Breast cancer resistance protein (BCRP) is a recently discovered ATP-binding cassette drug transporter. Hence, the full spectrum of therapeutic agents that interact with BCRP remains to be elucidated. Because human immunodeficiency virus protease inhibitors (HPIs) are well known P-glycoprotein (P-gp) substrates, and there is an overlap in substrate specificity between P-gp and BCRP, this study was performed to investigate whether HPIs are substrates and/or inhibitors of BCRP. First, the effect of HPIs on BCRP efflux activity in human embryonic kidney (HEK) cells stably expressing wild-type BCRP (482R) and its two mutants (482T and 482G) was studied by measuring intracellular mitoxantrone fluorescence using flow cytometry. We found that ritonavir, saquinavir, and nelfinavir were effective inhibitors of wild-type BCRP (482R) with IC 50 values of 19.5 Ϯ 0.8 M, 19.5 Ϯ 7.6 M, and 12.5 Ϯ 4.1 M, respectively. Ritonavir, saquinavir, and nelfinavir inhibited 482T and 482G with IC 50 values that were approximately 2 times greater than that for 482R. Indinavir and amprenavir had no significant inhibition on BCRP activity. Direct efflux of radiolabeled HPIs in HEK cells was measured to determine whether the HPIs are substrates of BCRP. None of the HPIs were found to be transported by BCRP. Together, ritonavir, saquinavir, nelfinavir, indinavir, and amprenavir are not substrates for BCRP. However, ritonavir, saquinavir, and nelfinavir are effective inhibitors of the transporter. These results suggest that BCRP may play an important role in drug-drug interactions involving coadministration of the HPIs with drugs that are substrates of the transporter.

Many antiviral drugs (e.g. fialuridine; FIAU) produce clinically significant mitochondrial toxicity that limits their dose or prevents their use in the clinic. Because the majority of nucleoside drugs is too hydrophilic to cross the highly impermeable mitochondrial membrane, we have hypothesized that they must be transported into the mitochondria to produce their toxicity. To test this hypothesis, we have sought to determine whether the nucleoside transporters, human equilibrative nucleoside transporter 1 (hENT1) or human concentrative nucleoside transporter 1 (hCNT1), when stably expressed in Madin-Darby canine kidney cells as yellow fluorescent fusion protein (YFP), are localized to the mitochondria. By using organelle-selective dyes and confocal microscopy, we have found that hENT1-YFP is localized to the mitochondria as well as the plasma membrane, whereas hCNT1-YFP was found predominantly on the plasma membrane. hENT1-YFP was not localized to the nuclear envelope, endosomes, lysosomes, or Golgi complex. Western blotting confirmed the presence of hENT1-YFP or endogenous hENT1 in mitochondria isolated from hENT1-YFP-expressing cells and human livers, respectively. In agreement with these localization data, [ 14 C]FIAU was efficiently transported into the mitochondria of cells expressing hENT1-YFP but not of cells expressing hCNT1-YFP. The mitochondrial toxicity of FIAU to Madin-Darby canine kidney cells was enhanced by hENT1-YFP, even when hENT1 activity on the plasma membrane was selectively blocked by 10 nM nitrobenzylthioinosine. Moreover, FIAU (50 M) produced significant mitochondrial toxicity (ϳ70% decrease in mitochondrial DNA synthesis) when it was directly incubated with mitochondria isolated from hENT1-expressing cells. In conclusion, we have identified for the first time that hENT1 is expressed on the mitochondrial membrane and that this expression enhances the mitochondrial toxicity of nucleoside drugs such as FIAU. Mitochondrial expression of hENTs may explain the clinically significant mitochondrial toxicity caused by the anti-HIV nucleoside drugs such as zidovudine, stavudine, and didanosine.Fialuridine (FIAU), 1 a uridine analog, was developed for the treatment of hepatitis B. In a phase II trial, administration of this antiviral nucleoside to patients with hepatitis B resulted in severe multisystem toxicity because of widespread mitochondrial damage including hepatotoxicity, pancreatitis, neuropathy, or myopathy. Of the seven patients exhibiting severe hepatotoxicity, five died and two survived after liver transplantation (1). Although the exact mechanism of mitochondrial toxicity of FIAU has not been determined (2), all of the evidence points toward inhibition of mitochondrial DNA polymerase ␥ by the phosphorylated metabolites of FIAU that accumulate within the mitochondrial compartment (3, 4). Other antiviral nucleoside drugs also cause mitochondrial toxicity such as the anti-HIV dideoxynucleosides (e.g. DDI) (5) and the anticancer drugs (e.g. fludarabine) (6). The mitochondrial toxicity of ...

To test the hypothesis that human concentrative and equilibrative nucleoside transporters (hCNT1 and hENT1) are present on the apical and basolateral membrane, respectively, we constructed a Madin-Darby canine kidney (MDCK) cell line that simultaneously and stably expresses recombinant hCNT1 and hENT1 gene products tagged with CFP and YFP fluorescent proteins, respectively. Using a confocal microscope, both hCNT1-CFP and hENT1-YFP were found to be distributed uniformly on the plasma membrane of undifferentiated MDCK cells. Upon differentiation of the MDCK cells on Transwell filter inserts, hCNT1-CFP was visualized exclusively on the apical membrane, whereas hENT1-YFP appeared predominantly on the basolateral membrane. As differentiation proceeded, there was an increase in alkaline phosphatase activity, and activity of hENT1 in the apical compartment decreased while hCNT1 activity remained constant. These results suggest that, on differentiation, hENT1 is sorted to the basolateral membrane. This was confirmed when the hCNT1-mediated uptake of [ 3 H]uridine from the apical compartment of the differentiated cells was found to be ϳ20-fold higher and that for hENT1 was ϳ4-fold lower than the corresponding uptake from the basal compartment. As observed in vivo, the net transport of [ 3 H]adenosine was from the apical to the basal compartment, whereas that for 14 C-deoxyadenosine was from the basal to the apical compartment. In summary, we have shown for the first time that hCNT1 and hENT1 are expressed in polarized MDCK cells on the apical and basolateral membrane, respectively, allowing vectorial transport in both directions depending on the relative activity (ratio of maximal transporter activity to affinity) of each transporter for their substrates.Nucleoside transporters (NTs) 1 are important in mediating the transport of nucleosides and nucleoside drugs (e.g. antiviral and anticancer drugs) across cell membranes (1). Physiologically, the sodium-dependent concentrative nucleoside transporters mediate the influx of nucleosides. Human concentrative nucleoside transporter 1 (hCNT1) is pyrimidine-specific, whereas hCNT2 is purine-specific. Both hCNT1 and hCNT2 transport uridine and adenosine (2) and are insensitive to inhibition by nitrobenzylthioinosine (NBMPR). hCNT1 and hCNT2 are expressed on specialized cells such as intestine and kidney epithelia (3, 4). The equilibrative transporters mediate both the influx and efflux of nucleosides and exhibit broad substrate specificity, accepting both purine and pyrimidine nucleosides as permeants. Human equilibrative nucleoside transporter 1 (hENT1 or es) is inhibited by NBMPR concentrations as low as 0.1 nM (IC50 ϳ0.4 nM), whereas hENT2 (ei) transporter is insensitive to inhibition as high as 1 M (IC 50 ϳ2.8 M) (1, 4, 5). One or both of the equilibrative transporters are expressed in most, if not all, cell types.Although functional measurement of transporter activity has helped elucidate the tissue expression of the concentrative and equilibrative nucleoside transporters,...

Background-Arylamine N-acetyltransferases in humans (NAT1 and NAT2) catalyse the acetylation of arylamines including food derived heterocyclic arylamine carcinogens. Other substrates include the sulphonamide 5-aminosalicylic acid (5-ASA), which is an NAT1 specific substrate; N-acetylation of 5-ASA is a major route of metabolism. NAT1 and NAT2 are both polymorphic. Aims-To investigate NAT expression in apparently healthy human intestines in order to understand the possible role of NAT in colorectal cancer and in the therapeutic response to 5-ASA. Methods-The intestines of four organ donors were divided into eight sections. DNA was prepared for genotyping NAT1 and NAT2 and enzymic activities of NAT1 and NAT2 were determined in cytosols prepared from each section. Tissue was fixed for immunohistochemistry with specific NAT antibodies. Western blotting was carried out on all samples of cytosol and on homogenates of separated muscle and villi after microdissection. Results-NAT1 activity of all cytosols was greater than NAT2 activity. NAT1 and NAT2 activities correlated with the genotypes of NAT1 and NAT2 and with the levels of NAT1 staining determined by western blotting. The ratio of NAT1:NAT2 activities showed interindividual variations from 2 to 70. NAT1 antigenic activity was greater in villi than in muscle. NAT1 was detected along the length of the villi in the small intestine. In colon samples there was less NAT1 at the base of the crypts with intense staining at the tips. Conclusions-The interindividual variation in NAT1 and NAT2 in the colon could aVect how individuals respond to exposure to specific NAT substrates including carcinogens and 5-ASA. (Gut 1998;42:402-409)

Any treatment of a pregnant woman with medication (drugs) de facto results in the treatment of her unborn child, even when her unborn child is not the target of drug therapy. This is because, in most instances, the placenta is not a complete barrier to the passage of drugs from the maternal to the fetal compartment. This barrier is in part due to the presence of various efflux transporters in the placenta. The placenta is also richly endowed with influx transporters. In this article, we will review the physiological characteristics of the placenta and how it functions as a barrier to passage of drugs into the fetal compartment. In addition, we will review placental transporters that are important in modulating the exposure of the fetus to drugs and, therefore, the efficacy and toxicity of such drugs towards the fetus.

The human equilibrative nucleoside transporter, hENT1, which is sensitive to inhibition by nitrobenzylthioinosine (NBMPR), is expressed in a wide variety of tissues. hENT1 is involved in the uptake of natural nucleosides, including regulation of the physiological effects of extracellular adenosine, and transports nucleoside drugs used in the treatment of cancer and viral diseases. Structure-function studies have revealed that transmembrane domains (TMD) 3 through 6 of hENT1 may be involved in binding of nucleosides. We have hypothesized that amino acid residues within TMD 3-6, which are conserved across equilibrative transporter sequences from several species, may have a critical role in the binding and transport of nucleosides. Therefore, we explored the role of point mutations of two conserved glycine residues, at positions 179 and 184 located in transmembrane domain 5 (TMD 5), using a GFP-tagged hENT1 in a yeast nucleoside transporter assay system. Mutations of glycine 179 to leucine, cysteine, or valine abolished transporter activity without affecting the targeting of the transporter to the plasma membrane, whereas more conservative mutations such as glycine to alanine or serine preserved both targeting to the plasma membrane and transport activity. Similar point mutations at glycine 184 resulted in poor targeting of hENT1 to the plasma membrane and little or no detectable functional activity. Uridine transport by G179A mutant was significantly lower (p < 0.05) and less sensitive (p < 0.05) to inhibition by NBMPR when compared to the wild-type transporter (IC(50) 7.7 +/- 0.8 nM versus 46 +/- 14.6 nM). Based on these data, we conclude that when hENT1 is expressed in yeast, glycine 179 is critical not only to the ability of hENT1 to transport uridine but also as a determinant of hENT1 sensitivity to NBMPR. In contrast, glycine 184 is likely important in targeting the transporter to the plasma membrane. This is the first identification and characterization of a critical amino acid residue of hENT1 that is important in both nucleoside transporter function and sensitivity to inhibition by NBMPR.

Three models, linked in series, can be used to analyze combined pharmacokinetic (PK) and pharmacodynamic (PD) data arising from non--steady-state experiments. A PK model relates dose to plasma drug concentration (Cp); a link model relates Cp to drug concentration at the effect site (Ce); and a PD model relates Ce to drug effect (E). All three submodels can be stated parametrically. Recently the use of a nonparametric PD submodel has been proposed (CLIN PHARMACOL THER 1984;35:733-41). In this article we use an extended nonparametric approach that represents both the PK and PD models nonparametrically, but retains a parametric link model. Cp data from several PK models and E data from several PD models were simulated. After the addition of noise to both the Cp and E data, they were analyzed by both the parametric and extended nonparametric methods. The methods were compared by how well they estimated the PD model. To assess robustness, the effect of misspecification of the PK submodel on the goodness of estimation of both methods was also compared. In the absence of model misspecification, the parametric method usually estimates the PD model better than the nonparametric method. However, this difference in the performances diminishes and even reverses when the PK model is misspecified. Because one can rarely be certain that model misspecification is absent, the nonparametric approach may offer a distinct advantage for routine analysis of PK/PD data.

No abstract