EX-VIVO ACTIVITY OF METHOTREXATE VERSUS NOVEL ANTIFOLATE INHIBITORS OF DIHYDROFOLATE-REDUCTASE AND THYMIDYLATE SYNTHASE AGAINST CHILDHOOD LEUKEMIA-CELLS

Leukemic cells of 27 children [14 patients with initial acute lymphobl astic leukemia (iALL), 8 patients with relapsed ALL (rALL), and 5 pati ents with acute nonlymphoblastic leukemia (ANLL)] were evaluated for t heir sensitivity to methotrexate (MTX) and five novel antifolate drugs , which have the potential to circumvent MTX resistance. The novel ant ifolates include a polyglutamatable [edatrexate (EDX)] and a lipophili c (trimetrexate) inhibitor of dihydrofolate reductase and two polyglut amatable inhibitors (ZD1694 and GW1843U89) and one lipophilic inhibito r (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 d rug-free period; and (b) 18 h of continuous exposure, For human CEM le ukemia cell lines with well-defined mechanisms of resistance to MTX, i n 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 wi de interpatient variability in MTX sensitivity was observed within the three leukemia groups, the median drug concentration required to inhi bit TS activity to 50% of untreated controls (TSI50) for a 3-h exposur e to MTX was similar for iALL and rALL cells but was up to 9-fold high er in ANLL cells. After a 3-h exposure, EDX, ZD1694, and GW1843U89 dis played a markedly (10-150-fold) increased potency over MTX in all leuk emia groups with comparable TSI50 values for ANLL and iALL cells. Comp ared with a 3-11 MTX exposure, continuous exposure resulted in lower T SI50 values for iALL (14-fold), rALL (14-fold), and ANLL cells (85-fol d). In comparison to MTX, the TSI50 values in these groups were also l ower 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 an d AG337 displayed markedly less potency as compared with that of long- term exposure. In conclusion, the efficacy of novel antifolates agains t childhood leukemia cells can be tested with the in situ TS inhibitio n assay. These novel antifolates displayed a greater efficacy than MTX against childhood leukemia cells and may have potential for the circu mvention of MTX resistance in ANLL cells.