Hiroshi Fushiki

The Selective Anaplastic Lymphoma Receptor Tyrosine Kinase Inhibitor ASP3026 Induces Tumor Regression and Prolongs Survival in Non–Small Cell Lung Cancer Model Mice

Activation of anaplastic lymphoma receptor tyrosine kinase (ALK) is involved in the pathogenesis of several carcinomas, including non–small cell lung cancer (NSCLC). Echinoderm microtubule–associated protein like 4 (EML4)-ALK, which is derived from the rearrangement of ALK and EML4 genes, has been validated as a therapeutic target in a subset of patients with NSCLC. Here, we investigated the effects of ASP3026, a novel small-molecule ALK inhibitor, against ALK-driven NSCLC. ASP3026 inhibited ALK activity in an ATP-competitive manner and had an inhibitory spectrum that differed from that of crizotinib, a dual ALK/MET inhibitor. In mice xenografted with NCI-H2228 cells expressing EML4-ALK, orally administered ASP3026 was well absorbed in tumor tissues, reaching concentrations >10-fold higher than those in plasma, and induced tumor regression with a wide therapeutic margin between efficacious and toxic doses. In the same mouse model, ASP3026 enhanced the antitumor activities of paclitaxel and pemetrexed without affecting body weight. ASP3026 also showed potent antitumor activities, including tumor shrinkage to a nondetectable level, in hEML4-ALK transgenic mice and prolonged survival in mice with intrapleural NCI-H2228 xenografts. In an intrahepatic xenograft model using NCI-H2228 cells, ASP3026 induced continuous tumor regression, whereas mice treated with crizotinib showed tumor relapse after an initial response. Finally, ASP3026 exhibited potent antitumor activity against cells expressing EML4-ALK with a mutation in the gatekeeper position (L1196M) that confers crizotinib resistance. Taken together, these findings indicate that ASP3026 has potential efficacy for NSCLC and is expected to improve the therapeutic outcomes of patients with cancer with ALK abnormality. Mol Cancer Ther; 13(2); 329–40. ©2014 AACR.

YM155, a selective survivin suppressant, inhibits tumor spread and prolongs survival in a spontaneous metastatic model of human triple negative breast cancer.

Metastatic triple negative breast cancer [TNBC, with negative expression of estrogen and progesterone receptors and no overexpression of HER2/neu (ErbB-2)] remains a major therapeutic challenge because of its poor overall prognosis and lack of optimal targeted therapies. Survivin has been implicated as an important mediator of breast cancer cell growth and dysfunctions in apoptosis, and its expression correlates with a higher incidence of metastases and patient mortality; thus, survivin is an attractive target for novel anti-cancer agents. In previous studies, we identified YM155 as a small molecule that selectively suppresses survivin expression. YM155 inhibits the growth of a wide range of human cancer cell lines. Tumor regression induced by YM155 is associated with decreased intratumoral survivin expression, increased apoptosis and a decreased mitotic index. In the present study, we evaluated the antitumor efficacy of YM155 both in vitro and in vivo using preclinical TNBC models. We found that YM155 suppressed survivin expression, including that of its splice variants (survivin 2B, δEx3 and 3B), resulting in decreased cellular proliferation and spontaneous apoptosis of human TNBC cells. In a mouse xenograft model, continuous infusion of YM155 led to the complete regression of subcutaneously established tumors. Furthermore, YM155 reduced spontaneous metastases and significantly prolonged the survival of animals bearing established metastatic tumors in the MDA-MB-231-Luc-D3H2-LN orthotopic model. These results suggest that the survivin-suppressing activity of YM155 may offer a novel therapeutic option for patients with metastatic TNBC.

Brain penetration of telmisartan, a unique centrally acting angiotensin II type 1 receptor blocker, studied by PET in conscious rhesus macaques☆

INTRODUCTION Telmisartan is a widely used, long-acting antihypertensive agent. Known to be a selective angiotensin II type 1 (AT(1)) receptor (AT(1)R) blocker (ARB), telmisartan acts as a partial agonist of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and inhibits centrally mediated effects of angiotensin II in rats following peripheral administration, although the brain penetration of telmisartan remains unclear. We investigated the brain concentration and localization of telmisartan using (11)C-labeled telmisartan and positron emission tomography (PET) in conscious rhesus macaques. METHODS Three male rhesus macaques were bolus intravenously administered [(11)C]telmisartan either alone or as a mixture with unlabeled telmisartan (1mg/kg). Dynamic PET images were acquired for 95min following administration. Blood samples were collected for the analysis of plasma concentration and metabolites, and brain and plasma concentrations were calculated from detected radioactivity using the specific activity of the administered drug preparation, in which whole blood radioactivity was used for the correction of intravascular blood radioactivity in brain. RESULTS Telmisartan penetrated into the brain little but enough to block AT(1)R and showed a consistently increasing brain/plasma ratio within the PET scanning period, suggesting slow clearance of the compound from the brain compared to the plasma clearance. Brain/plasma ratios at 30, 60, and 90min were 0.06, 0.13, and 0.18, respectively. No marked localization according to the AT(1)R distribution was noted over the entire brain, even on tracer alone dosing. CONCLUSIONS Telmisartan penetrated into the brain enough to block AT(1)R and showed a slow clearance from the brain in conscious rhesus macaques, supporting the long-acting and central responses of telmisartan as a unique property among ARBs.

Synthesis, SAR study, and biological evaluation of novel quinoline derivatives as phosphodiesterase 10A inhibitors with reduced CYP3A4 inhibition

A novel class of phosphodiesterase 10A inhibitors with potent PDE10A inhibitory activity and reduced CYP3A4 inhibition was designed and synthesized starting from 2-[4-({[1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl]oxy}methyl)phenyl]quinoline (1). Replacement of pyridine ring of 1 with N-methyl pyridone ring drastically improved CYP3A4 inhibition, and further optimization of these quinoline analogues identified 1-methyl-5-(1-methyl-3-{[4-(quinolin-2-yl)phenoxy]methyl}-1H-pyrazol-4-yl)pyridin-2(1H)-one (42b), which showed potent PDE10A inhibitory activity and a good CYP3A4 inhibition profile. A PET study with (11)C-labeled 42b indicated that 42b exhibited good brain penetration and specifically accumulated in the rodent striatum. Further, oral administration of 42b dose-dependently attenuated phencyclidine-induced hyperlocomotion in mice with an ED50 value of 2.0mg/kg and improved visual-recognition memory impairment at 0.1 and 0.3mg/kg in mice novel object recognition test.

Abstract A227: Antitumor activities of ASP3026 against EML4-ALK-dependent tumor models.

EML4-ALK is an oncogenic fusion kinase, which was first identified in non-small cell lung cancer (NSCLC), and is regarded as an attractive therapeutic target for treating a subpopulation of NSCLC patients. Crizotinib, which is inhibitor for MET and ALK, was recently approved by FDA (26 August 2011) for patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) that is anaplastic lymphoma kinase (ALK)-positive as detected by an FDA-approved test. We synthesized and screened chemical compounds utilizing an ALK kinase inhibition assay aimed at the EML4-ALK target for drug discovery, and found ASP3026, a novel and selective inhibitor for the ALK kinase. ASP3026 potently inhibited ALK kinase activity and was more selective than crizotinib in a Tyr-kinase panel. In an anchorage independent in vitro cell growth assay, ASP3026 inhibited the growth of NCI-H2228, a human NSCLC tumor cell line endogenously expressing EML4-ALK variant 3 and that of 3T3 cells expressing EML4-ALK variant 1, 2 and 3. The plasma and tumor concentrations of ASP3026 in mice xenografted with NCI-H2228 tumor were determined using high-performance liquid chromatography-tandem mass spectrometry. Significant tumor penetration was observed. The antitumor activities were evaluated using mice bearing subcutaneous NCI-H2228 tumor xenografts. ASP3026, (daily oral dosing for 14 days) induced dose dependent anti-tumor effects starting at 1 mg/kg with marked regression at 10, 30 and 100 mg/kg. Body weights were unaffected. Crizotinib, (twice daily oral dosing) was less potent, with growth inhibition at 10 mg/kg, and tumor regression at 30 mg/kg. A dose of 100 mg/kg of crizotinib was poorly tolerated. Resistance mutations in ALK kinase domain against crizotinib were reported following sequence analysis of tumor cells derived from crizotinib-relapsed patients. The position of the mutation is the so-called gatekeeper mutation and is thought to be one of the causes of crizotinib relapse. In an EML4-ALK driven tumor model with gatekeeper mutation, ASP3026 showed potent anti-tumor effects while crizotinib was ineffective even at 100 mg/gk qd. In summary, ASP3026 has a broad safety margin and inhibitory activity at the gatekeeper mutation. Therefore, ASP3026 may still effective in EML4-ALK fusion positive NSCLC patients, that have relapsed to crizotinib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A227.

Abstract 2678: First demonstration of in vivo PET imaging for ALK inhibitor using [11C]ASP3026, a novel brain-permeable type of ALK inhibitor.

The recent breakthrough identifying the onco-driver fusion mutant of ALK kinase and its inhibitor crizotinib commercially termed as Xalkori has brought significant benefit to a portion of non-small cell lung cancer (NSCLC) patients. However, a number of clinical issues in ALK-positive lung cancer remain, involving resistance to crizotinib caused by secondary mutation, amplification of the ALK gene, activation of alternative pathways, and metastatic resistance, etc. Among these mechanisms, brain metastasis is a critical issue because of its poor prognosis. We have recently identified ASP3026 as a novel type of ALK inhibitor under development, and have reported that ASP3026 shows antitumor activities in several crizotinib-refractory models including gate keeper mutants. Here, we report the first PET imaging of an ALK inhibitor using [11C]ASP3026. The study has revealed that ASP3026 shows a brain tumor permeability in an intracranial xenograft model of H2228-luc ALK fusion positive cells. In this model, significant growth inhibition of H2228 intracranial tumor was observed by treatment with ASP3026 (10 mg/kg, q.d.), but not with crizotinib (10 mg/kg, q.d.) as determined by a bioluminescent imaging technique. Pharmacokinetic measurements of ASP3026 and crizotinib indicated that ASP3026 showed a four-fold better brain penetration than crizotinib on AUC0-24 base analysis, with a brain/plasma ratio=0.72 and 0.18 for ASP3026 and crizotinib, respectively. Further, we synthesized positron-labeled [11C]ASP3026 and performed PET imaging to clarify penetration of ASP3026 into cranial tumors. Quantitative analysis of [11C]ASP3026-PET data indicated that ASP3026 showed higher uptake into cranial tumors (SUV=3.0) than brain parenchyma (SUV=0.8). Moreover, comparison of pharmacokinetic profiles in several tumor models showed that tumor uptake of ASP3026 was higher than that of surrounding tissue, suggesting that tumor accumulation of ASP3026 was dependent on the microenvironment of tumor. Taken together, these results suggest that ASP3026 has favorable properties that may be useful for the treatment of brain metastases in ALK-positive NSCLC patients. Thus, PET imaging using 11C-labeled ASP3026 may allow the tumor penetration of ASP3026 to be clarified in any primary or metastatic tumor site. Citation Format: Hiroshi Fushiki, Rika Saito, Makoto Jitsuoka, Itsuro Shimada, Yutaka Kondoh, Hideki Sakagami, Yukiko Funatsu, Akihiro Noda, Yoshihiro Murakami, Sousuke Miyoshi, Yoko Ueno, Satoshi Konagai, Takatoshi Soga, Shintaro Nishimura, Masamichi Mori, Sadao Kuromitsu. First demonstration of in vivo PET imaging for ALK inhibitor using [11C]ASP3026, a novel brain-permeable type of ALK inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2678. doi:10.1158/1538-7445.AM2013-2678

Abstract 866: ASP3026, a selective ALK inhibitor, induces tumor regression in a crizotinib-refractory model and prolongs survival in an intrapleurally xenograft model

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL EML4-ALK translocation has been validated as a therapeutic target in a subset of non-small cell lung cancer (NSCLC) patients. In clinical settings, crizotinib has shown promising response rates in patients with EML4-ALK positive NSCLC, while disease relapse has been observed in several sites, including the lungs. Our previous studies found that ASP3026, a selective ALK inhibitor, had higher kinase selectivity than crizotinib and induced regression of tumors expressing EML4-ALK with a gate-keeper mutation that confers resistance to crizotinib. Here, we evaluated the antitumor activity of ASP3026 and crizotinib against orthotopic lung and intrapleural tumors in mice using luciferase-expressing NCI-H2228, a human NSCLC tumor cell line endogenously expressing EML4-ALK. In an orthotopic lung model, implanted cells were monitored using bioluminescent imaging (BLI) of the chest area. ASP3026 and crizotinib were administered orally at once daily doses ranging from 3 to 30 mg/kg (starting after confirmation of tumor growth). ASP3026 at 30 mg/kg induced tumor regression by 27 days after the start of administration; in contrast, crizotinib at 30 mg/kg induced regression during the first 7 days of administration, although tumors subsequently regrew despite continuous drug administration. After tumor regrowth had been established by the first cycle treatment of crizotinib, substantial regression was achieved by subsequent administration of ASP3026 at 30 mg/kg against the crizotinib-refractory tumors. In an intrapleural xenograft model, disease-related mortality was observed in tested animals, with a median survival time (MST) of 39 days in vehicle-treated control mice and 71 days in crizotinib-treated animals at 30 mg/kg. In contrast, no mice receiving ASP3026 treatment at 30 mg/kg died during the experimental period (90 days). These effects were accompanied by change in the bioluminescent emissions, with crizotinib-treated mice showing increased emissions following the initial reduction, while ASP3026-induced reduction continuing throughout the experimental period. The mean bioluminescent emission in the group treated with ASP3026 was significantly lower than in the crizotinib-treated group, indicating that ASP3026-treated animals exhibited lower tumor burden than crizotinib-treated ones.Taken together, these results suggest that ASP3026 is effective in producing shrinkage of tumors refractory to crizotinib and shows superior efficacy to crizotinib against intrapleural metastatic tumors with respect to prolonging survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 866. doi:1538-7445.AM2012-866

Investigation of Mechanisms for MK-801-Induced Neurotoxicity Utilizing Metabolomic Approach

Single treatment of rats with the noncompetitive N-methyl-D-aspartate receptor antagonist MK-801 induces neuronal cell degeneration and death in the retrosplenial/posterior cingulate cortex (RS/PC) region, along with local cerebral glucose utilization. However, the relationship between this neuronal cell degeneration and death and glucose utilization remains unclear. To investigate the mechanism of MK-801-induced neurotoxicity and its relation to glucose utilization, changes in endogenous metabolites in the RS/PC region of MK-801 treated rats were assessed using metabolomics. Inverse correlation between citrulline and arginine levels suggested increased nitric oxide (NO) production. In addition, decreased levels of purine metabolites suggested enhanced xanthine oxidase activity accompanied with reactive oxygen species (ROS) production. Histopathological analysis confirmed that the production of ROS in the RS/PC region was increased by MK-801 and that the nonspecific NO synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) prevented MK-801-induced neuronal cell death. These results suggest that NO increases oxidative stress-related cell death. Increased levels of metabolites of glucose metabolism suggested enhanced energy production via glycolysis. To confirm the relationship between NO and glucose utilization, positron emission tomography (PET) imaging with [(18)F] fluoro-2-deoxy-d-glucose ([(18)F] FDG) was conducted. [(18)F] FDG-PET imaging accompanied by co-treatment of L-NAME with MK-801 demonstrated that L-NAME ameliorated MK-801-induced glucose utilization.In conclusion, MK-801 induces NO and ROS production in the RS/PC region, which might subsequently induce oxidative stress and in turn neuronal cell death. In addition, MK-801-induced NO production increased glucose utilization and affected glucose metabolism, the imbalance of which might generate additional oxidative stress related to neuronal cell death.

Abstract 918: ASP3026, a selective ALK inhibitor, shows anti-tumor activity in a mouse model xenografted with NCI-H2228 intracranially.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC EML4-ALK translocation has been validated as a therapeutic target in a subset of non-small cell lung cancer (NSCLC) patients. Crizotinib, an FDA-approved ALK inhibitor, is effective against several types of human cancers with ALK abnormalities including EML4-ALK, RANBP2-ALK and ALK mutations. However, long-term treatment is often limited by the development of resistant tumors and distant metastases. Multiple brain metastases are a critical issue because of their poor prognosis despite the standard radiotherapy. The reason for metastases and development of tumor in the brain could be attributable to the poor penetration of crizotinib into central nervous system, as previously reported. ASP3026 is a selective ALK inhibitor which shows antitumor activities in several crizotinib-refractory models including gate keeper mutants. Here, we established an intracranial xenograft model by implanting NCI-H2228 cells directly in the brain of immunocompromised mice. Xenografted cells develop intracranial tumors which grow in murine brain and eventually become lethal, resembling the clinical tumors that metastasized in the brain. To determine the anti-tumor activity against tumors in the brain, mice were treated with ASP3026 or crizotinib for 2 weeks and held to observe survival duration. Ten, 30 and 100 mg/kg daily oral administration of ASP3026 dose dependently inhibited tumor growth in brain, and tumor regression was achieved in 30 and 100 mg/kg groups confirmed by MR imaging. These results were supported by the observation that Ktrans and IAUC (90s) were significantly decreased in 30 and 100 mg/kg groups using dynamic contrast enhanced MR imaging (DCE-MRI). Furthermore, ASP3026 significantly prolonged the survival of tumor bearing mice compared to vehicle treatment group. On the other hand, 30mg/kg daily oral administration of crizotinib inhibited the tumor growth during the treatment period but did not produce significant survival benefit. Taken together, these results indicate that ASP3026 shows better efficacy than crizotinib and improves overall survival in an intracranial mouse xenograft model, suggesting that ASP3026 may have potential to benefit EML4-ALK positive NSCLC patients with brain metastases. Citation Format: Satoshi Konagai, Hiroshi Fushiki, Hideki Sakagami, Yoko Ueno, Masamichi Mori, Itsuro Shimada, Yutaka Kondoh, Sousuke Miyoshi, Shintaro Nishimura, Sadao Kuromitsu. ASP3026, a selective ALK inhibitor, shows anti-tumor activity in a mouse model xenografted with NCI-H2228 intracranially. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 918. doi:10.1158/1538-7445.AM2013-918

Preclinical Evaluation of an Anti-Nectin-4 ImmunoPET Reagent in Tumor-Bearing Mice and Biodistribution Studies in Cynomolgus Monkeys

PurposeNectin-4 is selectively overexpressed in a variety of cancers and is currently under clinical investigation as a therapeutic target. A monoclonal antibody against nectin-4 (AGS-22M6) was evaluated as an Immuno-positron emission tomography (ImmunoPET) reagent. Its ability to assay nectin-4 expression as well as detect nectin-4 positive tumors in the liver and bone was evaluated using mouse models.ProceduresThe biodistribution of [89Zr]AGS-22M6 was evaluated in mice bearing tumors with varying levels of nectin-4 expression. An isogenic breast cancer tumor line was used to model metastatic liver and bone disease in mice. The biodistribution of [18F]AGS-22M6 in cynomolgus monkeys was evaluated.ResultsA positive correlation was demonstrated between tumor nectin-4 expression and [89Zr]AGS-22M6 uptake. Tumors in the liver and bone were detected and differentiated based on nectin-4 expression. [18F]AGS-22M6 showed limited uptake in cynomolgus monkey tissues.Conclusions[89Zr]AGS-22M6 is a promising ImmunoPET reagent that can assay nectin-4 expression in both primary and metastatic lesions.