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−.

Description: RICBT using cyclosporine (CSP) and short-term mycophenolate mofetil (MMF) was performed on adult patients with high-risk hematological malignancies, from which data on complication measures and transplantation indications was obtained. From November 2003 to January 2005, RICBT was performed a total of 16 times on 14 patients. All patients were adults diagnosed with high-risk hematological malignancies, and 12 patients were not in remission. The average age of the patients was 57 years (range: 31–72 years). The average body weight of the patients was 59.1 kg (range: 48–72). The HLA match of two patients was 5/6 and 12 patients was 4/6. The average nucleated cell count was 2.55 x 107 cells/kg (range: 2.12–3.84 x 107 cells/kg). The conditioning regimen consisted of fludarabine and/or busulfan or cyclophosphamide and TBI (total body irradiation), and the intensity of therapy was adjusted based on age, disease, and systemic status. CSP and MMF were administered for GVHD (graft-versus-host disease) prophylaxis. The targeted duration of CSP and MMF administration was set at 100 and 28 days, respectively. Primary graft failure occurred in two patients. RICBT was repeated, and while the second treatment was successful in one patient, the other patient died of brain hemorrhage. Granulocyte recovery was observed in 13 patients on an average of 21 days, and platelet recovery was noted in 11 patients on an average of 43 days. Acute GVHD was assessed in 13 patients and confirmed in 12 patients, but a condition of grade II or above was seen in only three patients. Chronic GVHD was assessed in 12 patients and confirmed in six patients (limited type in five patients and extensive type in one patient). For the 16 treatments, the 100-day TRM (transplantation related mortality) was 19% (3 patients). Of the 14 patients, five remained alive and the statistically calculated one-year survival rate was 37%. Cause of death was recurrence in three patients, acute GVHD in one patient, viral infection in three patients, cerebral hemorrhage in one patient, and sudden death in one patient. Non-recurrence deaths included four patients with a past history of allogeneic transplantation, two patients with serious organ damage, and two patients with diabetic complications (two patients died of multiple causes). Of eight patients, excluding the six patients with past histories of allogeneic transplantation, serious organ damage, or diabetic complications, the 100-day TRM was 0% and the statistically calculated one-year survival rate was 54.7%. Two patients with VZV (varicella-zoster virus) infections died on days 221 and 228. While the risk of graft failure is generally high for RICBT, the success rate for the present RICBT technique was extremely high. This technique also made it possible to induce proper GVHD without steroidal agents. The low recurrence rate appeared to indicate the GVL (graft versus leukemia) effects of RICBT. The high GVL effects and low 100-day TRM observed appeared to be attributable to short-term MMF administration. We believe viral infections must be prevented for a lengthy period after the cessation of immunosuppressant therapy. Our results suggest that a past history of allogeneic transplantation, severe organ damage, or diabetic complications may constitute risk factors.

Description: Multicentric Castleman’s disease (MCD) is an atypical lympho- proliferative disorder which is closely associated with dysregulated overproduction of interleukin-6 (IL-6). In the previous report, we showed that the humanized anti-IL-6 receptor monoclonal antibody, tocilizumab (formerly called MRA), was therapeutically effective for the patients with MCD and the safety profile was acceptable relative to the clinical benefit. We also found that serum IgE levels were elevated in some patients with Castleman’s disease, which also decreased by IL-6 blocking therapy, suggesting that IL-6 may be involved in IgE production in vivo. To examine whether or not IL-6 serves as a regulator for IL-4, a major class switching factor for IgE, in vivo, we analyzed serum IL-4 as well as IgE levels in patients with MCD before and after the blockade of IL-6 actions utilizing humanized anti-IL-6 receptor monoclonal antibody in the 4-month clinical trial. Twenty-eight patients with MCD were enrolled and received intravenously 8 mg/kg of tocilizumab every two weeks for a total of 4 months. In fifteen of twenty-eight patients, 5–20 mg/day of prednisolone was used 4 weeks prior to study dosing, but the dosage of each patient was not changed during treatment. Serum IL-4 and IgE levels as well as inflammatory markers were monitored. This study complied with all provisions of the Declaration of Helsinki and was conducted in accordance with Good Clinical Practice guidelines. All patients gave written informed consent before participating in this study. Tocilizumab treatment improved the systemic inflammatory manifestations and laboratory markers such as CRP and fibrinogen as we previously reported. Before tocilizumab treatment, serum IL-4 and IgE levels were elevated (median 14.9 pg/mL, range 2.8–228 pg/mL and median 995 IU/mL, range 12–8000 IU/mL, respectively, n=28). Serum IL-4 levels gradually decreased by tocilizumab treatment (median 8.0 pg/mL, range 2.0–257 pg/mL at 6 weeks, n=28, p

Description: Although the Abelson (Abl) tyrosine kinase inhibitor imatinib mesylate has improved the treatment of breakpoint cluster region–Abl (Bcr-Abl)–positive leukemia, resistance is often reported in patients with advanced-stage disease. Although several Src inhibitors are more effective than imatinib and simultaneously inhibit Lyn, whose overexpression is associated with imatinib resistance, these inhibitors are less specific than imatinib. We have identified a specific dual Abl-Lyn inhibitor, NS-187 (elsewhere described as CNS-9), which is 25 to 55 times more potent than imatinib in vitro. NS-187 is also at least 10 times as effective as imatinib in suppressing the growth of Bcr-Abl–bearing tumors and markedly extends the survival of mice bearing such tumors. The inhibitory effect of NS-187 extends to 12 of 13 Bcr-Abl proteins with mutations in their kinase domain but not to T315I. NS-187 also inhibits Lyn without affecting the phosphorylation of Src, Blk, or Yes. These results suggest that NS-187 may be a potentially valuable novel agent to combat imatinib-resistant Philadelphia-positive (Ph+) leukemia.

Description: In Japan Adult Leukemia Study Group (JALSG) clinical trial, imatinib was used as initial therapy for 171 patients with chronic myelogenous leukemia. We measured levels of BCR-ABL transcripts in the blood of all patients in this trial before and 1, 2, 3, 6 and 12 months after the treatment. Levels of BCR-ABL transcripts were measured by a quantitative real-time polymerase-chain-reaction assay. Results were expressed copy number of BCR-ABL per 1 microgram of RNA. Median number of BCR-ABL transcript was 106,478 at diagnosis and 61328, 24213, 4981, 331 and 98 at 0, 1, 2, 3, 6, 12 months after the treatment, respectively. Three log reductions were obtained 0%, 35.5% and 50.9% of patients at 3, 6 and 12 months of the treatment. Half reduction time between 0 and 1 month (0M–1M) is 1.3 M. In the next month (1M–2M), it is 0.75M and those between 2–3 and 3–4.5 months are 0.44 and 0.51. Thereafter, reduction speed is down because half reduction time is 1.5 between 4.5 and 6 months. After 6 months half time is 4.1months. This reduction curve shows the. Exponential decline value per day are 1.8%, 3.1% and 5.2% during first, second and third month, respectively. Thereafter exponential decline value is decreasing as 4.4% (3M–4.5M), 1.5% (4.5M–6M) and 0.6% (6–12M). In the previous study reported that, successful therapy leads to a biphasic exponential decline of leukemic cells (Nature435:1267–1270, 2005). The first slope is determined by calculating the exponential decline between 0 and 3 months; mean value of 0.05 per day was observed. While the second slope showed 0.008 per day. Our observation suggests that imatinib leads to a triphasic exponential decline of leukemic cells; between 0 and 2 months, much slower declining curve in comparison with between 2–6 months.

Description: Chemical modifications of imatinib mesylate made with the guidance of molecular modeling yielded several promising compounds. Among them, we selected a compound denoted NS-187 (elsewhere described as CNS-9) on the basis of its affinity to Abl, and also to Lyn, which may be involved in imatinib-resistance (Figure). The most striking structural characteristic of NS-187 is its trifluoromethyl (CF3) group at position 3 of the benzamide ring. The presence of the CF3 group strengthened the hydrophobic interactionss of the molecule with the hydrophobic pocket of Abl. Another possible merit of the CF3 group is that it may fix the conformation of the drug by hindering its rotation at the 4-position of the benzamide ring; as a result, a CF3-bearing molecule may be more potent than more flexible compounds such as imatinib. In fact, NS-187 was 25–55 times more potent than imatinib in vitro and and at least 10 times more potent than in vivo. NS-187 also inhibited the phosphorylation and growth of all Bcr-Abl mutants tested except T315I at physiological concentrations. Another special feature of NS-187, in addition to its increased affinity to Abl is its unique spectrum of inhibitory activity against protein kinases. At a concentration of 0.1 μM, NS-187 inhibited only four of 79 tyrosine kinases, that is, Abl, Arg, Fyn, and Lyn. Notably, at 0.1 μM NS-187 did not inhibit PDGFR, Blk, Src or Yes. The IC50 values of NS-187 for Abl, Src and Lyn were 5.8 nM, 1700 nM and 19 nM, respectively, and those of imatinib were 106 nM, 〉10,000 nM and 352 nM, respectively. These findings indicate that NS-187 acts as a Bcr-Abl/Lyn inhibitor. In this respect, NS-187 may stand out among other novel Abl tyrosine kinase inhibitors, because BMS-354825 inhibits all members of the Src family, while AMN-107 inhibits none of the Src-family kinases. Our proposed docking models of the NS-187/Abl complex support the notion that NS-187 is more specific for Lyn than for Src. The amino acid at position 252 is either Gln or Cys in Src-family proteins. NS-187 inhibited the Gln252-bearing proteins Abl, Fyn and Lyn but had lower activity against the Cys252-bearing Src and Yes. This is probably because Gln, unlike Cys, readily forms hydrogen bonds. The distinguishing characteristic of NS-187, its high affinity for and specific inhibition of Abl and Lyn, may be useful in the treatment of Bcr-Abl-positive leukemia patients. Figure Figure

Description: CD19 is one of the most representative B-cell markers, and is widely used in diagnostic immunophenotyping in diffuse large B-cell lymphoma (DLBCL). However, it is known that the frequency of CD19 expression in DLBCL is less than that of CD20, and the clinical significance of CD19 expression has not yet been thoroughly examined. To clarify the clinical behavior of CD19-negative (CD19−) DLBCL, we have compared the clinical features, immunophenotype, and prognosis in relation to CD19 expression. The diagnosis of DLBCL was made according to the WHO Classification. We examined CD19 expression by means of immunohistochemistry using frozen sections with a monoclonal antibody, Leu12 (Becton Dickinson). Between 1987 and 2002, 227 cases of de novo DLBCL were examined the expression of CD19 in our laboratory. Anthracycline-containing chemotherapies were selected as the first-line treatment in 192 cases (85%). None was treated with rituximab. CD19 was expressed in 205 cases (90%), and 226 cases (99%) were positive for CD20. In 22 cases of CD19-negative (CD19−) DLBCL, the median age was 63 (39–79), and the male/female ratio was 11/11. According to CD19 expression, our DLBCL cases showed the following clinical features: male/female (CD19+ DLBCL 111/94, CD19− DLBCL 11/11: NS), age〉60 (70%, 55%: NS), PS〉1 (20%, 36%: P=0.07), sLDH〉1xN (44%, 73%: P=0.01), extranodal involvement〉1 site (12%, 18%: NS), stage III/IV (41%, 54%: NS), B symptom present (29%, 45%: NS). CD19− DLBCL expressed BCL2 protein less frequently than CD19+ DLBCL (P=0.03). The expression of CD5, CD10, CD21, BCL6, and MUM1 did not show a significant difference between CD19+ DLBCL and CD19− DLBCL. CD19− DLBCL showed significantly worse survival than CD19+ DLBCL (P=0.04, log-rank test). These findings suggest that the loss of CD19 expression in DLBCL is associated with high serum LDH level and poor prognosis. Simultaneous examination of CD19 and CD20 in diagnosis of DLBCL is recommended. Overall survival for patients with CD19+ DLBCL and with CD19− DLBCL. Overall survival for patients with CD19+ DLBCL and with CD19− DLBCL.

Description: Multipotent adult progenitor cells (MAPCs) are bone marrow-derived stem cells that have extensive in vitro expansion capacity and can differentiate in vivo and in vitro into tissue cells of all 3 germinal layers: ectoderm, mesoderm, and endoderm. The origin of MAPCs within bone marrow is unknown. MAPCs are believed to be derived from the bone marrow stroma compartment as they are isolated within the adherent cell component. Numerous studies of bone marrow chimeras in the human and the mouse point to a host origin of bone marrow stromal cells. Mesenchymal stem cells (MSCs), which coexist with stromal cells, have also been proven to be of host origin after allogeneic bone marrow transplantation in numerous studies. We report here that following syngeneic bone marrow transplants into lethally irradiated C57BL6 mice, MAPCs are of donor origin.

Description: Cyclin D is dysregulated in at least two-thirds of multiple myeloma (MM) tumors. In addition, recent reports showed that the dysregulation of cyclin D1 is frequent in the absence of a t(11;14) translocation in MM. However, as we also reported (Int J Oncol, 2004), there appears to be no obvious correlation between the expression of cyclin D1 and the proliferation index (PI) or Ki67 expression. Therefore, we thought that the down-regulation of cyclin D2 might offset the expression of cyclin D1 in myeloma cells with cyclin D1 overexpression in cDNA microarray, since primary myeloma cells or myeloma cell lines express cyclin D3 ubiquitously. Here we transfected cyclin D1 gene into a myeloma cell line (RPMI8226), originally not expressing cyclin D1, using a retrovirus-mediated gene transfer system. In this method we inserted a 1.1 kb fragment containing the open reading frame of cyclin D1 removing from a tet-cyclin D1 plasmid (kindly provided by Dr. Reed SI, MCB, 1994) into a retrovirus vector (pQCXIP). First, we analyzed the expression of cyclin D1 in the bulk culture of cyclin D1 transfectant. We detected the expression of cyclin D1 by western blot, and found that the limited numbers of transfectant expressed cyclin D1 protein by immunohistocytochemical staining. Subsequently, we separated the two types of cyclin D1 transfectant by limiting dilution. Both transfectants showed the expression of cyclin D1 mRNA in RT-PCR, however, one of the two did not show the expression of cyclin D1 protein in western blot and immunohistocytochemical staining. Interestingly, we clearly detected the down-regulation of cyclin D2 mRNA in the transfectant with cyclin D1 protein expression by RQ-PCR. Furthermore, we detected an increase of cells in S phase in the transfectant with cyclin D1 protein by flow cytometry. Unlike in the study of Lamb J et al. (Cell, 2003), we could not observe the induction of IL-6 by the transfection of cyclin D1 gene. Although the mechanism of the impairment of cyclin D1 translation is unclear, here we suggest that the lack of correlation between the expression of cyclin D1 and PI might be due to the impairment of cyclin D1 translation or the offset of the expression of cyclin D1 by the down-regulation of cyclin D2. We are now analyzing the effects of velcade and IMiDs on these transfectants, since we suspect that these differences would affect the response to chemotherapy for MM. Furthermore, we are going to analyze the difference of gene expression between these transfectants using cDNA microaray. Therefore, these transfectants could be useful materials to analyze the cyclin D1 dysregulation in myeloma cells.