Satoshi Konagai

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.

Abstract 1728: ASP8273, a novel mutant-selective irreversible EGFR inhibitor, inhibits growth of non-small cell lung cancer (NSCLC) cells with EGFR activating and T790M resistance mutations

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BACKGROUND: Reversible EGFR TKIs, gefitinib and erlotinib, have shown antitumor efficacy in NSCLC patients with activating mutations in EGFR kinase domain. But the clinical efficacy of these agents is limited by the development of acquired drug resistance, which is most commonly caused by T790M resistance mutation in EGFR. This mutation has been detected in approximately 50% to 60% of patients. The 2nd generation irreversible EGFR inhibitors inhibit EGFR with T790M, but their clinical efficacy to NSCLC patients with T790M appears to be limited due to severe adverse effects caused by concomitant WT EGFR inhibition. Therefore, an EGFR TKI which inhibits T790M mutant EGFR selectively with less activity against WT EGFR may be beneficial. Here we report ASP8273, a novel, small molecule EGFR TKI that inhibits the kinase activity of EGFR containing the activating and T790M resistance mutations with less activity against WT EGFR. METHODS: The inhibitory effect and the selectivity of ASP8273 were evaluated against mutant EGFR (L858R, del ex19, L858R/T790M and del ex19/T790M) and WT EGFR using in vitro enzymatic and cell-based assay. Binding mode of ASP8273 to EGFR was assessed by mass spectrometry. Antitumor activity of ASP8273 was evaluated in xenograft models using PC-9 (del ex19), HCC827 (del ex19), NCI-H1975 (L858R/T790M) and PC-9ER (Erlotinib Resistant)(del ex19/T790M) NSCLC cells. RESULTS: ASP8273 inhibited mutant EGFR containing del ex19 or L858R activating mutations as well as the T790M resistance mutation with lower IC50 values than WT EGFR. Mass spectrometry analysis revealed that ASP8273 is covalently bound to a mutant EGFR(L858R/T790M) via C797 in the kinase domain of EGFR. In NCI-H1975 cells, ASP8273 induced long-lasting inhibition of EGFR phosphorylation for 24 h after washout of compound. In assays using endogenously EGFR-dependent cells, ASP8273 inhibited the growth of PC-9(del ex19), HCC827(del ex19), NCI-H1975(del ex19/T790M) and PC-9ER(del ex19/T790M) with IC50 values of 8-33 nM, more potently than that of NCI-H1666(WT) with IC50 value of 230 nM. In mouse xenograft studies, ASP8273 induced tumor regression in NCI-H1975 (L858R/T790M), HCC827 (del ex19) and PC-9 (del ex19) xenograft models by repeated oral dosing in a dose-dependent manner. Dosing schedules did not affect the efficacy of ASP8273. In an NCI-H1975 xenograft model, complete regression of tumor was achieved after 14-days of ASP8273 treatment. Complete regression was maintained in 50% of mice more than 85 days after cessation of ASP8273 treatment. CONCLUSIONS: ASP8273 inhibits the growth of NSCLC cells with EGFR activating and T790M resistance mutations with evidence of tumor regression. Therefore, ASP8273 may show therapeutic efficacy in NSCLC patients with EGFR mutations. Clinical trials of ASP8273 in NSCLC patients are planned in the US/EU and Asia. Citation Format: Hideki Sakagami, Satoshi Konagai, Hiroko Yamamoto, Hiroaki Tanaka, Takahiro Matsuya, Masamichi Mori, Hiroyuki Koshio, Masatoshi Yuri, Masaaki Hirano, Sadao Kuromitsu. ASP8273, a novel mutant-selective irreversible EGFR inhibitor, inhibits growth of non-small cell lung cancer (NSCLC) cells with EGFR activating and T790M resistance mutations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1728. doi:10.1158/1538-7445.AM2014-1728

Abstract 2586: ASP8273 selectively inhibits mutant EGFR signal pathway and induces tumor shrinkage in EGFR mutated tumor models

Activating mutations of epidermal growth factor receptor (EGFR) are associated with the high sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) like erlotinib. However, acquired resistance limits the clinical efficacy of EGFR-TKIs, which is the most commonly caused by T790M mutation in EGFR. Second generation EGFR-TKIs such as afatinib are able to inhibit T790M mutation but the clinical efficacy in T790M positive patients is limited due to severe side effects associated with wild type (WT) EGFR inhibition. ASP8273 is a mutant-selective irreversible EGFR inhibitor currently in clinical trials (ClinicalTrials.gov Identifier: NCT02113813, NCT02192697). We have previously reported that ASP8273 covalently binds to mutant EGFR via C797 and inhibits kinase activity of mutant EGFR, which results in anti-tumor activity in xenograft models. To further explore the selectivity and the activity of ASP8273 on mutant EGFR, we evaluated effects of ASP8273 and other EGFR-TKIs on EGFR signal pathway, cell growth and anti-tumor activity. Phosphorylation of EGFR, ERK and Akt was determined by Western blot after treatment with EGFR-TKIs at 10, 100 and 1000nM. Apoptosis induction was analyzed by detecting caspase activity after 24h treatment with EGFR-TKIs. Anti-tumor activity of ASP8273 was evaluated in mice xenografted with HCC827 (deletion in exon 19 [del ex19]), NCI-H1975 (T790M/L858R), A431 (WT), and a patient derived LU1868 (T790M/L858R). ASP8273 selectively inhibited phosphorylation of EGFR and its down-stream signal pathway, ERK and Akt from 10nM in HCC827 and NCI-H1975 while inhibitory effects were only detected at 1000nM in A431.In NCI-H1650 (del ex19), ASP8273 inhibited cell growth with an IC50 value of 70nM while other EGFR-TKIs were only partially effective. ERK and Akt phosphorylation were diminished after ASP8273 treatment at 1000nM, however, other EGFR-TKIs only partially reduced the phosphorylation levels of these proteins. ASP8273 potently enhanced the caspase activity in NCI-H1650 after 24h treatment, which is concordant with signal and cell growth inhibitory effect. In HCC827 and NCI-H1975 xenograft models, ASP8273 induced tumor regression at 10, 30 and 100mg/kg without affecting body weight. ASP8273 also produced tumor growth inhibition from 10mg/kg in the NSCLC patient derived tumor xenograft, LU1868 which express T790M/L858R. On the other hand, ASP8273 did not produce significant tumor growth inhibition at 10 and 30mg/kg in A431 xenograft model. ASP8273 selectively inhibited mutant EGFR compared to WT EGFR in preclinical models, showing activity in mutant EGFR cell line which is resistant to other EGFR-TKIs including AZD9291 and CO-1686. These results indicate the potential of ASP8273 to induce tumor shrinkage in patients with mutant EGFR positive tumors including those that do not respond to other EGFR-TKIs despite EGFR mutation. Citation Format: Satoshi Konagai, Hideki Sakagami, Hiroko Yamamoto, Hiroaki Tanaka, Takahiro Matsuya, Shinya Mimasu, Yusuke Tomimoto, Masamichi Mori, Hiroyuki Koshio, Masaaki Hirano, Sadao Kuromitsu, Masahiro Takeuchi. ASP8273 selectively inhibits mutant EGFR signal pathway and induces tumor shrinkage in EGFR mutated tumor models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2586. doi:10.1158/1538-7445.AM2015-2586

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 2821: Anti-tumor activity of ASP3026, – A novel and selective ALK inhibitor -

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. 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 inhibited ALK kinase activity at an IC 50 value of 3.5 nmol/L, and showed more selective ALK inhibition in a Tyr-kinase panel than PF02341066. 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, with an IC 50 value of 64.8 nmol/L. This growth inhibition was accompanied with the decrease in phosphorylation of EML4-ALK protein, indicating that ASP3026 exerts its anti-proliferative activity through ALK kinase inhibition. The plasma and tumor concentrations of ASP3026 in mice xenografted with NCI-H2228 tumor after a 5-day repeated oral dosing of ASP3026 (10 mg/kg once daily) were determined using high-performance liquid chromatography-tandem mass spectrometry. Tmax values were 4 h in plasma and tumors. Cmax values at the corresponding doses were, respectively, 875 nmol/mL and 15500 nmol/g. A decrease of phophorylated EML4-ALK was confirmed 4 hours after a single administration of ASP3026 at 10 mg/kg by Western-blot analysis. The antitumor activities were evaluated using mice bearing subcutaneous NCI-H2228 tumor xenografts. ASP3026, administered as twice daily oral dosing for 14 days, induced dose dependent anti-tumor effects starting at 1 mg/kg with strong regression at 10, 30 and 100 mg/kg. No influence on body weights was observed in all dose range of ASP3026 treated-mice. In contrast, PF02341066 at twice daily oral dosing resulted in growth inhibition of NCI-H2228 xenografted tumors at 10 mg/kg, and tumor regression at 30 mg/kg. In addition, 100 mg/kg of PF02341066 was intolerable in this model. These results suggest that ASP3026 is a novel and selective ALK inhibitor, which is orally active, and will possibly target NSCLC patients possessing the EML4-ALK fusion. We are starting phase I clinical trials of ASP3026 in the near future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2821. doi:10.1158/1538-7445.AM2011-2821

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

Abstract B188: Preclinical antitumor activity of ASP8273, a mutant-selective irreversible EGFR inhibitor in an AXL-overexpressing NSCLC model

Background: Activating mutations of epidermal growth factor receptor (EGFR) are associated with the high sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) like erlotinib. However, acquired resistance limits the clinical efficacy of EGFR-TKIs. Several mechanisms of the acquired resistance have been described, including gatekeeper T790M mutation and MET amplification. Overexpression of AXL, a receptor type tyrosine kinase and/or its ligand, GAS-6 have been identified in a subset of NSCLC patients with acquired resistance, and inhibition of AXL has been shown to restore sensitivity to erlotinib in nonclinical resistant models, suggesting that AXL is also involved in EGFR-TKI resistance of NSCLC. ASP8273 is a mutant-selective irreversible EGFR inhibitor currently in clinical trials (ClinicalTrials.gov Identifier: NCT02113813, NCT02192697, NCT02500927). We have previously reported that ASP8273 selectively inhibited kinase activities of EGFR activating and T790M mutations compared to wild type EGFR, and induced tumor regression in HCC827 and NCI-H1975 xenografted mice. In this study, we evaluated the antitumor activity of ASP8273 against AXL overexpressing PC-9 cells. Method: PC-9 vec. cells and PC-9 AXL cells were constructed by the infection of pMXs-puro vector and AXL coding vector, respectively into PC-9, a NSCLC cell line with EGFR exon 19 del activating mutation. Inhibitory effects of ASP8273 and other EGFR-TKIs, including erlotinib on EGFR and AXL signaling and cell proliferation were investigated in PC-9 vec. and PC-9 AXL cells. In vivo antitumor effect was also examined in subcutaneously implanted with PC-9 AXL. Result: ASP8273 at 1 μmol/L inhibited cell growth associated with the inhibition of EGFR, AXL, AKT, and ERK phosphorylation in PC-9 AXL. On the other hand, EGFR-TKIs at 1 μmol/L did not show cell growth inhibition in PC-9 AXL cells, whereas those showed cell growth inhibition in PC-9 vec. cells. EGFR-TKIs inhibited the phosphorylation of EGFR but not AXL, AKT, and ERK in PC-9 AXL cells. In an in vivo xenograft model using PC-9 AXL, once-daily oral administration of ASP8273 at 50 mg/kg induced statistically significant tumor growth inhibition although the other EGFR-TKIs did not. Conclusion: These results suggest that overexpression of AXL confers resistance to EGFR-TKIs in NSCLC cells and ASP8273 may show antitumor activity against EGFR-TKIs-resistant NSCLC patients with AXL expression. Citation Format: Naoki Kaneko, Hiroaki Tanaka, Satoshi Konagai, Hiroko Yamamoto, Hideki Sakagami, Tomohiro Eguchi, Takahiro Matsuya, Masamichi Mori, Hiroyuki Koshio, Tadashi Terasaka, Masaaki Hirano, Sadao Kuromitsu, Masahiro Takeuchi. Preclinical antitumor activity of ASP8273, a mutant-selective irreversible EGFR inhibitor in an AXL-overexpressing NSCLC model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B188.

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