ALBERT

Description: Central nervous system (CNS) relapse accompanying prolonged administration of imatinib mesylate, an Abl-specific tyrosine kinase inhibitor, 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 into the cerebrospinal fluid due to presence of P-glycoprotein (P-gp) at blood-brain barrier. To overcome imatinib-resistance mechanisms such as bcr-abl gene amplification, point mutations within ABL kinase domain, and activation of Lyn, we recently developed a specific dual BCR-ABL/Lyn inhibitor, INNO-406 (formerly NS-187), which is 25–55 times more potent than imatinib in vitro and at least 10 times more potent in vivo (Blood106: 3948–3954, 2005). The aim of this study was to investigate the efficacy of INNO-406 in treating CNS Ph+ leukemia. The intracellular accumulation of [14C]INNO-406 in P-gp overexpressing LLC-GA5-COL150 cells was much less than that in parental LLC-PK1 cells. The addition of 10 mM cyclosporin A (CsA) increased the intracellular accumulation of [14C]INNO-406 in both LLC-PK1 cells and LLC-GA5-COL150 cells. The peak concentration of INNO-406 in the brain when 30 mg/kg INNO-406 was administered p.o. was 50 ng/ g (87 nM), representing only 10% of plasma drug level. These findings suggested that INNO-406 is also a substrate of P-gp, as is imatinib. However, the residual concentration of INNO-406 in the CNS was enough to inhibit the growth of Ph+ leukemic cells according to the in vitro data. To increase the concentration of INNO-406 in CNS, we next examined the combined effects of CsA. In the brain, the concentration of INNO-406 was doubled following prior administration of 50 mg/kg CsA. Since pharmacokinetic studies suggested the possible effects of INNO-406 against CNS Ph+ leukemia, we investigated in vivo anti-CNS Ph+ leukemia effects of INNO-406 alone and combination of INNO-406 and CsA using immunodeficient mice (nude or NOD/SCID) which received Ph+ leukemic cells into the cerebral ventricle. INNO-406 alone inhibited growth of leukemic cells harboring either wild type or mutated BCR-ABL such as E255K and M351T in CNS. Furthermore, CsA significantly enhanced anti-CNS Ph+ leukemia effects of INNO-406 in vivo not only against cells harboring wild type BCR-ABL but also against cells harboring BCR-ABL/M351T (Figure). In conclusion, INNO-406 was found to inhibit Ph+ leukemic cell growth in CNS in spite of efflux of the compound by P-gp, and CsA augmented the anti-CNS Ph+ leukemia effects of INNO-406. Phase I clinical study on INNO-406 was initiated in the U.S.A. in July 2006. The efficacy and safety of INNO-406 in the treatment of leukemias is expected to be verified by early-phase clinical trials. Figure Figure

Description: Central nervous system (CNS) is one of the major cites for extramedullary relapse of Ph+ leukemias, which have been treated with imatinib mesylate (IM). The reason for this is that IM is a substrate for P-glycoprotein (P-gp) at the blood brain barrier and effluxed by it. We have already shown in the last annual meeting that INNO-406 had much stronger anti-tumor effects against the murine CNS leukemia model compared with IM, and INNO-406 is also effluxed from the murine CNS by P-gp. In this study, we investigated the combination effect of INNO-406 and P-gp inhibitors, verapamil or cyclosporin-A (CsA). First, we examined the growth-suppressive effect of INNO-406 and the combination with the P-gp inhibitors against the BCR-ABL positive leukemic cell line, K562 and the P-gp-overexpressing K562, K562/D1-9 cell line. K562/D1-9 showed 10 times higher resistant to both IM and INNO-406 compared with K562. Furthermore, both verapamil and CsA synergistically augmented the effect of INNO-406. Next, we investigated the pharmacokinetics of INNO-406 when orally administrated with CsA to mice. Mice were administrated p.o. with 50mg/kg of CsA 2 hours before INNO-406. We found that the concentration of INNO-406 in the CNS elevated by 2 times when combined with CsA, while the plasma concentration was decreased to two thirds of that when singly administrated with INNO-406. It was suggested that the decreased plasma concentration of INNO-406 seen here resulted from the increased uptake into the CNS by CsA inhibiting P-gp at the blood brain barrier. These changes of the drug distribution to the murine tissues may alter the anti-leukemic effect of INNO-406, thus we planned to investigate the combination effect of INNO-406 and CsA in the murine models of both CNS and systemic leukemia. We found that CsA significantly augmented the anti-tumor effect of INNO-406 in the CNS leukemia model. Moreover, in spite of the decreased plasma concentration of INNO-406, the combination with CsA also prolonged the survival phase of the mice in the systemic leukemia model, more significantly than single treatment of INNO-406 (Figure). This may be explained by which CsA increased the intracellular uptake of INNO-406, resulted from the direct inhibition of drug efflux via P-gp expressed in the leukemic cells. Phase I clinical study on INNO-406 is now underway in MD Anderson Cancer Center and in Frankfurt University. From the results of this study, we expected the effective application of INNO-406 in combination with P-gp inhibitor to the patients suffering from refractory Ph+ leukemia as well as CNS relapse. Figure Figure

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.