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.