Cytotoxic Effect of R-Etodolac (SDX-101) in Combination with Purine Analogues or Monoclonal Antibodies on Ex-Vivo B-Cell Chronic Lymphocytic Leukemia Cells.

Background: SDX-101 (R-etodolac) is the R isomer of the non-steroidal anti-inflammatory drug Etodolac. SDX-101 decreased the in vitro survival of B-cell chronic lymphocytic leukemia (B-CLL) cells and showed synergistic activity with chlorambucil. In multiple myeloma cells, SDX-101 showed cytotoxic activity and displayed a synergistic cytotoxic effect with dexamethasone. Currently, SDX-101 is being tested in phase II clinical trial for treatment of refractory B-CLL in combination with chlorambucil.

Aims: To evaluate the ex-vivo cytotoxic effect of SDX-101 in combination with agents proven to be effective first line treatment in B-CLL: fludarabine (FA), cladribine (2-chlordeoxyadenosine, 2-CdA), anti-CD-52 (alemtuzumab, ALT) or anti-CD-20 (rituximab, RTX).

Methods: Tumor cells were obtained from 20 untreated patients with B-CLL. Cytotoxicity of SDX-101 and other study drugs was assessed using a propidium iodide-based flow cytometry assay. Apoptosis was assessed measuring activity of caspase-3 and DNA-content (sub-G1 fraction) by flow cytometry, and by measuring protein expression of p53 protein family members (p53 and p73), and BCl-2 family members (pro-apoptotic Bax, Bak and Bid; anti-apoptotic Bcl-2 and Mcl-1). The percentage of apoptotic cells and the expression of apoptosis-regulating proteins were measured at 0 h and 24 h. IC50 values were defined as the concentration of agents that achieved 50% decrease in cell viability. The combination index (CI) was used to estimate sub-additive (CI value 1.2), additive (0.8–1.2) or synergistic (<0.8) interactions.

Results: Dose-ranging experiments indicated that the SDX-101 IC50 in CLL cells was 800 μM, and that 400 μM of SDX-101 reduced CLL viability by 11% on average after 24 hrs. An SDX-101 dose of 400 μM was selected for combination studies with FA (used at 3.5 μM, yielding a viability reduction of 9.9%), 2-CdA (0.175 μM, 10.1%), RTX (20μg/ml, 6.1%), and ALT (20μg/ml, 8.4%). The combination of SDX-101+FA provided a drop of 20.9% in cell viability (p<0.03, vs. single drugs alone). The SDX-101+2-CdA combination reduced viability by 27.8% (p<0.006 vs. single drugs), SDX-101+RTX reduced viability by 22.3% (p<0.01 vs. single drugs), and that of SDX-101+ALT combination reduced it by 18.7% (n.s.). The CIs for those combinations were 0.89, 1.17, 0.95, and 1.25, respectively. Apoptosis analysis by sub-G1 fraction and caspase-3 activity confirmed the viability results for each tested combination. Analysis of apoptosis-regulating proteins showed, that SDX-101 combined with RTX or 2-CdA distinctly up-regulated Bax (p<0.02, vs. single drugs), whereas other combinations did not significantly increase its expression. The expression of p73 protein showed 2–4 fold increase after treatment with SDX-101 alone, and all combinations further up-regulated p73, especially that with RTX (up to 6-fold increase in p73 expression). Interestingly, although SDX-101 (400 μM at 24 hr) increased protein expression of Mcl-1, SDX-101 combination with RTX, 2-CdA or FA caused a 2-3-fold decrease of its expression.

Conclusion: SDX-101 in combination with low doses of either 2-CdA, FA or RTX exerts an additive cytotoxic effect on ex-vivo B-CLL cells, indicating a possible clinical benefit of such a combination treatment. Up-regulation of pro-apoptotic protein expression (p73 and Bax), and down-regulation of anti-apoptotic protein (Mcl-1) seem to play a mechanistic role in these additive cytotoxic effects.