Abstract
Resistance to, or intolerance of, imatinib mesylate in patients with CML has encouraged the development of other Bcr-Abl inhibitors. AMN107 is a novel oral aminopyrimidine ATP-competitive inhibitor of the protein tyrosine kinase activity of Bcr-Abl. Following oral administration to animals, AMN107 is well absorbed, has a good pharmacokinetic profile, and is well tolerated. The activity of AMN107, relative to imatinib, in both imatinib sensitive (KBM5 and KBM7) and corresponding imatinib-resistant (KBM5-STI571R1.0 and KBM7-STI571R1.0) cell lines was studied. KBM5 cells contain multiple copies of the Ph chromosome but lack the normal c-ABL. KBM7 cell line is near haploid. KBM5 and KBM7 cells differ in their response to imatinib exposure: no apoptosis with G0/G1 cell cycle arrest in KBM5 cells in contrast to apoptosis in KBM7 cells. KBM5-STI571R1.0 and KBM7-STI571R1.0 have an imatinib IC50 (concentration that kills 50% of the cells) about twenty times higher then the value in the corresponding parental cell line. KBM5- STI571R1.0 have a marginal increase in the BCR-ABL gene copy number, modest increase in p210 protein expression, but a highly imatinib-resistant Bcr/Abl tyrosine kinase activity associated with the acquisition of a single C-T change at ABL nucleotide 944 (T315I). KBM7-STI571R1.0 cells have no mutations within the ATP-binding domain of the Bcr/Abl, but have amplification of BCR/ABL gene and increased levels of expression of Bcr/Abl p210 protein, with decreased inhibition of the Bcr/Abl tyrosine kinase activity by imatinib, and loss of apoptotic response to imatinib. AMN107 showed higher potency in KBM5 cells than imatinib in suppressing cell growth (MTS assay) with IC50 values of 11.3 nM and 480.5 nM respectively. With KBM5-STI571R1.0, IC50 were 2418.3 nM and 6361.4 nM for AMN107 and imatinib, respectively. The IC50 for AMN107 and imatinib treatment in KBM7 cell line were 4.3 nM and 259.0 nM, respectively. In KBM7-STI571R1.0 IC50 were 97.2 nM and 2497.3 nM for AMN107 and imatinib, respectively. In experiments focused on cell cycle analysis and caspase-3 activity, the cell cycle distribution and apoptotic responses induced by imatinib and AMN107 appeared generally similar in all four ell lines, but at significantly lower concentrations of AMN107 (corresponding to findings in MTS assay). At a concentration of 2.5 mM, imatinib inhibited completely Bcr-Abl phosphorylation in both KBM5 and KBM7 cells, while AMN107 achieved the same effect at 125.0 nM In imatinib-resistant cells imatinib did not completely inhibit Bcr-Abl phosphorylation even when doses as high as 10.0 mM were applied, while AMN107 inhibited Bcr-Abl phosphorylation at 2.5 mM. The survival of SCID mice bearing KBM5 cells treated with AMN107 was significantly extended when compared with controls. Irradiated female five weeks old SCID mice were injected intraperitonealy (ip) with 2.4 x 107 KBM5 cells on Day 0. Treatment with AMN107 started on Day 20 and was given daily ip for 20 days. T/C survival ratios were 144%, 159%, and 182% for groups treated with 10, 20, and 30 mg/kg, respectively. AMN107 has significant activity against imatinib-sensitive and -</DEL><INS cite=mailto:fgiles dateTime=2004-07-28T15:36>i</INS><INS cite=mailto:fgiles dateTime=2004-07-28T15:35>matini</INS><INS cite=mailto:fgiles dateTime=2004-07-28T15:36>b </INS>resistant CML and warrants investigation in patients with CML.
A C-terminal protein-binding domain in the retinoblastoma protein regulates nuclear c-Abl tyrosine kinase in the cell cycle