ALBERT

Description: Central nervous system (CNS) relapse accompanying the prolonged administration of imatinib mesylate has recently become apparent as an impediment to the therapy of Philadelphia chromosome–positive (Ph+) leukemia. CNS relapse may be explained by limited penetration of imatinib mesylate into the cerebrospinal fluid because of the presence of P-glycoprotein at the blood-brain barrier. To overcome imatinib mesylate–resistance mechanisms such as bcr-abl amplification, mutations within the ABL kinase domain, and activation of Lyn, we developed a dual BCR-ABL/Lyn inhibitor, INNO-406 (formerly NS-187), which is 25 to 55 times more potent than imatinib mesylate in vitro and at least 10 times more potent in vivo. The aim of this study was to investigate the efficacy of INNO-406 in treating CNS Ph+ leukemia. We found that INNO-406, like imatinib mesylate, is a substrate for P-glycoprotein. The concentrations of INNO-406 in the CNS were about 10% of those in the plasma. However, this residual concentration was enough to inhibit the growth of Ph+ leukemic cells which expressed not only wild-type but also mutated BCR-ABL in the murine CNS. Furthermore, cyclosporine A, a P-glycoprotein inhibitor, augmented the in vivo activity of INNO-406 against CNS Ph+ leukemia. These findings indicate that INNO-406 is a promising agent for the treatment of CNS Ph+ leukemia.

Description: Second-generation ABL tyrosine kinase inhibitors (TKIs) such as dasatinib, nilotinib and INNO-406 (formerly NS-187) have been developed to override imatinib-resistance mechanisms. We directly compared the growth-inhibitory effects in imatinib-sensitive and -resistant CML cell lines and the inhibitory profile for SRC family kinases (SFKs) among ABL TKIs. Dasatinib was the most potent inhibitor in all six CML cell lines evaluated (K562, BV173, KU812, MEG01, KT-1, MYL). Despite both nilotinib and INNO-406 being 2-phenylaminopyrimidine derivatives, INNO-406 demonstrated 2.5 times more activity than nilotinib against BV173 and KU812, although in the other four cell lines, there was no significant difference between these two TKIs. In three imatinib-resistant cell lines including K562/D1-9 (P-glycoprotein overexpressing), K562-IMR (BCR-ABL overexpressing) and MYL-R (BCR-ABL and LYN overexpressing), dasatinib also showed the greatest potency. INNO-406 was 3.1 times more effective than nilotinib against MYL-R, confirming a possible effect of INNO-406 against LYN. Nilotinib showed more potency than INNO-406 against K562/D1-9, suggesting less affinity to P-gp. None of the ABL TKIs inhibited the growth of Ba/F3/T315I. Dasatinib showed at least six-fold greater potency than nilotinib and INNO-406 against most Ba/F3 harboring BCR-ABL mutants. However, dasatinib was not effective against T315A, F317L and F317A, which have been detected in dasatinib-resistant CML patients. Interestingly, nilotinib and INNO-406 inhibited T315A, F317L and F317V (Table 1). To determine why dasatinib was ineffective against the T315A, F317L and F317V, the X-ray crystal structures of the dasatinib/ABL (PDB ID: 2GQG) and INNO-406/ABL (PDB ID: 2E2B) complexes were closely explored. While the P-loop of ABL closely locates INNO-406 and tightly grips INNO-406, it locates remotely from dasatinib and does not directly interact with dasatinib. The T315A, F317L and F317A mutations cause decreased steric and hydrogen-bonding interactions. Accordingly, the P-loop is likely to compensate these decreased interactions for INNO-406 but not for dasatinib. Next we compared the inhibitory effect of dasatinib and INNO-406 against SFKs. Dasatinib inhibited all eight SFKs at very low concentrations, while INNO-406 inhibited only LCK and LYN. In conclusion, dasatinib showed the strongest potency against BCR-ABL with less selectivity over SFKs. Nilotinib showed weaker affinity for SFKs compared to the other compounds, but was highly specific for ABL and may be useful for P-glycoprotein overexpressing leukemic cells. INNO-406 had intermediate affinity between dasatinib and nilotinib and inhibited LCK and LYN, in addition to ABL. Both nilotinib and INNO-406 were potent inhibitors of the dasatinib-resistant T315A, F317L and F317V BCR-ABL mutations. These findings should be useful for treating imatinib-resistant patients with second-generation ABL TKIs. IC50 values (nM) for cellular proliferation imatinib dasatinib nilotinib INNO-406 Ba/F3/T315I 〉 2000 〉 2000 〉 2000 〉 2000 Ba/F3/T315A 〉 2000 〉 2000 949.2 422.5 Ba/F3/F317L 〉 2000 〉 2000 929.8 293.5 Ba/F3/F317V 1053.7 〉 2000 286.9 284.0