ALBERT

Description: In CML, the presence of primary and acquired resistance to imatinib mesylate (IM) suggests the importance of developing new therapeutic agents that might be useful in case of suboptimal response to IM. In CML Bcr-Abl activates NF-k-B, which is involved in tumorigenesis. NF-k-B is activated by proteasomes, which also regulate the degradation of its inhibitor Ik-B. Proteasome blockers are thus optimal candidates for CML treatment. Several HIV protease inhibitors (PIs) and in particular saquinavir, at therapeutic concentrations, significantly inhibit human proteasome activities, NF-k-B activation and degradation of its inhibitor IkB. This supports a direct antitumor activity of PIs. The IM sensitive CML line K562 and the IM-resistant CML lines K562-R and KCL22-R have been exposed for 24, 48 and 72 hours to various concentrations of saquinavir alone or associated with imatinib 2μM. Viability was then evaluated by trypan blue exclusion and apoptosis by flow cytometry, using annexin V-FITC conjugates. Saquinavir inhibited proliferation and promoted apoptosis in cell lines in a dose and time related manner. Saquinavir IC50 after 72 hour culture was 43.9 μM in K562 and 6.0 and 9.6 μM in K562-R and KCL22-R, respectively. The addition of IM 2 μM caused a significant increase of the antiproliferative and pro-apoptotic effect of saquinavir. In the presence of IM, the saquinavir IC50 after 72 hours was 1.8 μM in K562 and 0.39 and 6.68 μM in K562-R and KCL22-R lines, respectively. Percentage of apoptotic cells in K562, K562-R and KCL22-R lines after 72 hour-exposure to different concentrations of saquinavir. CL 1 μM 5 μM 10 μM 20 μM 30 μM 40 μM CL =control K562 12.8 15.5 16.1 13.6 19.3 20.4 26.6 K562-R 12.7 15.6 16.4 28.4 70.2 80.7 55.3 KCL22-R 3.6 4.1 4.3 6.1 26.5 34.7 47.3 The effect of saquinavir on the CFU-GM growth from peripheral blood (PB) of three CML patients was also investigated: one 15 year old male in chronic phase at diagnosis, one 45 year old female in chronic phase during interferon treatment, one 60 year old male in blastic phase. Saquinavir inhibited CFU-GM growth not only in chronic phase at diagnosis and during INF treatment, but also in IM-resistant blastic phase. Saquinavir inhibits PB CFU-GM growth in CML patients saquinavir concentrations CML chronic phase at diagnosis CML chronic phase in INF treatment CML blastic phase Percentages of inhibition of CFU-GM growth in comparison with control cultures, performed without saquinavir. 10 μM 41% 60% 24% 20 μM 82% 60% 71% In conclusion, saquinavir exerts a clear dose-related inhibitory and pro-apoptotic effect on Bcr-Abl positive cell lines. It acts on K562-R at concentrations lower than in the parental line and similar to those achievable in vivo and maintained in HIV patients for prolonged periods of time and usually well tolerated. This activity is further enhanced by the association of IM. The observation that saquinavir inhibits PB CFU-GM growth in CML patients even in blastic phase further supports the possibility of utilizing this compound in vivo in a combined therapeutic approach for CML.