Uptake of 2-β- d-ribofuranosylthiazole-4-carboxamide (Tiazofurin) and analogues by the facilitated transport mechanism of erythrocytes

Tiazofurin (TR), a new antitumor agent, enters human erythrocytes by utilizing their facilitated nucleoside transport system. TR competes with endogenous nucleosides for this transport mechanism, thereby reducing nucleoside uptake into the cells. Pre-incubation of erythrocytes for 10 min at 22°C with 100 μM and 500 μM TR reduced the transport of 14C-uridine into the cells by 27% and 74%, respectively. Simultaneous exposure of cells to TR and [ 14C]uridine did not alter the inhibitory effect of TR. Furthermore, the transport inhibitory effect of TR was lost when cells were washed twice with Hanks basal salt solution following a 10-min pre-incubation with TR. The K m and V max (±S.E.) for radiolabeled TR transport into erythrocytes are 170 ± 26 μM and 55 ± 13 nmol/h per 10 6 cells, respectively, which is similar to the kinetic constants measured for uridine transport into erythrocytes ( K m = 168 ± 37 μM and V max = 61 ± 16 nmol/h per 10 6 cells). The K i (±S.E.) of TR for uridine transport is 178 ± 11 μM and for thymidine transport is 102 ± 59 μM. Three analogues of TR (its selenium isostere (SR), and Ara (Ara-TR) and Xylo (Xylo-TR) derivatives) were compared with TR for their ability to compete with and inhibit uridine transport, as these analogues were not available in a radiolabeled form for direct measurement of their transport into the cell. SR had similar kinetic characteristics of inhibition of uridine transport to TR ( K i = 145 ± 15 μM) but Ara-TR had a K i = 1.04 ± 0.13 mM while Xylo-TR inhibited uridine transport with a K i = 1.57 ± 0.67 mM. Thus, TR is transported into erythrocytes with the same velocity and affinity for the carrier as uridine and competitively inhibits nucleoside transport into the cell. Of 3 other C-nucleoside derivatives examined, SR is of similar potency to TR but Ara-TR and Xylo-TR are much less effective at competing with uridine for the nucleoside transporter.