ALBERT

Description: We have identified a specific dual Bcr-Abl/Lyn inhibitor, NS-187 (elsewhere described as CNS-9), which is 25–55 times more potent than imatinib against wild type Bcr-Abl in vitro. To evaluate the potential of NS-187 as a therapeutic agent, we assessed its in vivo activity. When Balb/c mice were given NS-187 orally at a dose of 30 mg/kg, the pharmacokinetic parameters were as follows: Tmax, 2 h; Cmax, 586 ng/ml; AUC0-∝, 2999 ng•h/ml; T1/2, 1.0 h; and bioavailability value (BA), 33%. The maximal tolerated dose (MTD) of NS-187 in Balb/c or Balb/c-nu/nu mice was 200 mg/kg/day (100 mg/kg, twice daily). To test the effect of NS-187 on in vivo tumor growth, Balb/c-nu/nu mice were injected subcutaneously with Bcr-Abl-positive KU812 cells on Day 0 and given NS-187 or imatinib orally twice a day from Day 7 to Day 17. At 20 mg/kg/day, imatinib inhibited tumor growth slightly, while at 200 mg/kg/day, it inhibited tumor growth almost completely. In contrast, at only 0.2 mg/kg/day NS-187 significantly inhibited tumor growth, while at 20 mg/kg/day it completely inhibited tumor growth without any adverse effects. The body weights of the treated tumor-bearing mice were not significantly different from those of untreated mice, even at a dosage of 200 mg/kg/day NS-187. Thus, NS-187 was at least 10-fold more potent than imatinib in vivo with complete inhibition of tumor growth as the end-point. We also tested the ability of NS-187 to suppress tumor growth in another murine tumor model, namely, Balb/c-nu/nu mice intravenously transplanted with BaF3 cells harboring wild type Bcr-Abl. The mice were treated orally with NS-187 or imatinib for 11 days starting on Day 1. All eight untreated mice and all eight mice treated with 400 mg/kg/day imatinib had died by Day 25 due to leukemic cell expansion, and NS-187 significantly prolonged the survival of the mice in a dose-dependent manner. We next examined the ability of NS-187 to block the in vivo growth of BaF3 cells harboring one of the Abl point-mutants M244V, G250E, Q252H, Y253F, T315I, M351T and H396P in Balb/c-nu/nu mice. These mice were treated with NS-187 or imatinib for 11 days starting on Day 1. NS-187 at 200 mg/kg/day significantly prolonged the survival of mice inoculated with BaF3 cells harboring any of these mutants except T315I compared with untreated or imatinib-treated mice (see Figure for an example). Thus, NS-187 was more potent than imatinib and could override the point-mutation-based imatinib-resistance mechanism in vivo. The efficacy and safety of NS-187 for Ph+ leukemias is expected to be verified by early-phase clinical trials. Figure Figure

Description: Recently, we have successfully identified human cord blood (CB)-derived CD34-negative (CD34−) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) method (Blood101:2924,2003). These CD34− SRCs could home into the BM niche only by IBMI, because they expressed lower levels of homing receptors including CXCR4. These CD34− SRCs did not express CD38 as well as c-kit. It is well documented that the tyrosine kinase receptors c-kit and flt3 are expressed and function in early mouse and human hematopoiesis. In murine model, it was reported that Lin−CD34−Sca−1+c-kit+flt3− cells supported long-term multilineage hematopoietic reconstitution. In contrast, Lin−CD34−Sca-1+c-kit+flt3+ cells are progenitors for the common lymphoid progenitor. More recently, these Lin−CD34−Sca-1+c-kit+flt3+ hematopoietic stem cells (HSCs) have been revealed to lack erythro-megakaryocytic potential. In this study, we have investigated the function of flt3 in our identified human CB-derived CD34− SRCs. First, we studied the SRC activity of CB-derived Lin-CD34+Flt3+/− or CD34−Flt3+/− cells using IBMI. Both CD34+FLT3+/− cells repopulated all 13 recipient mice. The level of human CD45+ cells in murine BMs received transplants of CD34+Flt3+ cells (29.3~90.8%, median 62.8%) was higher than those received transplants of CD34+Flt3− cells (9.8~45.1%, median 17.7%). On the other hand, only CD34−Flt3− cells repopulated all 7 recipient mice and the level of human CD45+ cells in murine BMs was 11.7~63.3% (median 37.9%). To further evaluate the long-term repopulating potential of these three populations, including CD34+Flt3+/− and CD34−Flt3− cells, BM cells obtained from each primary recipient mice were accessed for their SRC activities by secondary transplantation by IBMI. While CD34+Flt3+ cells did not show secondary repopulating activity, CD34+Flt3− cells could repopulate 83% (5/6) of secondary recipients. Moreover, CD34−Flt3− cells did repopulate all 5 secondary recipient mice with higher repopulating rate. Next, we cocultured CD34−Flt3−cells with the murine stromal cell line, HESS-5 in the presence of SCF, TPO, IL-3, IL-6, and G-CSF. After one week, significant numbers of CD34+Flt3− and CD34+Flt3+ cells were generated. Then we sorted these two populations, CD34+Flt3+/− cells, and tested their SRC activities by IBMI. Seven out of 10 and 5 out of 10 mice received CD34+Flt3+/− cells were repopulated with human cells, respectively. These results indicated that human CB-derived Lin−CD34−Flt3− cells produced CD34+Flt3− as well as CD34+Flt3+ SRCs in vitro. Our present study has demonstrated that human CB-drived CD34− SRCs do not express Flt3 tyrosine kinase receptor as did murine CD34− KSL cells. Based on these data, we propose that the immunophenotype of very primitive long-term repopulating human HSC is Lin−CD34−CD38−c-kit-Flt3−.