Leflunomide Inhibits Proliferation of Neoplastic B-Cell Lines and Induces Apoptosis in Primary CLL Cells.

Leflunomide is a commercially available immunosuppressive agent approved for the treatment of rheumatoid arthritis. The compound is administered orally and is rapidly converted to the active compound A77 1726. The half-life of A77 1726 is long, with values reported as 10–15 days. Steady state plasma concentrations, in the treatment of rheumatoid arthritis, approach 250 μM. The proposed mechanism of A77 1726 is a reduction in lymphocyte proliferation due to inhibition of de novo pyrimidine synthesis and inhibition of tyrosine kinases. Given the need for chemotherapeutics with activity against neoplastic B-cell diseases that possess favorable pharmacokinetic and side effect profiles, we examined the in vitro antiproliferative effect of A77 1726 upon neoplastic B-cell lines, and its in vitro cytotoxic effect upon primary CLL cells. Raji, Ramos, 697, WaC3CD5 and Daudi B-cell lines were treated with A77 1726 (0, 1, 10, 50, 100, 200 and 300 μM) for 24, 48 and 96 hrs in RPMI media supplemented with 10% fetal bovine serum. The antiproliferative effect of was determined using the MTT assay. A77 1726 IC₅₀ values for each cell line are: Raji (36 μM), Ramos (18 μM), 697 (29 μM), WaC3CD5 (83 μM) and Daudi (13 μM). Cell cycle analysis of Raji cells using propidium iodide (PI) staining with FACS analysis, showed a reduction of the fraction of cells in G2 from 19 % to 4.9 % with A77 1726 (200 μM) treatment. A77 1726 binds to albumin, diminishing its effective concentration. Human albumin (3 gm/dl) reduced the effectiveness of A77 1726 (200 μM) upon both Raji and WaC3CD5 cell lines. In the presence of albumin, the number of viable Raji cells increased from 32% to 74%, and for WaC3CD5 cells the value increased from 19% to 78%, as compared to the untreated control cells. Both cell lines multiplied slower in the presence of human albumin, thus the observed antiproliferative effect of A77 1726 was proportionally reduced. A77 1726 reduces de novo pyrimidine synthesis by inhibiting the enzyme dihydroorotate dehydrogenase. The reduction in de novo pyrimidine synthesis can be surmounted by the exogenous addition of uridine to the culture media. The Raji and WaC3CD5 cell lines were incubated with A77 1726 with and without uridine to determine the role of pyrimidine synthesis in A77 1726′s antiproliferative effect upon the cell lines. For the Raji cell line, addition of 50 μM uridine to A77 1726 (200 μM) treated cells increased the number of viable cells from 22% to 62% of the untreated control. For the WaC3CD5 cell line, the addition of uridine did not decrease the antiproliferative effect of A77 1726. These data agree with prior studies that indicated an antiproliferative effect of A77 1726 beyond its inhibition of de novo pyrimidine synthesis. CLL cells do not reproduce in vitro; however, we hypothesized that the pyrimidine-independent effect of A77 1726 may be cytotoxic to CLL cells in vitro. Five negatively selected primary CLL samples were treated with A77 1726 (0, 50, 100, 200 and 300 μM) for 120 hrs and cell viability was determined with the MTT assay. Significant (greater than 40 % of control value) cytotoxicity occurred with 200 μM A77 1726 in 3 of 5 samples. Treatment with 300 μM A77 1726 led to significant killing in all the samples; the mean viability, as compared to untreated control, was 36% (SD 21, N=5). Addition of uridine did not reverse the observed cytotoxic effect of A77 1726 upon CLL cells. Annexin V-FITC/PI staining with FACS analysis confirmed the cytotoxic effect of A77 1726 on CLL cells. Further study of leflunomide in animal models of neoplastic B-cell disorders is warranted.