title: Ex vivo activity of methotrexate versus novel antifolate inhibitors of dihydrofolate reductase and thymidylate synthase against childhood leukemia cells.
abstract: Leukemic cells of 27 children [14 patients with initial acute lymphoblastic leukemia (iALL), 8 patients with relapsed ALL (rALL), and 5 patients with acute nonlymphoblastic leukemia (ANLL)] were evaluated for their sensitivity to methotrexate (MTX) and five novel antifolate drugs, which have the potential to circumvent MTX resistance. The novel antifolates include a polyglutamatable [edatrexate (EDX)] and a lipophilic (trimetrexate) inhibitor of dihydrofolate reductase and two polyglutamatable inhibitors (ZD1694 and GW1843U89) and one lipophilic inhibitor (AG337) of thymidylate synthase (TS). Drug activity was assessed via the determination of in situ inhibition of TS activity after exposing leukemic cells to antifolate drugs for: (a) 3 h, followed by a 15-h drug-free period; and (b) 18 h of continuous exposure. For human CEM leukemia cell lines with well-defined mechanisms of resistance to MTX, in situ TS inhibition correlated with the growth-inhibitory effects of MTX and the novel antifolates (r = 0.86-0.93; P < 0.01). Although a wide interpatient variability in MTX sensitivity was observed within the three leukemia groups, the median drug concentration required to inhibit TS activity to 50% of untreated controls (TSI50) for a 3-h exposure to MTX was similar for iALL and rALL cells but was up to 9-fold higher in ANLL cells. After a 3-h exposure, EDX, ZD1694, and GW1843U89 displayed a markedly (10-150-fold) increased potency over MTX in all leukemia groups with comparable TSI50 values for ANLL and iALL cells. Compared with a 3-h MTX exposure, continuous exposure resulted in lower TSI50 values for iALL (14-fold), rALL (14-fold), and ANLL cells (85-fold). In comparison to MTX, the TSI50 values in these groups were also lower for EDX (1.6-3.5-fold), ZD1694 (2.1-4.3-fold), and GW1843U89 (15-35-fold). On short-term exposure, the lipophilic drugs trimetrexate and AG337 displayed markedly less potency as compared with that of long-term exposure. In conclusion, the efficacy of novel antifolates against childhood leukemia cells can be tested with the in situ TS inhibition assay. These novel antifolates displayed a greater efficacy than MTX against childhood leukemia cells and may have potential for the circumvention of MTX resistance in ANLL cells.
authors.affiliation: Department of Medical Oncology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Mauritz
authors.forename: M W
authors.initials: MW
authors.lastname: Bekkenk
authors.forename: M G
authors.initials: MG
authors.lastname: Rots
authors.forename: R
authors.initials: R
authors.lastname: Pieters
authors.forename: E
authors.initials: E
authors.lastname: Mini
authors.forename: C H
authors.initials: CH
authors.lastname: van Zantwijk
authors.forename: A J
authors.initials: AJ
authors.lastname: Veerman
authors.forename: G J
authors.initials: GJ
authors.lastname: Peters
authors.forename: G
authors.initials: G
authors.lastname: Jansen
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000627:therapeutic use*; D002648:Child; D019008:Drug Resistance, Neoplasm; D005493:Folic Acid Antagonists / Q000627:therapeutic use*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy*; D008727:Methotrexate / Q000627:therapeutic use*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D014407:Tumor Cells, Cultured
chemical_list: D000964:Antimetabolites, Antineoplastic; D005493:Folic Acid Antagonists; D013940:Thymidylate Synthase; D008727:Methotrexate