Role of P-glycoprotein in the distribution of the HIV protease inhibitor atazanavir in the brain and male genital tract.

The blood-testis barrier and blood-brain barrier are responsible for protecting the male genital tract and central nervous system from xenobiotic exposure. In HIV-infected patients, low concentrations of antiretroviral drugs in cerebrospinal fluid and seminal fluid have been reported. One mechanism that may contribute to reduced concentrations is the expression of ATP-binding cassette drug efflux transporters, such as P-glycoprotein (P-gp). The objective of this study was to investigate in vivo the tissue distribution of the HIV protease inhibitor atazanavir in wild-type (WT) mice, P-gp/breast cancer resistance protein (Bcrp)-knockout (Mdr1a-/-, Mdr1b-/-, and Abcg2-/- triple-knockout [TKO]) mice, and Cyp3a-/- (Cyp) mice. WT mice and Cyp mice were pretreated with a P-gp/Bcrp inhibitor, elacridar (5 mg/kg of body weight), and the HIV protease inhibitor and boosting agent ritonavir (2 mg/kg intravenously [i.v.]), respectively. Atazanavir (10 mg/kg) was administered i.v. Atazanavir concentrations in plasma (Cplasma), brain (Cbrain), and testes (Ctestes) were quantified at various times by liquid chromatography-tandem mass spectrometry. In TKO mice, we demonstrated a significant increase in atazanavir Cbrain/Cplasma (5.4-fold) and Ctestes/Cplasma (4.6-fold) ratios compared to those in WT mice (P<0.05). Elacridar-treated WT mice showed a significant increase in atazanavir Cbrain/Cplasma (12.3-fold) and Ctestes/Cplasma (13.5-fold) ratios compared to those in vehicle-treated WT mice. In Cyp mice pretreated with ritonavir, significant (P<0.05) increases in atazanavir Cbrain/Cplasma (1.8-fold) and Ctestes/Cplasma (9.5-fold) ratios compared to those in vehicle-treated WT mice were observed. These data suggest that drug efflux transporters, i.e., P-gp, are involved in limiting the ability of atazanavir to permeate the rodent brain and genital tract. Since these transporters are known to be expressed in humans, they could contribute to the low cerebrospinal and seminal fluid antiretroviral concentrations reported in the clinic.

Expression of ATP-binding cassette membrane transporters in a HIV-1 transgenic rat model.

P-glycoprotein (P-gp, product of Mdr1a and Mdr1b genes), multidrug resistance associated proteins (Mrps), and breast cancer resistance protein (Bcrp), all members of the ATP-binding cassette (ABC) membrane-associated drug transporters superfamily, can significantly restrict the entry of antiretroviral drugs (ARVs) into organs which exhibit a barrier function such as the central nervous system (CNS) and the male genital tract (MGT). In vitro, HIV-1 viral proteins such as glycoprotein-120 (gp120) and transcriptional transactivator (tat) have been shown to alter the expression of these transporters and ARVs permeability. The objective of this study was to compare mRNA expression of these transporters, in vivo, in several tissues obtained from HIV-1 transgenic rats (Tg-rat) (8 and 24 weeks) with those of age-matched wild-type rats. At 24 weeks, significant changes in several drug transporter mRNA expressions were observed, in particular, in brain, kidney, liver and testes. These findings suggest that HIV-1 viral proteins can alter the expression of ABC drug transporters, in vivo, in the context of HIV-1 and further regulate ARVs permeability in several organs including the CNS and MGT, two sites which have been reported to display very low ARVs permeability in the clinic.

Expression of membrane drug efflux transporters in the sigmoid colon of HIV-infected and uninfected men.

The use of antiretroviral therapy (ART) as pre-exposure prophylaxis (PrEP) has gained global attention as a promising HIV prevention strategy in men who have sex with men. Permeability of these agents in the rectal mucosa may be partially regulated by interactions with drug efflux transporters, P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs) and/or breast cancer resistance protein (BCRP). The objective of this work was to investigate the expression of drug efflux transporters in recto-sigmoid colon tissues of HIV-infected and uninfected men, and evaluate the association of ART and/or HIV infection with drug transporter expression. MDR1/P-gp, MRPs (1-4) and BCRP mRNA and protein expression were detected in sigmoid colon biopsies of HIV-uninfected individuals. Biopsies from HIV-infected, ART-naïve participants revealed a significant downregulation of P-gp and MRP2 protein levels compared to HIV-uninfected individuals. Biopsies from HIV-infected ART-treated patients showed 1.9-fold higher P-gp protein expression and 1.5-fold higher MRP2 protein expression compared to the ones obtained from the HIV-infected ART-naïve patients. This is a first report demonstrating that HIV infection or ART could alter expression of drug efflux transporters in gut mucosa which in turn could affect the permeability of PrEP antiretroviral agents across this barrier, a highly vulnerable site of HIV transmission.

Regulation of breast cancer resistant protein by peroxisome proliferator-activated receptor α in human brain microvessel endothelial cells.

Breast cancer resistance protein (BCRP/ABCG2), an ATP-binding cassette (ABC) membrane-associated drug efflux transporter, is known to localize at the blood-brain barrier (BBB) and can significantly restrict xenobiotic permeability in the brain. The objective of this study is to investigate the regulation of BCRP functional expression by peroxisome proliferator-activated receptor alpha (PPARα), a ligand-activated transcription factor primarily involved in lipid metabolism, in a cerebral microvascular endothelial cell culture system (hCMEC/D3), representative of human BBB. We demonstrate that PPARα-selective ligands (i.e., clofibrate, GW7647) significantly induce BCRP mRNA and protein expression in a time- and concentration-dependent manner, whereas pharmacological inhibitors (i.e., MK886, GW6471) prevent this induction. Using [(3)H]mitoxantrone, an established BCRP substrate, we observe a significant reduction in its cellular accumulation by monolayer cells treated with clofibrate, suggesting increased BCRP efflux activity. In addition, we show a significant decrease in BCRP protein expression and function when PPARα is down-regulated by small interfering RNA. Applying chromatin immunoprecipitation and quantitative real-time polymerase chain reaction, we observe that clofibrate treatment increases PPARα binding to the peroxisome proliferator response element within the ABCG2 gene promoter. This study provides the first evidence of direct BCRP regulation by PPARα in a human in vitro BBB model and suggests new targeting strategies for either improving drug brain bioavailability or increasing neuroprotection.

Expression of ATP-binding cassette membrane transporters in rodent and human sertoli cells: relevance to the permeability of antiretroviral therapy at the blood-testis barrier.

The blood-testis barrier (BTB), composed primarily of Sertoli cells, is responsible for protecting developing germ cells from xenobiotic exposure. ATP-binding cassette (ABC) membrane-associated drug efflux transporters, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and the multidrug resistance-associated proteins (Mrps), have been shown to restrict antiretroviral drug permeability at blood-tissue barriers such as the blood-brain barrier. However, it remains unclear whether these transporters are functional at the level of Sertoli cells and can regulate anti-HIV drug permeability at the BTB. This study investigated the functional expression of ABC transporters in a mouse Sertoli cell line system (TM4) and in primary cultures of human Sertoli cells (HSECs). Expression of multidrug resistance Mdr1a/1b/MDR1/P-gp, Mrp1/MRP1, and Mrp4/MRP4 is confirmed by quantitative polymerase chain reaction and immunoblotting analysis in TM4 cells and HSECs. Immunofluorescence studies revealed plasma membrane localization of P-gp, Mrp1/MRP1, and Mrp4/MRP4 in both cell systems. However, Bcrp expression and localization was only detected in rodent cells. Accumulation of 1) rhodamine-6G (R-6G), a fluorescent P-gp substrate, 2) [³H]atazanavir, a HIV protease inhibitor and known P-gp substrate, 3) 2'7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein (BCECF), a fluorescent Mrp substrate, and 4) [³H]mitoxantrone, a BCRP substrate, by TM4 monolayer cells in the presence of established inhibitors demonstrates that these transporters are functional. In addition, several anti-HIV drugs significantly enhance the accumulation of R-6G, [³H]atazanavir, BCECF, and [³H]mitoxantrone by TM4 cells. This study provides the first evidence of ABC transporter expression and activity in Sertoli cells and suggests that these transporters could play an important role in restricting antiretroviral drug permeability at the BTB.

Characterization of mENT1Delta11, a novel alternative splice variant of the mouse equilibrative nucleoside transporter 1.

Mammalian cells require specific transport mechanisms for the cellular uptake and release of endogenous nucleosides such as adenosine, and nucleoside analogs used in chemotherapy. We have identified a novel splice variant of the mouse equilibrative nucleoside transporter, mENT1, that results from the exclusion of exon 11 during pre-RNA processing. This variant encodes a truncated protein (mENT1Delta11) missing the last three transmembrane domains of the full-length mENT1. The mENT1Delta11 transcript and protein were found to be differentially distributed among tissues relative to full-length mENT1. PK15-NTD (nucleoside transport deficient) cells were transfected with mENT1 or mENT1Delta11 and assessed for nucleoside transport function. No significant differences were observed between the mENT1 and mENT1Delta11 in terms of transport function or inhibitor binding affinity. PK15-mENT1Delta11 transfected cells bound the ENT1 probe [3H]nitrobenzylthioinosine (NBMPR) with high affinity and mediated the cellular accumulation of both [3H]2-chloroadenosine and [3H]uridine. The only significant differences between the mENT1 variants were that mENT1Delta11 could not be photolabeled with [3H]NBMPR and that mENT1Delta11 was insensitive to the transporter-modifying effects of N-ethylmaleimide. These data suggest that the last three transmembrane domains of mENT1 are not necessary for transport activity, but this region does contain the cysteines responsible for the sensitivity of mENT1 to sulfhydryl reagents, and the residues important for covalent modification of the protein with NBMPR. These results provide important guidelines for future mutagenesis studies aimed at elucidating the tertiary structure of the ENT1 protein and the domains involved in inhibitor binding and substrate translocation.

Hypoxanthine uptake and release by equilibrative nucleoside transporter 2 (ENT2) of rat microvascular endothelial cells.

The cardioprotective actions of adenosine are terminated by its uptake into endothelial cells with subsequent metabolism through hypoxanthine to uric acid. This process involves xanthine oxidase-mediated generation of reactive oxygen species (ROS), which have been implicated in the vascular dysfunction observed in ischemia-reperfusion injury. The equilibrative nucleoside transporter, ENT2, mediates the transfer of hypoxanthine into cells. We hypothesize that ENT2 also mediates the cellular release of hypoxanthine, which would limit the amount of intracellular hypoxanthine available for xanthine oxidase-mediated ROS production. Rat microvascular endothelial cells (MVECs) were isolated from skeletal muscle by lectin-affinity purification. The transport of [(3)H]hypoxanthine was assessed using an oil-stop method, and hypoxanthine metabolites were identified by thin-layer chromatography. MVECs accumulated hypoxanthine with a K(m) of 300 microM and a V(max) of 2.8 pmol microl(-1) s(-1). ATP-depleted cells loaded with [(3)H]hypoxanthine released the radiolabel with kinetics similar to that obtained for [(3)H]hypoxanthine influx. The uptake and release of [(3)H]hypoxanthine were both blocked by ENT2 inhibitors with similar order of potency. Thus, ENT2 mediates both the influx and efflux of hypoxanthine. Inhibition of ENT2 in MVECs might be expected to increase the amount of intracellular hypoxanthine available for metabolism by xanthine oxidase and enhance the intracellular production of ROS.

Differential regulation of mouse equilibrative nucleoside transporter 1 (mENT1) splice variants by protein kinase CK2.

Nucleosides are accumulated by cells via a family of equilibrative transport proteins (ENTs). An alternative splice variant of the most common subtype of mouse ENT (ENT1) has been identified which is missing a protein kinase CK2 (casein kinase 2) consensus site (Ser(254)) in the central intracellular loop of the protein. We hypothesized that this variant (mENT1a) would be less susceptible to modulation by CK2-mediated phosphorylation compared to the variant containing the serine at position 254 (mENT1b). Each splice variant was transfected into nucleoside transporter deficient PK15 cells, and stable transfectants assessed for their ability to bind the ENT1-selective probe [(3)H]nitrobenzylthioinosine (NBMPR) and to mediate the cellular uptake of [(3)H]2-chloroadenosine, with or without treatment with the CK2 selective inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB). mENT1a had a higher affinity for NBMPR relative to mENT1b - measured both directly by the binding of [(3)H]NBMPR, and indirectly via inhibition of [(3)H]2-chloroadenosine influx by NBMPR. Furthermore, incubation of mENT1b-expressing cells with 10 microM TBB for 48 h decreased both the K(D) and B(max) of [(3)H]NBMPR binding, as well as the V(max) of 2-chloroadenosine uptake, whereas similar treatment of mENT1a-expressing cells with TBB had no effect. PK15 cells transfected with hENT1, which has Ser(254), was similar to mENT1b in its response to TBB. In conclusion, inhibition of CK2 activity, or deletion of Ser(254) from mENT1, enhances transporter affinity for the inhibitor, NBMPR, and reduces the number of ENT1 proteins functioning at the level of the plasma membrane.