CELLULAR PHARMACOKINETICS OF ZD1694 IN CULTURED HUMAN LEUKEMIA-CELLS SENSITIVE, OR MADE RESISTANT, TO THIS DRUG

We have analysed the cellular metabolism of a novel thymidylate syntha se (TS) inhibitor, ZD1694, in MOLT-3 and K562 human leukaemia cell lin es sensitive to or made resistant to ZD1694 by continuous exposure of the cells to ZD1694 with stepwise escalation of the drug concentration . The initial cellular uptake of [H-3]ZD1694 was greater in K562 cells than in MOLT-3 cells and the drug accumulated approximately 3-fold mo re in the former cells following incubation with 0.1 mu M ZD1694 at 37 degrees C for 24 h. TS and dihydrofolate reductase activities were no t significantly different between the two cell lines. After a 30-min i ncubation with the drug at 37 degrees C, 85% of the total drug (2.3 pm ol/mg protein) in K562 cells was found as tri- to pentaglutamates, whe reas MOLT-3 cells accumulated less drug in this time (0.83 pmol/mg pro tein) and polyglutamates of chain length greater than triglutamate wer e not found to a significant extent. When the incubation time was exte nded to 24 h, the polyglutamate profile in K562 cells was progressivel y shifted towards those of long glutamate chain length and 59% of the total cellular drug (204 pmol/mg protein) was identified as the penta form. In contrast, even distribution between tri- and pentaglutamate w as observed in MOLT-3 cells. Total cellular polyglutamates were approx imately 3-fold higher in K562 cells than in MOLT-3 cells, and this may explain the 2.5-fold difference in the sensitivity to ZD1694 between the two cell lines. Continuous exposure of MOLT-3 and K562 cells to ZD 1694 up to 1 mu M or 0.1 mu M resulted in 1600- and 4200-fold resistan t sublines, respectively (MOLT-3/ZD1694 . C and K562/ZD1694 . C). The resistant MOLT-3 cells showed a markedly lower cellular accumulation a nd poor retention of [H-3]ZD1694 with no significant change of initial drug uptake by 10 min and with a little increase of TS activity. HPLC analysis demonstrated that more than 90% of the H-3 co-eluted with th e monoglutamate (parent drug) in the resistant MOLT-3 cells, indicatin g extremely diminished polyglutamation in the cells. On the other hand , cellular uptake of [H-3]ZD1694 was extensively impaired in K562/ZD16 94 . C cells and cellular accumulation of the drug was only 2.5% of th at in the parent cells following 24 h incubation with the drug. Neithe r an increase of TS or dihydrofolate reductase activity nor a change i n the polyglutamate formation profile was observed in the resistant K5 62 cells. These results indicate that the cellular ability to produce the polyglutamate metabolites of ZD1694 must influence the sensitivity of the tumour cells to this drug, and development of mechanisms invol ved in the ZD1694 resistance may relate to the intrinsic biochemical p roperties of the cells.