Development of FF-10101, a Novel Irreversible FLT3 Inhibitor, Which Overcomes Drug Resistance Mutations
Authors: Nakatani Toshiyuki , Uda Ken , Yamaura Takeshi , Takasaki Masaru , Akashi Akimi , Chen Fangli , Ishikawa Yuichi , Hayakawa Fumihiko , Hagiwara Shinji , Kiyoi Hitoshi , Naoe Tomoki 
Published: October 2019

Abstract
Background: FLT3 is one of the most frequently mutated genes in acute myeloid leukemia (AML). Internal tandem duplication (ITD) of juxtamembrane domain sequence and missense point mutations at D835 residue within kinase domain are major mutations of FLT3 in AML. These mutations induce constitutive activation of FLT3 and its downstream pathway, resulting in aberrant cell proliferation of AML cells. FLT3 is, therefore, believed to be an attractive drug target for AML. Several FLT3 inhibitors were evaluated in clinical trials and they demonstrated clinical efficacy; however, drug-resistant secondary mutations such as F691L and D835 mutations with FLT3-ITD were often appeared. Therefore development of novel FLT3 inhibitors is required to overcome resistance to current FLT3 inhibitors. Here we report that a novel irreversible FLT3 inhibitor, FF-10101 is a promising agent for AML therapy.
Methods: Bone marrow samples from patients with AML were subjected to Ficoll-Hypaque density gradient centrifugation. Informed consent was obtained from all patients, and approval was obtained from the ethics committee of our institute. In vitro growth inhibitory assay was performed with leukemia cell lines and 32D transfectants in liquid culture and primary AML cells in semisolid culture. Cell viability was determined by MTS assay or ATP quantification assay. For in vivo efficacy study, leukemia xenograft mouse model was prepared by tale vein injection of MOLM-13 cells or primary AML cells. Efficacies of tested compounds were evaluated by detection of human CD45-positive cells in bone marrow cells obtained from femurs at the end of studies.
Results: Kinase profiling assay with 216 human recombinant kinases revealed that FF-10101 selectively and potently inhibited kinase activities of wtFLT3 and FLT3 D835Y with IC50 values of 0.20 nM and 0.16 nM, respectively. In MV4-11 cells, FF-10101 treatment decreased phosphorylation of FLT3 and its downstream molecules in a dose-dependent manner. FF-10101 treatment for 2 days demonstrated growth inhibitory effect on FLT3-dependent human cell lines, MV4-11, MOLM-13, MOLM-14 and 32D transfectants expressing FLT3-ITD with equal to or greater potency than Quizartinib, a highly potent FLT3 inhibitor currently under clinical development for AML (FF-10101 IC50=0.83 nM-2.4 nM, Quizartinib IC50=0.95 nM-4.5 nM). Cell cycle arrest was observed followed by increased sub-G1 population in MV4-11 cells treated with 1 nM FF-10101. Importantly, FF-10101 retained growth inhibitory activities against 32D transfectants expressing drug resistance mutations such as FLT3-ITD/D835Y, FLT3-ITD/Y842C or FLT3-ITD/Y842H with IC50 values of 0.66-3.1 nM, although Quizartinib demonstrated weak inhibitory effects with IC50 values of 85-150nM. In mice xenografted with MOLM-13, oral administration of 5 mg/kg FF-10101 once daily for 8 days significantly decreased MOLM-13 cells in bone marrow as compared to vehicle administration (p<0.001). Next, anti-leukemic effect of FF-10101 was assessed by using primary AML cells. In vitro cell growth assay, 1 week treatment of FF-10101 significantly reduced primary AML cells harboring FLT3-ITD. Growth inhibitory effect was also observed in primary AML cells harboring FLT3 D835H mutation, although Quizartinib had little effect. When 10 mg/kg FF-10101 was orally administrated twice daily to mice xenografted with primary AML cells with FLT3-ITD, AML cells in bone marrow were significantly reduced with comparable efficacy of Quizartinib. Furthermore, FF-10101 demonstrated more potent efficacy than Quizartinib in mice xenografted with primary AML cells harboring FLT3 D835H mutation. FF-10101 also retained its efficacy against mice xenografted with residual AML cells in Quizartinib-treated mice inoculated with primary AML cells harboring FLT3 D835H.
Conclusions: We have developed a novel irreversible FLT3 inhibitor, FF-10101. FF-10101 showed potent anti-leukemic effect on cell lines and primary AML cells by selective inhibition of FLT3 both in vitro and in vivo. Notably, FF-10101 also has potency against drug resistance mutations. These results strongly indicate that FF-10101 is a promising agent for AML patients with FLT3 mutations. Phase I study of FF-10101 for AML patients is planned for 2016.

Disclosures
Nakatani: FUJIFILM Corporation: Employment. Uda:FUJIFILM Corporation: Employment. Yamaura:FUJIFILM Corporation: Employment. Takasaki:FUJIFILM Corporation: Employment. Ishikawa:GlaxoSmithKline K.K.: Research Funding. Hagiwara:FUJIFILM Corporation: Employment. Kiyoi:Eisai Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; MSD K.K.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Teijin Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Japan Blood Products Organization: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Naoe:FUJIFILM Corporation: Patents & Royalties, Research Funding; Celgene K.K.: Research Funding; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties; Toyama Chemical CO., LTD.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding.