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Supplementary Figure 1 from Tumor Regression in Mice by Delivery of Bcl-2 Small Interfering RNA with Pegylated Cationic Liposomes

Supplementary Figure 2 from Tumor Regression in Mice by Delivery of Bcl-2 Small Interfering RNA with Pegylated Cationic Liposomes

Supplementary Table 1 from Tumor Regression in Mice by Delivery of Bcl-2 Small Interfering RNA with Pegylated Cationic Liposomes

The pharmacokinetics and antitumor activity of pegylated small interfering RNA (siRNA)/cationic liposome complexes were studied after systemic administration to mice. We designed pegylated-lipid carriers for achieving increased plasma concentrations of RNA and hence improved accumulation of RNA in tumors by the enhanced permeability and retention effect. We compared the pharmacokinetics of siRNA complexed with liposomes incorporating pegylated lipids with longer (C-17 or C-18), shorter (C-12 to C-16), or unsaturated (C-18:1) acyl chains. When longer acyl chains were used, the plasma concentrations of siRNA obtained were dramatically higher than when shorter or unsaturated chains were used. This may be explained by the higher gel-to-liquid-crystalline phase-transition temperature (Tc) of lipids with longer acyl chains, which may form more rigid liposomes with reduced uptake by the liver. We tested a siRNA that is sequence specific for the antiapoptotic bcl-2 mRNA complexed with a pegylated liposome incorporating a C-18 lipid (PEG-LIC) by i.v. administration in a mouse model of human prostate cancer. Three-fold higher accumulation of RNA in the tumors was achieved when PEG-LIC rather than nonpegylated liposomes was used, and sequence-specific antitumor activity was observed. Our siRNA/PEG-LIC complex showed no side effects on repeated administration and the strength of its antitumor activity may be attributed to its high uptake by the tumors. Pegylation of liposomes improved the plasma retention, uptake by s.c. tumors, and antitumor activity of the encapsulated siRNA. PEG-LIC is a promising candidate for siRNA cancer therapy. [Cancer Res 2008;68(21):8843–51]

The trajectory of glomerular filtration rate (GFR) in relation to glomerular hyperfiltration (GHF) has been unknown. It was evaluated retrospectively in 23,982 GHF-free health examinees who were followed for 2−10 yr (mean: 5.1 yr). GFR was estimated by the serum creatinine concentration, and GHF was defined as age- and sex-specific estimated GFR (eGFR) ≥ 95% of the Japanese general population. The temporal profile of eGFR was plotted in a GHF-centered way, which was fitted to a random coefficient linear mixed model. Of the 23,982 subjects, 797 and 23,185 subjects developed or did not develop GHF, respectively, so that they were termed as the GHF(+)and GHF(−)groups. At baseline, median eGFR was significantly elevated in the GHF(+)group compared with in the GHF(−)group: 94.1 versus 77.3 mL/min/1.73 m2( P < 0.001). Elevation of basal eGFR lasted for a mean (SD) of 3.3 (1.9) yr in the GHF(+)group; mean eGFR then rose to the GHF range, which was 108.5 mL/min/1.73 m2. The eGFR decline after the peak was steeper in the GHF(+)group than in the GHF(−)group: −0.984 versus −0.497 mL/min/1.73 m2/yr ( P < 0.001). Baseline eGFR, but no other variable, well predicted incident GHF, with an area under the receiver operating characteristic curve of 0.87 (95% confidence interval: 0.86–0.88). In conclusion, GHF occurs as a chronic, multiphasic phenomenon: initially with a sustained GFR elevation for years, followed by a GFR surge to the GHF range, which was accompanied by accelerated GFR declining.

Aims To compare impact of elevated HbA1c and fasting plasma glucose (FPG) on incident chronic kidney disease (CKD) in a non-diabetic cohort. Methods Data from diabetes- and CKD-free 25,109 health examinees were retrospectively analysed with a mean observation period of 5.3 years. Prediabetes was diagnosed by the ADA and WHO criteria, and CKD by estimated glomerular filtration rate (eGFR) Results For incident CKD (n = 2483), high HbA1c but not FPG was an independent risk: adjusted hazard ratio (AHR, 95%CI) for HbA1c 1% and FPG 1 mmol/L, 1.91 (1.70–2.16) and 0.85 (0.60–1.20), respectively. Prediabetes by the ADA and WHO criteria were both risk for CKD with AHR (95%CI), 1.21 (1.12–1.32) and 1.31 (1.16–1.48), respectively. Prediabetes diagnosed by ‘elevated HbA1c irrespective of FPG’, either by the ADA and the WHO definition, was a risk with AHR (95%CI), 1.48 (1.36–1.61) and 1.51 (1.31–1.74), respectively. In contrast, prediabetes diagnosed by ‘raised FPG irrespective of HbA1c’ was not a CKD risk. Conclusions Elevated HbA1c, but not FPG, identified CKD risk in non-diabetic individuals.

Objective We aimed to clarify the onset of diabetes. Design Data from 27,392 nondiabetic health examinees were retrospectively analyzed for a mean of 5.3 years. Trajectories of fasting plasma glucose (FPG), body mass index (BMI), and the single point insulin sensitivity (Si) estimator (SPISE), an index of Si, 10 years before diagnosis of prediabetes (PDM; n = 4781) or diabetes (n = 1061) were separately assessed by a mixed effects model. Diabetes and PDM were diagnosed by the American Diabetes Association definition on the basis of FPG and glycosylated hemoglobin A1c values. Results In individuals who developed diabetes, mean FPG and BMI were significantly higher (P < 0.01 each) and SPISE lower than those who did not at −10 years: FPG 101.5 mg/dL vs 94.5 mg/dL, BMI 24.0 kg/m2 vs 22.7 kg/m2, and SPISE 7.32 vs 8.34, P < 0.01 each. These measurements, in subjects who developed prediabetes, were slightly but definitely different from those who did not, already at −10 years: FPG 91.8 mg/dL vs 89.6 mg/dL, BMI 22.6 kg/m2 vs 22.1 kg/m2, and SPISE 8.44 vs 8.82, P < 0.01 each. In both cases, the differences were progressively greater toward year 0, the time of diabetes, or PDM diagnosis. Conclusions FPG was significantly elevated in those who developed diabetes at least 10 years before diagnosis of diabetes, and this was also the case in those who developed PDM. Glucose dysregulation precedes diagnosis of diabetes at least for 20 years., FPG already increased at 10 years before diagnosis of diabetes and also at 10 years before diagnosis of prediabetes. Diabetes may begin at least >20 years before its diagnosis.

A hypothesis that postchallenge hyperglycemia in subjects with low body weight (BW) may be due, in part, to small glucose volume (GV) was tested. We studied 11,411 nondiabetic subjects with a mean BW of 63.3 kg; 5,282 of them were followed for a mean of 5.3 yr. In another group of 1,537 nondiabetic subjects, insulin sensitivity, secretion, and a product of the two (index of whole body insulin action) were determined. Corrected 2 h-plasma glucose (2hPGcorr) during a 75-g oral glucose tolerance test in subjects with BW ≤ 59 kg was calculated as 2hPGcorr = δPG2h · ECW/[16.1 (males) or 15.3 (females)] + fasting PG (FPG), where δPG2h is plasma glucose increment in 2 h; ECW is extracellular water (surrogate of GV); FPG is fasting plasma glucose; and 16.1 and 15.3 are ECW of men and women, respectively, with BW = 59 kg. Multivariate analyses for BW with adjustment for age, sex, and percent body fat were undertaken. BW was, across its entire range, positively correlated with FPG ( P < 0.01). Whereas BW was correlated with 2hPG and δPG in a skewed J-shape, with inflections at around 60 kg ( P for nonlinearity < 0.01 for each). Nonetheless, in those with BW ≤ 59 kg, insulin sensitivity, secretion, and action were unattenuated, and incident diabetes was less compared with heavier counterparts. BW was linearly correlated with 2hPGcorr, i.e., the J-shape correlation was mitigated by the correction. In conclusion, postchallenge hyperglycemia in low BW subjects is in part due to small GV rather than impaired glucose metabolism.

Aim Risk profile for incident chronic kidney disease (CKD) in Japanese subjects has not been established. Our aim was to identify risk factors for CKD in Japanese. Methods Consecutive 171 536 health examinees (median age 49 years and estimated glomerular filtration rate (eGFR) 78.2 mL/min per 1.73 m2) without CKD were re-examined after a median period of 6.2 years. Results of Cox proportional hazards models in randomly assigned two thirds (Derivation cohort) were verified in the rest (Validation cohort). CKD was defined as eGFR

Aim To clarify the natural course of prediabetes and develop predictive models for conversion to diabetes. Methods A retrospective longitudinal study of 2105 adults with prediabetes was carried out with a mean observation period of 4.7years. Models were developed using multivariate logistic regression analysis and verified by 10-fold cross-validation. The relationship between [final BMI minus baseline BMI] (δBMI) and incident diabetes was analyzed post hoc by comparing the diabetes conversion rate for low ( 2 ) and high δBMI (≥ −0.31kg/m 2 ) subjects after matching the two groups for the covariates. Results Diabetes developed in 252 (2.5%/year), and positive family history, male sex, higher systolic blood pressure, plasma glucose (fasting and 1h- and 2h-values during 75g OGTT), hemoglobin A1c (HbA1c) and alanine aminotransferase were significant, independent predictors for the conversion. By using a risk score (RS) that took account of all these variables, incident diabetes was predicted with an area under the ROC curve (95% CI) of 0.80 (0.70–0.87) and a specificity of prediction of 61.8% at 80% sensitivity. On division of the participants into high- ( n =248), intermediate- ( n =336) and low-risk ( n =1521) populations, the conversion rates were 40.1%, 18.5% and 5.9%, respectively. The conversion rate was lower in subjects with low than high δBMI (9.2% vs 14.4%, p =0.003). Conclusions Prediabetes conversion to diabetes could be predicted with accuracy, and weight reduction during the observation was associated with lowered conversion rate.

The quantitative impact of weight gain on prediabetic glucose dysregulation remains unknown; only one study quantitated the impact of weight loss. We quantified the impact of weight gain on the evolution and regression of prediabetes (PDM).In 4234 subjects without diabetes, using logistic regression analysis with a 4.8-year follow-up period, we analyzed the relationship between (1) δBMI (BMIMean (±s.d.) δBMI was 0.17 (±1.3) kg/mIn Japanese adults, an increase in the BMI by even 1 kg/m

To clarify if prediabetes defined by the International Expert Committee (PrediabetesIEC) and/or the American Diabetes Society (PrediabetesADA) is a risk for incident glomerular hyperfiltration (GH). 24,524 health examinees without diabetes, chronic kidney disease (CKD), GH and antihypertensive treatment at baseline, and repeated examinations at least twice during a mean of 5.3 years were retrospectively analysed. Diabetes was defined as fasting plasma glucose (FPG) ≥ 7.0 mmol/L and/or HbA1c ≥ 47 mmol/mol, CKD by estimated glomerular filtration rate (eGFR)

To develop diabetes risk score (RS) based on the current definition of diabetes, we retrospectively analyzed consecutive 4,159 health examinees who were non-diabetic at baseline. Diabetes, diagnosed by fasting plasma glucose (FPG) ≥7.0 mmol/L, 2hPG ≥11.1 mmol/L and/or HbA1c ≥6.5% (48 mmol/mol), developed in 279 of them during the mean period of 4.9 years. A full RS (RSFull), a RS without 2hPG (RS-2hPG) and a non-invasive RS (RSNI) were created on the basis of multivariate Cox proportional model by weighted grading based on hazard ratio in half the persons assigned. The RSs were verified in the remaining half of the participants. Positive family history (FH), male sex, smoking and higher age, systolic blood pressure (SBP), FPG, 2hPG and HbA1c were independent predictors for RSFull. For RS-2hPG, 7 independent predictors, exclusive of 2hPG and smoking but inclusive of elevated triglycerides (TG) comparing to RSFull, were selected. FH, male sex, and higher age, SBP and HbA1c were independent predictors in RSNI. In the validation cohort, C-statistic (95%CI) of RSFull, RS-2hPG and RSNI were 0.80 (0.76-0.84), 0.75 (0.70-0.78) and 0.68 (0.63-0.72), respectively, which were significantly different from each other (P

Risk profile for incident chronic kidney disease (CKD) in Japanese subjects has not been established. Our aim was to identify risk factors for CKD in Japanese.Consecutive 171 536 health examinees (median age 49 years and estimated glomerular filtration rate (eGFR) 78.2 mL/min per 1.73 mIn the Derivation cohort, CKD developed in 1002 (5.8%) subjects. Seven variables such as lower eGFR, male gender, higher uric acid concentration, lower red cell count and higher age and systolic blood pressure were identified as significant risks for CKD, with lowered eGFR being an overwhelmingly strong risk: adjusted hazard ratio for those with the baseline eGFR70 mL/min per 1.73 mSeven risk factors for incident CKD were identified in Japanese health examinees. However, lowered baseline eGFR outweighed other risks to the degree that eGFR alone was suffice for CKD prediction.

Imatinib mesylate (Gleevec™) has improved the treatment of Bcr-Abl-positive leukemia. However, resistance is often reported in patients with advanced-stage disease. Chemical modifications of imatinib made with the guidance of molecular modeling have yielded several promising compounds that could override imatinib resistance. Among them, we selected a compound denoted NS-187. The most striking structural characteristic of NS-187 is its trifluoromethyl group at position 3 of the benzamide ring, which strengthens the hydrophobic interactions and fixes the conformation of the compound. NS-187 was 25–55 times more potent than imatinib against wild-type Bcr-Abl in vitro. At physiological concentrations, NS-187 also inhibited the phosphorylation and growth of all Bcr-Abl mutants tested except T315I. In addition to Bcr-Abl, NS-187 also inhibited Lyn, which might be involved in imatinib resistance, without affecting the phosphorylation of Src, Blk, or Yes. This indicates that NS-187 acts as a dual Bcr-Abl/Lyn inhibitor. Our proposed docking models of the NS-187/Abl complex support the notion that NS-187 is more specific for Lyn than for Src. In Balb/c-nu/nu mice, which were injected subcutaneously with Bcr-Abl-positive KU812 cells, NS-187 showed at least tenfold more potency than imatinib. 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 or several mutations of Bcr-Abl (M244V, G250E, Q252H, Y253F, E255K, T315I, M351T, and H396P). NS-187 prolonged the survival of mice injected with leukemic cells expressing wild-type or all mutated Bcr-Abl except T315I, and its efficacy correlated well with its in vitro effects. NS-187 also inhibited leukemic cells harboring wild-type Bcr-Abl growth in the central nervous system, which sometimes becomes a sanctuary for leukemic cells under imatinib treatment. These results suggest that NS-187 may be a potentially valuable novel agent to combat imatinib-resistant Bcr-Abl-positive leukemia. A phase I study of NS-187 will start in 2006.

Advanced-phase chronic myeloid leukemia patients treated with imatinib often relapse due to point mutations in the Abl kinase domain. We herein examine the in vitro and in vivo effects of a Bcr-Abl/Lyn dual tyrosine kinase inhibitor, NS-187, on seven mutated Bcr-Abl proteins. NS-187 inhibited both Tyr393-phosphorylated and Tyr393-unphosphorylated Abl, resulting in significant in vitro growth inhibition of cells expressing six of seven mutated Bcr-Abl kinases, though not T315I. Furthermore, NS-187 prolonged the survival of mice injected with leukemic cells expressing all mutated Bcr-Abl tested except T315I, and its efficacy correlated well with its in vitro effects.

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.

NS-9 is a complex of polyinosinic-polycytidylic acid and a novel cationic liposome, LIC-101. The complex has strong cytotoxic activity against tumor cells derived from epithelial or fibroblastic cells. We have investigated the mechanism of the cytotoxic activity of NS-9 using knockdown cells in which the expression of proteins of interest was inhibited by RNA interference. NS-9 showed strong cytotoxic activity against knockdown cells with reduced expression of double-stranded RNA-dependent protein kinase, RNase L, or IFN-α/β receptor, but showed no cytotoxic activity against IFN regulatory factor-3 (IRF3) knockdown cells. In IRF3-knockdown cells, NS-9 also did not induce either the DNA fragmentation or the rRNA degradation observed in negative control cells. We conclude that IRF3 plays a crucial role in the cytotoxic activity of NS-9 against tumor cells, whereas RNA-dependent protein kinase, RNase L, or type I IFNs are not important for its activity.

Purpose: The RNA interference effect is an alternative to antisense DNA as an experimental method of down-regulating a specific target protein. Although the RNA interference effect, which is mediated by small interfering RNA (siRNA) or micro-RNA, has potential application to human therapy, the hydrodynamic method usually used for rapid administration of oligonucleotides is unsuitable for use in humans. In this study, we have investigated the antitumor activity of a synthetic siRNA, B717, which is sequence specific for the human bcl-2 oncogene, complexed with a novel cationic liposome, LIC-101. Experimental Design: In a mouse model of liver metastasis, we administered B717/LIC-101 by bolus intravenous injection, adjusting the rate and volume of administration to what is feasible in human therapy. In a mouse model bearing prostate cancer in which the cells were inoculated under the skin, B717/LIC-101 was administered subcutaneously around the tumor. Results: The B717/LIC-101 complex inhibited the expression of bcl-2 protein and the growth of tumor cell lines in vitro in a sequence-specific manner in the concentration range of 3 to 100 nmol/L. Furthermore, the complex had a strong antitumor activity when administered intravenously in the mouse model of liver metastasis. B717 (siRNA) was shown to be delivered to tumor cells in the mouse liver, but only when complexed with LIC-101. The complex also inhibited tumor cell growth in the mouse model bearing prostate cancer. Conclusions: By combining siRNA with our cationic liposome, we overcame the difficulty of administering siRNA to animals in ways that can be applied in human therapy. Although our siRNA/liposome complex is not yet in clinical trials, it is expected to provide a novel siRNA therapy for cancer patients.

Intestinal non-Hodgkin's lymphoma (NHL), especially the T-cell type, is well known to be associated with celiac disease (CD), an enteropathic disorder with a propensity for certain racial and genetic backgrounds. CD is typically characterized by gastrointestinal (GI) symptoms, anti-transglutaminase antibodies in the sera, and microscopical findings of the intestinal mucosa, which resolve with a gluten-free diet (GFD). In Asian populations, including the Japanese, CD and the associated NHL have been supposed to be quite rare, and studies concerning the frequency of CD or its relationship with NHL are scarce. We describe a Japanese middle-aged man with intestinal diffuse large B-cell lymphoma associated with CD. Following multi-combined chemotherapy, the patient's lymphoma has been in a state of complete response, and his GI symptoms have improved with a GFD. This case suggests that the possibility of CD and its association with intestinal NHL should be kept in mind, even in Asian populations.

Background & Aims The interferon (IFN) system plays a critical role in innate antiviral response. We presume that targeted induction of IFN in human liver shows robust antiviral effects on hepatitis C virus (HCV) and hepatitis B virus (HBV). Methods This study used chimeric mice harboring humanized livers and infected with HCV or HBV. This mouse model permitted simultaneous analysis of immune responses by human and mouse hepatocytes in the same liver and exploration of the mechanism of antiviral effect against these viruses. Targeted expression of IFN was induced by treating the animals with a complex comprising a hepatotropic cationic liposome and a synthetic double-stranded RNA analog, pIC (LIC-pIC). Viral replication, IFN gene expression, IFN protein production, and IFN antiviral activity were analyzed (for type I, II and III IFNs) in the livers and sera of these humanized chimeric mice. Results Following treatment with LIC-pIC, the humanized livers of chimeric mice exhibited increased expression (at the mRNA and protein level) of human IFN-λs, resulting in strong antiviral effect on HBV and HCV. Similar increases were not seen for human IFN-α or IFN-β in these animals. Strong induction of IFN-λs by LIC-pIC occurred only in human hepatocytes, and not in mouse hepatocytes nor in human cell lines derived from other (non-hepatic) tissues. LIC-pIC-induced IFN-λ production was mediated by the immune sensor adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1R domain-containing adaptor molecule-1 (TICAM-1), suggesting dual recognition of LIC-pIC by both sensor adaptor pathways. Conclusions These findings demonstrate that the expression and function of various IFNs differ depending on the animal species and tissues under investigation. Chimeric mice harboring humanized livers demonstrate that IFN-λs play an important role in the defense against human hepatic virus infection.

We report two patients with diffuse large B-cell lymphoma with isolated oculomotor nerve palsy. This was the initial manifestation of lymphoma in one patient but in a second, the palsy appeared as a part of a generalized recurrence of lymphoma. In addition, we reviewed the clinical findings of 12 previously reported patients. Isolated oculomotor nerve palsy was most frequently associated with the large B-cell lymphoma cell type, and was not frequently associated with periorbital pain. The pupil was spared in half the patients irrespective of infiltration of the oculomotor nerve. MRI and cerebrospinal fluid cytological examinations are helpful in determining invasion of lymphoma to the oculomotor nerve, cavernous sinus, and surrounding leptomeninges.

A 48-year-old woman with Caroli's disease accompanied by hepatolithiasis, choledocholithiasis, and cholangiocarcinoma is reported. Magnetic resonance imaging was useful for the diagnosis of the cholangiocarcinoma. The relationship between cholangiocarcinoma and hepatolithiasis, choledocholithiasis, and chronic cholangitis in Caroli's disease is discussed.

A cationic liposome such as LIC-101, which can mediate the rapid entry of siRNA into a majority of cells, would appear to be the ideal vector for in vivo delivery of siRNA. Our siRNA/LIC-101 complex has bcl-2-inhibitory and antiproliferative activity against various tumor cell lines. Furthermore, siRNA/LIC-101 can be safely administered to animals and it shows strong antitumor activity in mouse models of cancer. This in vivo result takes cancer therapy that includes siRNA one step closer to clinical use, with potential for application to various gene-targeting therapies. Finally, we hope that our cationic liposome can be developed as a uniform drug delivery system for various RNA medicines of the future, including siRNA, siRNA expression vectors, and ribozymes, in non-viral RNA delivery.

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

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

The penetration of imatinib mesylate (Gleevec™) into the central nervous system (CNS) is poor. Hence the CNS becomes a sanctuary site for patients who are on prolonged imatinib therapy. P-glycoprotein (P-gp) plays an important role in limiting the distribution of imatinib to the CNS, and it is well known that imatinib is a substrate of P-gp. We have recently identified a specific dual Bcr-Abl/Lyn inhibitor, NS-187, which can override imatinib-resistance. NS-187 was 25–55 and at least 10 times more potent than imatinib in vitro and in vivo, respectively. The purpose of this study was to investigate whether NS-187 can inhibit the growth of Ph+ leukemic cells in the CNS. In our preliminary pharmacokinetic study, the intracranial concentration of NS-187 was 10% of its serum concentration, suggesting the involvement in P-gp. To determine whether NS-187 is effluxed by P-gp, we examined the growth-inhibitory effects of NS-187 alone and in combination with a P-gp inhibitor, verapamil or cyclosporin A, on K562 cells and on a multidrug-resistant (MDR) K562/D1-9 cell line overexpressing P-gp. The K562/D1-9 cell line was 10 times more resistant to NS-187 than the parental K562 cell line, and P-gp inhibitors abolished this resistance, indicating that the action of NS-187, like that of imatinib, is affected by the P-gp-related MDR system. Even though NS-187 was found to be a substrate for P-gp, it inhibited the growth of K562/D1-9 cells at a concentration which could be achieved in the brain. we therefore tested the anti-tumor effects of NS-187 in murine CNS leukemia models. mice were inoculated into right cerebral ventricle with 1×105 BaF3/wt bcr-ablGFP cells (Balb/c-nu/nu mice) or 1×106 K562GFP cells (NOD/SCID mice). Five days after inoculation, mice were randomized into groups of 4 and orally administrated twice a day with vehicle, imatinib or NS-187 for 14 consecutive days. Sixteen days after inoculation, three mice from each group were sacrificed and their brains were examined under a fluorescent stereoscopic microscope. NS-187 inhibited the proliferation of leukemic cells in the brain, whereas imatinib did not. Moreover, NS-187 significantly prolonged the survival of the mice in a dose-dependent manner in both murine models compared with imatinib (Figure). In conclusion, NS-187 can inhibit Ph+ leukemic cell growth in the CNS in spite of efflux of the compound by P-gp. Figure Figure