title: Cellular pharmacokinetics of ZD1694 in cultured human leukaemia cells sensitive, or made resistant, to this drug.
abstract: We have analysed the cellular metabolism of a novel thymidylate synthase (TS) inhibitor, ZD1694, in MOLT-3 and K562 human leukaemia cell lines 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 [3H]ZD1694 was greater in K562 cells than in MOLT-3 cells and the drug accumulated approximately 3-fold more in the former cells following incubation with 0.1 microM ZD1694 at 37 degrees C for 24 h. TS and dihydrofolate reductase activities were not significantly different between the two cell lines. After a 30-min incubation with the drug at 37 degrees C, 85% of the total drug (2.3 pmol/mg protein) in K562 cells was found as tri- to pentaglutamates, whereas MOLT-3 cells accumulated less drug in this time (0.83 pmol/mg protein) and polyglutamates of chain length greater than triglutamate were not found to a significant extent. When the incubation time was extended to 24 h, the polyglutamate profile in K562 cells was progressively 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 was observed in MOLT-3 cells. Total cellular polyglutamates were approximately 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 ZD1694 up to 1 microM or 0.1 microM resulted in 1600- and 4200-fold resistant sublines, respectively (MOLT-3/ZD1694.C and K562/ZD1694.C). The resistant MOLT-3 cells showed a markedly lower cellular accumulation and poor retention of [3H]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 3H co-eluted with the monoglutamate (parent drug) in the resistant MOLT-3 cells, indicating extremely diminished polyglutamation in the cells. On the other hand, cellular uptake of [3H]ZD1694 was extensively impaired in K562/ZD1694.C cells and cellular accumulation of the drug was only 2.5% of that in the parent cells following 24 h incubation with the drug. Neither an increase of TS or dihydrofolate reductase activity nor a change in the polyglutamate formation profile was observed in the resistant K562 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 involved in the ZD1694 resistance may relate to the intrinsic biochemical properties of the cells.
authors.affiliation: Department of Laboratory Medicine, National Defense Medical College, Saitama, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Takemura
authors.forename: W
authors.initials: W
authors.lastname: Gibson
authors.forename: R
authors.initials: R
authors.lastname: Kimbell
authors.forename: H
authors.initials: H
authors.lastname: Kobayashi
authors.forename: H
authors.initials: H
authors.lastname: Miyachi
authors.forename: A L
authors.initials: AL
authors.lastname: Jackman
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000493:pharmacokinetics* / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D004791:Enzyme Inhibitors / Q000493:pharmacokinetics* / Q000494:pharmacology; D005492:Folic Acid / Q000031:analogs & derivatives; D006801:Humans; D007424:Intracellular Fluid / Q000201:enzymology; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy* / Q000378:metabolism*; D011099:Polyglutamic Acid / Q000378:metabolism; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy* / Q000378:metabolism*; D011799:Quinazolines / Q000493:pharmacokinetics* / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013876:Thiophenes / Q000493:pharmacokinetics* / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D014316:Tritium; D014407:Tumor Cells, Cultured
chemical_list: D000964:Antimetabolites, Antineoplastic; D004791:Enzyme Inhibitors; D011799:Quinazolines; D013876:Thiophenes; D014316:Tritium; D011099:Polyglutamic Acid; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; C068874:raltitrexed