibition was quantified by calcein and pheophorbide A efflux assays as described previously (23, 26). We used the growth inhibition assay in MDCKII cells overexpressing human P-gp/ABCB1 (7), BCRP/ABCG2 (17), and MRP1-3/ABCC1-3 (8) as a surrogate for substrate characteristics of etravirine as it has been described for other antiretroviral drugs (3,13,26). The induction assay, quantification of mRNA expression by real-time reverse transc...

“…Polymorphisms in the multidrug resistance gene (MDR1) that encodes the P glycoprotein transporter can influence intracellular concentrations of raltegravir and other agents and therefore potentially antiretroviral (ARV) efficacy (27). P glycoprotein concentrations increase with the luteal phase of the menstrual cycle (12); thus, we hypothesized that P glycoprotein may efflux more raltegravir across the cell membrane in the luteal phase (a time of higher progesterone) rather than proliferative phase of the menstrual cycle (a time of lower progesterone) and decrease intracellular raltegravir concentrations.…”

Section: Discussionmentioning

confidence: 99%

Correlation between Plasma, Intracellular, and Cervical Tissue Levels of Raltegravir at Steady-State Dosing in Healthy Women

Mitchell

Roemer

Nkwopara

et al. 2014

Raltegravir is an antiretroviral with potential value for preexposure prophylaxis (PrEP) against HIV, but the intracellular pharmacokinetics in genital tissue have not been described. In this study, healthy, HIV-uninfected nonpregnant women took 400 mg of raltegravir twice daily for 22 days. On day 8, 15, and 22, blood was collected 0, 4, 6, 8, and 12 h and cervical biopsy specimens taken 0, 6, and 12 h after raltegravir dosing. Plasma and intracellular raltegravir concentrations in peripheral blood mononuclear cells (PBMC) and cervical tissue were measured by tandem mass spectrometry. Linear mixed effects models evaluated correlations between different sample types, as well as differences in concentration between phases of the menstrual cycle. Ten women were enrolled: 9 completed all three visits and 1 completed two visits. The age (mean ؎ standard deviation) of participants was 30 ؎ 8 years. Trough plasma concentrations of raltegravir 12 h after a directly observed dose were above the HIV 95% inhibitory concentration (IC 95 ) of 33 nM (14.6 ng/ml) in 96% of measurements, compared to 67% of PBMC and 89% of cervical tissue trough values. Across all measurements, only 2% (3/135) of plasma values fell below the IC 95 , compared to 10% (13/135) for PBMC and 6% (5/81) for cervical tissue. There was no impact of menstrual phase on raltegravir concentrations. In conclusion, cervical tissue raltegravir concentrations were no greater than plasma concentrations, and ϳ10% of all cervical tissue trough values were below the IC 95 , making the current twice-daily formulation of raltegravir impractical for PrEP. P reexposure prophylaxis (PrEP) using antiretroviral medication has proven effective for infants and adults to prevent HIV acquisition. Studies have shown significant reduction in risk of HIV acquisition in high-risk infants born to HIV-infected women (1, 2), men who have sex with men (3), and HIV-1 serodiscordant heterosexual partners (4). The ideal agent for PrEP should have a long half-life and high concentrations in the genital tract at the site of viral exposure, should be nontoxic, and should not be part of first-line HIV regimens. Tenofovir was chosen as a potential PrEP agent in part because it fulfills almost all of these criteria (5). However, tenofovir is part of the first-line HIV treatment regimen in many parts of the world (6), raising concerns about the impact of development of drug resistance if HIV transmission occurs while taking PrEP.Raltegravir is an HIV integrase inhibitor which blocks strand transfer of viral DNA and integration into the host's genome and has minimal adverse effects (7). Genital fluid concentrations of raltegravir are higher than plasma concentrations, an attractive quality for a PrEP agent (8, 9). However, no studies have examined intracellular tissue concentrations of raltegravir in the genital tract, where the level of the drug would be critical to ensuring efficacy (10). Raltegravir is a substrate for P glycoprotein transporter, which is expressed in cervical tissue (11) and...