Alan R. P. Paterson

Mediated transport of nucleosides in human erythrocytes. Specific binding of the inhibitor nitrobenzylthioinosine to nucleoside transport sites in the erythrocyte membrane

Abstract Transport of purine and pyrimidine nucleosides in human erthrocytes, which occurs by facilitated diffusion, is inhibited by 6-((4-nitrobenzyl)thio)-9-β- d -ribofuranosylpurine (nitrobenzylthioinosine) and related compounds. The present studies with nitrobenzylthioinosine indicated that inhibition of nucleoside transport resulted from binding of this compound to nucleoside transport sites in the erythrocyte membrane. Two modes of retention of nitrobenzylthioinosine by intact erythrocytes were apparent: saturable binding with an apparent dissociation constant of 10 −9 M and non-saturable intracellular accumulation of inhibitor. High affinity binding of nitrobenzylthioinosine was significantly reduced by the related transport inhibitors, 6-(methylthio)-9-β- d -ribofuranosylpurine and 2-amino-6-((2-hydroxy-5-nitrobenzyl)thio)-9-β- d -ribofuranosylpurine. The high affinity binding sites were identified as transport sites by demonstrating that inhibition of uridine transport was proportional to the number of high affinity sites occupied by nitrobenzylthioinosine. The results presented indicate that nitrobenzylthioinosine interacts specifically with nucleoside transport elements of the human erythrocyte.

Abstract S1-03: Primary results from BETH, a phase 3 controlled study of adjuvant chemotherapy and trastuzumab ± bevacizumab in patients with HER2-positive, node-positive or high risk node-negative breast cancer

Background The humanized monoclonal antibody (mAb) trastuzumab (H) + chemotherapy (chemo) prolongs disease-free survival (DFS) in patients (pts) with HER2-positive breast cancer (BC) in the adjuvant setting. Vascular endothelial growth factor (VEGF-A), one central regulator of angiogenesis, is a downstream target of HER2. Tumors overexpressing HER2 also overexpress VEGF-A and exhibit increased angiogenic potential. Combining H with the anti-VEGF-A mAb bevacizumab (B) significantly decreased tumor volume vs B or H alone in HER2-positive xenograft models and demonstrated efficacy in phase 2 studies. In the phase 3 AVEREL study in pts with HER2-positive metastatic BC, adding B to H + docetaxel (T) led to a non-significant increase in a duration of PFS and objective response rates. Chemo plus H±B is now explored in this large phase 3 trial to assess the impact of VEGF-A blockade on residual or micrometastatic disease in the adjuvant setting. Methods BETH (NCT00625898) is a randomized, phase 3, open-label study evaluating the addition of B to 2 different H-chemo regimens. Pts had centrally-confirmed HER2-positive BC (FISH+ and/or IHC 3+), ECOG PS 0-1, unilateral invasive breast adenocarcinoma, total mastectomy or lumpectomy, and LVEF ≥55%. Prior therapy with anthracyclines, taxanes, carboplatin (C), H or B for any malignancy or radiotherapy, chemo, and/or targeted therapy for the currently diagnosed BC were not permitted. Pts were stratified by center, hormone receptor status (ER and/or PR-positive, ER/PR-negative), and axillary lymph node status (0, 1-3, 4+) before inclusion into 1 of 2 chemo cohorts, and then randomized. All pts were recruited by investigators from the Translational Research in Oncology (TRIO/CIRG), the National Surgical Adjuvant Breast and Bowel Project (NSABP) or a group of independent sites. Cohort 1 (3231 pts) included pts receiving 6 cycles of TCH±B followed by H±B for 1 yr after the first dose. Cohort 2 (278 pts) included pts from some independent sites electing to use anthracycline-based therapy and these pts received 3 cycles of TH±B followed by 3 cycles of 5-fluorouracil, epirubicin, cyclophosphamide followed by H±B to complete 1 yr of treatment. T was given at 8 mg/kg IV loading dose, 6 mg/kg IV q3w thereafter; B was given at 15 mg/kg IV q3w. The primary endpoint is invasive DFS (IDFS) for B-containing vs. non-B-containing regimens. Secondary endpoints are IDFS within chemo cohorts, DFS, overall survival, recurrence-free interval (RFI), distant RFI, safety including specific cardiac assessments, and the identification of predictive biomarkers for B. The sample size was determined to test the hypothesis of interest, both in the faster accruing cohort and overall. With 3509 pts enrolled, the trial will have 85% power to detect a HR of 0.70 favoring the addition of B overall, irrespective of chemo regimen. With ∼3000 pts in the faster-accruing cohort, the study will have 80% power to detect a hazard ratio (HR) of 0.70 at a 2-sided alpha of 0.05. Median duration of follow-up will be 36 months in Jun 13, cut-off date of the primary analysis. Initial efficacy, safety, and plasma marker analyses will be reported. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S1-03.

Stage-specific alteration of nucleoside membrane permeability and nitrobenzylthioinosine insensitivity in Plasmodium falciparum infected erythrocytes

In human erythrocytes, the intracellular presence of malarial parasites (Plasmodium falciparum) markedly changed the permeation characteristics of the nucleosides, adenosine and tubercidin, an adenosine analogue. We report parasite-induced changes in the kinetics of cellular uptake of the nucleosides and in the appearance in infected cells of a nucleoside permeation route of low sensitivity to the classical inhibitor of erythrocytic nucleoside transport, nitrobenzylthioinosine (NBMPR). These changes and a diminution in NBMPR effectiveness during parasite maturation to the trophozoite or schizont stage, suggest the presence in the infected cells of an altered or new nucleoside permeation mechanism of low sensitivity to NBMPR. The incorporation of adenosine into polynucleotides was also of low sensitivity to 10 microM NBMPR. Binding studies of [3H]NBMPR with both normal erythrocytes and those harbouring parasites at each morphological stage indicated that fewer high affinity NBMPR binding sites were present on cells containing mature parasites than on the uninfected cells. The apparent low sensitivity to NBMPR of nucleoside permeation in erythrocytes containing P. falciparum forms may enable therapeutic measures with cytotoxic nucleosides to be directed with selectivity toward parasite-containing cells.

Sodium-dependent, concentrative nucleoside transport in cultured intestinal epithelial cells

In a simple salts medium, monolayers of IEC-6 intestinal cells achieved concentrations of unmetabolized formycin B (an analog of inosine) about 6-fold higher than in the medium. Rates of formycin B influx were a saturable function of Na+ concentrations in the medium. Although IEC-6 cells possess sites with high affinity for nitrobenzylthioinosine, a potent inhibitor of equilibrative (facilitated diffusion) nucleoside transport systems in certain cell types, the inhibitor had only minor effects on formycin B uptake in IEC-6 cells, but reduced efflux of the analog from these cells. These findings indicate the joint presence in IEC-6 cells of nucleoside transporters of two types, one that is concentrative and Na+-dependent, and another that is sensitive to nitrobenzylthioinosine and apparently equilibrative.

Mediated transport of nucleosides by human erythrocytes specificity toward purine nucleosides as permeants

Abstract Transport of uridine and thymidine across the plasma membrane of human eruthrocytes is mediated by a facilitated diffusion mechanism with broad specificity toward the base portion and narrow specificity toward the sugar portion of pyrimidine nucleosides. Specificity of this mechanism was further investigated by measuring efflux of radioactivity when erythrocytes containing radioactive uridine were incubated in medium containing purine nucleosides. Adenosine, guanosine, inosine, and arabinosyladenine accelerated uridine efflux and were therefore considered substrates for the transport mechanism. 6-Thioinosine, 6-thioguanosine, and several S -substituted 6-thiopurine ribonucleosides inhibited efflux of radioactive uridine. Adenine nucleosides with sugar moieties other than ribose or arabinose inhibited or had no effect on uridine efflux.

TRANSPORT OF NUCLEOSIDES

Since nucleosides are not intermediates in the synthesis de novo of purine and pyrimidine nucleotides, the free nucleosides that occur in extracellular fluids are derived from nucleotides and polynucleotides. Cells utilize nucleosides from their environment: the passage of nucleosides across the plasma membrane is mediated by specific, enzyme-like elements of the membrane, and through the action of kinases, nucleosides are “trapped ” intracellularly in the form of 5‘-phosphates. There is evidence that exchange of purines between erythrocytes and liver’.’ occurs at the nucleoside level,3 and nucleosides may well be a common, metabolic “currency ” between cells; for example, the intercellular exchange of purines (termed “ metabolic cooperation”) which occurs when cultured fibroblasts possessing hypoxanthineguanine phosphoribosyltransferase are in contact with fibroblasts lacking this enzyme activity could occur at the nucleoside l e ~ e l . ~ . ~ I t is evident from the kinetic characteristics of nucleoside uptake by erythrocytes and various types of cultured cells that the uptake process is mediated: demonstrations of rate saturability, competition between permeants, and inhibition by specific compounds have ruled out permeation by simple diffusion. Studies of uridine and thymidine transport by human erythrocytes have provided clear demonstrations of permeation by a facilitated diffusion mechanism. Because uridine and thymidine are not cleaved or phosphorylated in these cells,6 it was possible to demonstrate (1) nonconcentrative uptake of these permeants and (2) the occurrence of ’‘ trans phenomena” such as the acceleration of nucleoside outflow from “loaded” cells by the inward flux of a different nucleoside;6-8 these are characteristics of facilitated diffu~ion.~ In cell-permeant systems in which internal permeant molecules are metabolized, rates of permeant uptake that are constant during an assay period started by addition of permeant are generally assumed to measure rates of transport. The assumption that transport is the rate-limiting event in the uptake process appears to be valid in several systems,”-’* although this is difficult to establish rigorously. Characteristics of mediated entry have been recognized in studies of nucleoside uptake by a variety of cell types other than erythrocytes, including these examples selected from a large literature: rate saturability,6*‘0*11 competition between permeants,’ %’ and inhibition by particular compounds.’0-’6*17 It has been a general finding that at low concentrations, nucleoside uptake is mediated ; however, it is less certain that facilitated diffusion is the general mechanism of nucleoside permeation in cells other than erythrocytes, because it is experimentally difficult to demonstrate certain critical features of facilitated diffusion when metabolic processes are competing for intracel-

Inhibitory effects of propentofylline on [3H]adenosine influx: A study of three nucleoside transport systems

The neuroprotective effects of adenosine are well-recognized. Recently, propentofylline, a xanthine derivative, has been shown to increase extracellular concentrations of adenosine in ischemic brain and to limit the extent of neuronal damage in experimental models of cerebral ischemia. Since the concentration of adenosine in brain is controlled, in part, by nucleoside transporter proteins, the action of propentofylline was proposed to be due to inhibition of mediated transfer of adenosine across cell membranes. To determine the likelihood of this mechanism, we examined the inhibitory effects of propentofylline on [3H]adenosine transport by the three best-characterized nucleoside transport processes, es, ei, and cif in cultured cell lines under conditions where only a single transporter type was operative. Propentofylline inhibited [3H]adenosine uptake by each of the three transport processes in a concentration-dependent manner. The greatest inhibitory potency was for es transporters (L1210/B23.1 cells), with an IC50 value of 9 microM, followed by ei transporters, with IC50 values of 170 microM (L1210/C2 cells) and 166 microM (Walker 256 cells). Propentofylline was a weak inhibitor of cif transporter, with an IC50 value of 6 mM. These results demonstrate that propentofylline is an inhibitor of adenosine transport processes and suggest that its neuroprotective effects may be due to an increase in extracellular concentrations of adenosine by virtue of inhibition of es transporter function.

Inhibition of Nucleoside Transport

In many types of animal cells, the movement of nucleoside molecules across the plasma membrane is a specific, transporter-mediated process [1, 2]. The existence of the nucleoside transport mechanism has been recognized from the kinetic properties of nucleoside fluxes, but nucleoside transporter elements of the plasma membrane have not yet been visualized or isolated, and their properties have only been studied indirectly. The potent, tightly bound inhibitor of nucleoside transport, nitrobenzylthioinosine (NBMPR),1 has been a valuable probe of transporter function and biology, enabling, for example, quantitation of transporter elements on cells [3] and providing an approach to their isolation from plasma membrane preparations [4]. This chapter discusses the nucleoside transport-inhibitory properties of NBMPR, N6-nitrobenzyldeoxyadenosine, and certain of their congeners, and of dilazep, a coronary vasodilator.

Nucleoside transport in human erythrocytes: nitrobenzylthioinosine binding and uridine transport activities have similar radiation target sizes

Intact human erythrocytes were irradiated in the frozen state with a high-energy electron beam. Nitrobenzylthioinosine-sensitive uridine influx, equilibrium exchange uridine influx and high-affinity nitrobenzylthioinosine binding were inactivated as a simple exponential function of the radiation dose, indicating an in situ target size of 122 000. The results suggest that the nitrobenzylthioinosine-binding site(s) and the permeation site(s) of the transporter are present on the same transporter element.

Nucleoside Transport Inhibition In Vitro and In Vivo

Among cultured cell lines, considerable diversity has been shown in the sensitivity of cellular nucleoside transport (NT) systems to nitrobenzylthioinosine (NBMPR), a potent inhibitor of facilitated diffusion NT systems. In cultured S49 lymphoma cells and human erythrocytes, NT systems are of high NBMPR sensitivity (IC50 1 μM). A number of cell lines, including leukemia L1210, appear to possess NT systems of both high and low sensitivity to NBMPR. We report the joint presence in cultured hepatoma cells (Novikoff UASJ-2.9, Morris 3924A, Reuber H-35) of NT systems with low NBMPR sensitivity and NBMPR binding sites, a finding which suggests that the latter do not interact with the former. Implications of these findings are discussed. Recognition of energy-requiring, concentrative NT systems in several cell types has added to the complexity of this area. We describe studies of formycin B permeation into cultured IEC-6 intestinal epithelial cells, which show the presence of (a) a concentrative NT system of low NBMPR sensitivity that requires an inward Na+ gradient, and (b) an NBMPR-sensitive transporter that mediates formycin B efflux.