Becky Chandler

The effects of protease inhibitors and nonnucleoside reverse transcriptase inhibitors on p-glycoprotein expression in peripheral blood mononuclear cells in vitro.

Several antiretroviral compounds have been shown to be substrates for the efflux protein P-glycoprotein (P-gp) although few studies have investigated the effects of drug on expression of this protein. Here, an in vitro system has been adopted to investigate the effects of protease inhibitors (PIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) on P-gp expression in peripheral blood mononuclear cells (PBMCs). PBMCs isolated from healthy volunteers were incubated with 10 or 100 microM PI (saquinavir, ritonavir, lopinavir, indinavir, nelfinavir, amprenavir) or 10 microM NNRTI (efavirenz, nevirapine) for 72 hours. Surface P-gp expression was measured by flow cytometry and compared with vehicle-incubated controls. Toxicity was assessed by MTT assay and the effects of each compound were compared between individuals with differing genotypes at position 3435 of exon 26 of MDR1, which was assigned by restriction fragment length polymorphism. Significant increases in median P-gp expression were observed following incubation with 10 microM nelfinavir (10.2 versus 6.7% P-gp-positive cells) and efavirenz (10.0 versus 6.7% P-gp-positive cells). No significant differences in induction were observed between genotypes (CC, CT, TT). Following incubation with 100 microM PI, significant upregulation of P-gp occurred except with amprenavir. However, nelfinavir, ritonavir, and lopinavir caused marked toxicity, indicating that at higher concentrations, the increase in P-gp may be at least partially related to a stress response. These results indicate the potential of some PIs and NNRTIs to induce P-gp expression in PBMCs in vitro.

Modulation of the intracellular accumulation of saquinavir in peripheral blood mononuclear cells by inhibitors of MRP1, MRP2, P-gp and BCRP.

Background:The efflux transporters P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRP) and breast cancer resistance protein (BCRP) limit the accumulation of antiretrovirals in cell lines but it is more important to know whether the expression of these transporters in peripheral blood mononuclear cells (PBMC) impacts cellular drug concentrations. Objectives:To study the transport and accumulation of saquinavir (SQV) in PBMC and the effects of specific inhibitors of MRP1, MRP2, P-gp and BCRP. Methods:Transport and accumulation of [3H]-SQV was measured in PBMC in the absence or presence of specific and non-specific inhibitors of MRP1, MRP2, P-gp and BCRP. Flow cytometric, western blot and real-time PCR assays were used to examine the relative expression of the drug efflux transporters in the same batches of PBMC. Results:MRP2 is present in PBMC. The expression of P-gp, MRP1, MRP2 (mRNA) and BCRP all displayed batch-to-batch variability. Specific and non-specific inhibitors of MRP1, P-gp and MRP2 significantly increased the baseline accumulation of SQV. Accumulation of SQV was not correlated with the expression of any single transporter. Conclusions:Multiple drug efflux transporters are important in the intracellular accumulation of SQV in PBMC. If drug efflux contributes towards virological failure, then all contributing transporters will need to be inhibited.

Functional Correlation of P-Glycoprotein Expression and Genotype with Expression of the Human Immunodeficiency Virus Type 1 Coreceptor CXCR4

ABSTRACT The aim of this study was to investigate the relationship between lymphocyte P-glycoprotein (P-gp) expression and genotype in vivo and the expression of lymphocyte receptors critical in the life cycle of human immunodeficiency virus type 1 (HIV-1), i.e., CD4, CCR5, and CXCR4. Using flow cytometry to quantify each membrane receptor/transporter, we demonstrate a highly significant correlation between P-gp protein expression and the expression of CXCR4 (rho = 0.874; P < 0.0001). Furthermore, confocal microscopy showed colocalized expression of CXCR4 and P-gp in the lymphocyte membrane. This significant relationship was also apparent at the mRNA level by use of reverse transcription-PCR (rho = 0.61; P < 0.005) and was present in both phytohemagglutinin-stimulated and unstimulated peripheral blood mononuclear cells. Genotypic analysis of the C3435T single-nucleotide polymorphism of P-gp confirmed significantly higher levels of P-gp in C (range, 2.45 to 11.00 relative fluorescence units [RFU])- than in T (range, 0.25 to 5.00 RFU)-homozygous individuals (P = 0.0088; 95% confidence interval [95% CI], 0.7 to 6.3 RFU). An equivalent association between CXCR4 levels and C (range, 12.7 to 44.1 RFU) versus T (range, 3 to 18.9 RFU) genotype was also demonstrated (P = 0.0019; 95% CI, 5.4 to 23.7). Functionally, although these correlates had no impact on HIV-1 production from either X4- or R5-tropic virus, expression correlated significantly with the activity of the HIV-1 protease inhibitor (PI) saquinavir for both P-gp (rho = 0.75; P = 0.0019) and CXCR4 (rho = 0.71; P = 0.0041). This study defines an association between P-gp (expression and genotype) and CXCR4 that may have implications for the selection of viral tropism and the access of drugs to protease for specific tropic types. The interplay between these two proteins may also influence the viral genotypes which escape effective chemotherapy and which therefore have the opportunity to evolve resistance to PIs.

The implications of P‐glycoprotein in HIV: friend or foe?

P‐glycoprotein (P‐gp), coded by the ABCB1 gene, has a wide tissue distribution. The drug transporter is known to limit the bioavailability of a plethora of drugs and xenobiotics including the human immunodeficiency virus (HIV) protease inhibitors. There remains a considerable degree of debate in the literature with respect to the role of ABCB1 polymorphisms in HIV‐treatment outcome and some studies have also implicated antiretroviral drugs as inducers of P‐gp. Recent evidence indicates a role for P‐gp in the inhibition of viral infectivity and/or release and cellular relationships with other infection‐related proteins (and cholesterol). It is becoming increasingly clear that future studies on P‐gp in HIV should consider both pharmacological and virological issues.

Intracellular accumulation of efavirenz and nevirapine is independent of P-glycoprotein activity in cultured CD4 T cells and primary human lymphocytes

BACKGROUND Interaction of antiretrovirals with drug transporters such as P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), breast cancer resistance protein (BCRP) and solute carrier organic anion transporter (SLCO) may influence the emergence of viral mutants by altering intracellular drug concentrations. Here we characterize the effect of transporter expression in a variety of cell types such as control CEM, CEM(VBL) (P-gp-overexpressing), CEM(E1000) (MRP1-overexpressing), MT4, control MDCKII, MDCKII(MDR1) (P-gp-overexpressing) and peripheral blood mononuclear cells (PBMCs) on the uptake of [(14)C]efavirenz and [(3)H]nevirapine. We also investigated the lipophilicity of [(14)C]efavirenz and [(3)H]nevirapine. METHODS The expression of P-gp, MRP1, MRP2, SLCO1A2, 1B1, 1B3, 2B1, 3A1 and 4A1 was assessed by PCR. Inhibitors of P-gp (XR9576, GF120918, dipyridamole) and MRP (MK571, frusemide, dipyridamole), and SLCO substrate or inhibitor (estrone-3-sulphate or montelukast, respectively) were used to study the role of drug transporters in the accumulation of [(14)C]efavirenz and [(3)H]nevirapine. Lipophilicity was measured by the octanol/saline partition coefficient. RESULTS CEM cells, MT4 cells and PBMCs express various SLCO isoforms, with SLCO3A1 detected in all of the cells. XR9576, dipyridamole and GF120918 had no effects on the accumulation of [(14)C]efavirenz, while MK571 and frusemide produced variable effects in the cells. The accumulation of [(14)C]efavirenz was significantly decreased in all the cells by montelukast and estrone-3-sulphate. CONCLUSIONS P-gp expression had no effect on the accumulation of [(14)C]efavirenz and [(3)H]nevirapine. MRP1/2 expression, lipophilicity and SLCO-like transporters (possibly SLCO3A1) may have greater influence on the accumulation of [(14)C]efavirenz than [(3)H]nevirapine.

Intracellular ‘boosting’ of darunavir using known transport inhibitors in primary PBMC

AIMS ABCB1, some ABCCs and SLCOs have been reported to affect the intracellular accumulation of various protease inhibitors in vitro and ex vivo. Darunavir is the most recently licensed protease inhibitor and we sought to investigate the ability of transport inhibitors to influence its intracellular accumulation in lymphocytes from healthy volunteers. METHODS The intracellular accumulation of radiolabelled darunavir was assessed using CEM cells and ABCB1-overexpressing CEM(VBL) cells. Apical and basolateral transport of radiolabelled darunavir through MDCKII monolayers was also studied. Finally the ability of known inhibitors to influence intracellular accumulation of darunavir in peripheral blood mononuclear cells (PBMC) was investigated. RESULTS CEM(VBL) cells (1.4 +/- 0.6, P < 0.001, 95% CI for the difference = 0.46, 0.80, n= 7) had significantly lower accumulation of darunavir compared with CEM cells (5.6 +/- 0.7, n= 7) and this was reversed by addition of tariquidar (30 nm, 4.6 +/- 0.8, P < 0.001, 95% CI =-0.64, -0.41, n= 4). In MDCKII-ABCBI cells, transport from the basal to the apical compartment was observed and this was also reversible with the addition of tariquidar. In PBMCs, dipyridamole (6.9 +/- 1.3, P < 0.01, 95% CI for the difference =-1.16, -0.30, (n= 8) significantly increased whilst montelukast (5.7 +/- 1.0, P < 0.01, 95% CI for the difference = 0.16, 0.79, n= 8) significantly decreased the intracellular accumulation of darunavir when compared with control (6.2 +/- 1.1, n= 8). CONCLUSIONS Darunavir is a substrate for efflux and influx transporters in PBMC and intracellular concentrations can be manipulated using known inhibitors.