Miyuki Yoshida

Selective ALK inhibitor alectinib with potent antitumor activity in models of crizotinib resistance

The clinical efficacy of the ALK inhibitor crizotinib has been demonstrated in ALK fusion-positive NSCLC; however, resistance to crizotinib certainly occurs through ALK secondary mutations in clinical use. Here we examined the efficacy of a selective ALK inhibitor alectinib/CH5424802 in models of crizotinib resistance. Alectinib led to tumor size reduction in EML4-ALK-positive xenograft tumors that failed to regress fully during the treatment with crizotinib. In addition, alectinib inhibited the growth of some EML4-ALK mutant-driven tumors, including the G1269A model. These results demonstrated that alectinib might provide therapeutic opportunities for crizotinib-treated patients with ALK secondary mutations.

Alectinib Shows Potent Antitumor Activity against RET-Rearranged Non–Small Cell Lung Cancer

Alectinib/CH5424802 is a known inhibitor of anaplastic lymphoma kinase (ALK) and is being evaluated in clinical trials for the treatment of ALK fusion–positive non–small cell lung cancer (NSCLC). Recently, some RET and ROS1 fusion genes have been implicated as driver oncogenes in NSCLC and have become molecular targets for antitumor agents. This study aims to explore additional target indications of alectinib by testing its ability to inhibit the activity of kinases other than ALK. We newly verified that alectinib inhibited RET kinase activity and the growth of RET fusion–positive cells by suppressing RET phosphorylation. In contrast, alectinib hardly inhibited ROS1 kinase activity unlike other ALK/ROS1 inhibitors such as crizotinib and LDK378. It also showed antitumor activity in mouse models of tumors driven by the RET fusion. In addition, alectinib showed kinase inhibitory activity against RET gatekeeper mutations (RET V804L and V804M) and blocked cell growth driven by the KIF5B-RET V804L and V804M. Our results suggest that alectinib is effective against RET fusion–positive tumors. Thus, alectinib might be a therapeutic option for patients with RET fusion–positive NSCLC. Mol Cancer Ther; 13(12); 2910–8. ©2014 AACR.

The Selective Class I PI3K Inhibitor CH5132799 Targets Human Cancers Harboring Oncogenic PIK3CA Mutations

Purpose: The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in cell proliferation and survival in human cancer. PIK3CA mutations, which are found in many cancer patients, activate the PI3K pathway, resulting in cancer development and progression. We previously identified CH5132799 as a novel PI3K inhibitor. Thus, this study aimed to clarify the biochemical and antitumor activity of CH5132799 and elucidate the correlation between CH5132799 response and genetic alterations in the PI3K pathway. Experimental Design: Kinase inhibitory activity was profiled in cell-free assays. A large panel of human breast, ovarian, prostate, and endometrial cancer cell lines, as well as xenograft models, were used to evaluate the antitumor activity of CH5132799, followed by analysis for genetic alterations. Effects on Akt phosphorylation induced by mTORC1 inhibition were tested with CH5132799 and compared with mTORC1 and PI3K/mTOR inhibitors. Results: CH5132799 selectively inhibited class I PI3Ks and PI3Kα mutants in in vitro kinase assays. Tumors harboring PIK3CA mutations were significantly sensitive to CH5132799 in vitro and were remarkably regressed by CH5132799 in in vivo mouse xenograft models. In combination with trastuzumab, tumors disappeared in the trastuzumab-insensitive breast cancer model with the PIK3CA mutation. Moreover, CH5132799 did not reverse a negative feedback loop of PI3K/Akt/mTOR signaling and induced regression against tumors regrown after long-term mTORC1 inhibitor treatment. Conclusions: CH5132799 is a selective class I PI3K inhibitor with potent antitumor activity against tumors harboring the PIK3CA mutations. Prediction of CH5132799 response on the basis of PIK3CA mutations could enable patient stratification in clinical settings. Clin Cancer Res; 17(10); 3272–81. ©2011 AACR.

Discovery and biological activity of a novel class I PI3K inhibitor, CH5132799

Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase and a promising therapeutic target for cancer. Using structure-based drug design (SBDD), we have identified novel PI3K inhibitors with a dihydropyrrolopyrimidine skeleton. Metabolic stability of the first lead series was drastically improved by replacing phenol with aminopyrimidine moiety. CH5132799, a novel class I PI3K inhibitor, exhibited a strong inhibitory activity especially against PI3Kα (IC(50)=0.014 μM). In human tumor cell lines with PI3K pathway activation, CH5132799 showed potent antiproliferative activity. CH5132799 is orally available and showed significant antitumor activity in PI3K pathway-activated human cancer xenograft models in mice.

Lead optimization of a dihydropyrrolopyrimidine inhibitor against phosphoinositide 3-kinase (PI3K) to improve the phenol glucuronic acid conjugation

Our lead compound for a phosphoinositide 3-kinase (PI3K) inhibitor (1) was metabolically unstable because of rapid glucuronidation of the phenol moiety. Based on structure-activity relationship (SAR) information and a FlexSIS docking simulation score, aminopyrimidine was identified as a bioisostere of phenol. An X-ray structure study revealed a hydrogen bonding pattern of aminopyrimidine derivatives. Finally, aminopyrimidine derivatives 33 showed strong tumor growth inhibition against a KPL-4 breast cancer xenograft model in vivo.

Metabolites of alectinib in human: their identification and pharmacological activity

Two metabolites (M4 and M1b) in plasma and four metabolites (M4, M6, M1a and M1b) in faeces were detected through the human ADME study following a single oral administration of [14C]alectinib, a small-molecule anaplastic lymphoma kinase inhibitor, to healthy subjects. In the present study, M1a and M1b, which chemical structures had not been identified prior to the human ADME study, were identified as isomers of a carboxylate metabolite oxidatively cleaved at the morpholine ring. In faeces, M4 and M1b were the main metabolites, which shows that the biotransformation to M4 and M1b represents two main metabolic pathways for alectinib. In plasma, M4 was a major metabolite and M1b was a minor metabolite. The contribution to in vivo pharmacological activity of these circulating metabolites was assessed from their in vitro pharmacological activity and plasma protein binding. M4 had a similar cancer cell growth inhibitory activity and plasma protein binding to that of alectinib, suggesting its contribution to the antitumor activity of alectinib, whereas the pharmacological activity of M1b was insignificant.

Modification of a dihydropyrrolopyrimidine phosphoinositide 3-kinase (PI3K) inhibitor to improve oral bioavailability.

Phosphoinositide 3-kinase (PI3K) is activated in various human cancer cells and well known as a cancer therapy target. We previously reported a dihydropyrrolopyrimidine derivative as a highly potent PI3K inhibitor that has strong tumor growth inhibition in a xenograft model. In this report, we describe further optimization to improve its bioavailability.

Abstract 754: Selective ALK inhibitor alectinib (CH5424802/RO5424802) with potent antitumor activity in models of crizotinib resistance, including intracranial metastases

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: EML4-ALK has been implicated as a driver oncogene and a therapeutic target in non-small cell lung cancer (NSCLC). The clinical efficacy of the ALK inhibitor crizotinib has been demonstrated in ALK fusion-positive NSCLC; however, resistance to crizotinib certainly occurs through ALK secondary mutations in clinical use. In addition, brain metastases are frequent sites of initial failure in patients because crizotinib has poor penetration of the central nervous system. This study aimed to clarify the efficacy of ALK inhibitor alectinib (CH5424802/RO5424802) in models of crizotinib-resistant ALK mutant tumors and intracranial tumors. Experimental Design: The antitumor activity of alectinib was evaluated in subcutaneous xenograft tumor models of Ba/F3 cells expressing mutated EML4-ALK, and in intracranial tumor implantation models of EML4-ALK-positive NSCLC NCI-H2228. Results: Alectinib had kinase inhibitory activity against some ALK mutations, including G1269A, and blocked tumor growth driven by a mutant of EML4-ALK in vitro and in vivo. In intracranial tumor implantation mouse models, alectinib resulted in regression of NCI-H2228 tumor in brain and provided a survival benefit. Alectinib is not a substrate for P-glycoprotein efflux transporter in vitro and showed a high brain-to-plasma ratio in rat. Additionally, alectinib led to tumor size reduction in the subcutaneous NCI-H2228 xenograft tumor that had failed to regress fully during treatment with crizotinib. Conclusions: Alectinib was effective against most EML4-ALK mutations and showed potent efficacy against intracranial ALK-positive tumor. Thus, alectinib might provide therapeutic opportunities for crizotinib-treated patients with ALK secondary mutations and brain metastases. Citation Format: Tatsushi Kodama, Toshiyuki Tsukaguchi, Masami Hasegawa, Miyuki Yoshida, Kenji Takanashi, Osamu Kondoh, Hiroshi Sakamoto. Selective ALK inhibitor alectinib (CH5424802/RO5424802) with potent antitumor activity in models of crizotinib resistance, including intracranial metastases. [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 754. doi:10.1158/1538-7445.AM2014-754

Optimization of the phenylurea moiety in a phosphoinositide 3-kinase (PI3K) inhibitor to improve water solubility and the PK profile by introducing a solubilizing group and ortho substituents

Phosphoinositide 3-kinase (PI3K) is a promising anti-cancer target, because various mutations and amplifications are observed in human tumors isolated from cancer patients. Our dihydropyrrolopyrimidine derivative with a phenylurea moiety showed strong PI3K enzyme inhibitory activity, but its pharmacokinetic property was poor because of lack of solubility. Herein, we report how we improved the solubility of our PI3K inhibitors by introducing a solubilizing group and ortho substituents to break molecular planarity.

Abstract 773: Alectinib shows potent antitumor activity against both ALK- and RET-rearranged non-small cell lung cancers

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA We have shown that alectinib is a potent ALK inhibitor and is being evaluated in clinical trials for the treatment of ALK fusion-positive non-small cell lung cancer (NSCLC). Recently, RET and ROS1 fusion genes have been implicated as driver oncogenes in 1-2% of NSCLC and have been developed into promising molecular targets for antitumor agents. Here, we investigate the additional target indication of alectinib by testing its ability to inhibit the activity of RET and ROS1 kinases. In enzyme assay, alectinib showed kinase inhibitory activity against RET as wells as ALK but did not show against ROS1. Alectinib inhibited the growth of LC-2/ad cells harboring CCDC6-RET and Ba/F3 cells expressing KIF5B-RET by suppressing RET phosphorylation. Alectinib also showed antitumor activity in mouse models of RET fusion-positive tumors (LC-2/ad cells and Ba/F3 cells expressing KIF5B-RET) and of ALK fusion-positive tumors (NCI-H2228 cells harboring EML4-ALK). In addition, alectinib showed kinase inhibitory activity against RET gatekeeper mutations (RET V804L and V804M) and blocked KIF5B-RET gatekeeper mutation-driven cell growth. Our results suggest that alectinib is effective against RET fusion-positive tumors, as observed in ALK fusion-positive tumors. Thus, alectinib might be a therapeutic option for patients with RET fusion-positive NSCLC. Citation Format: Tatsushi Kodama, Toshiyuki Tsukaguchi, Yasuko Satoh, Miyuki Yoshida, Yoshiaki Watanabe, Osamu Kondoh, Hiroshi Sakamoto. Alectinib shows potent antitumor activity against both ALK- and RET-rearranged non-small cell lung cancers. [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 773. doi:10.1158/1538-7445.AM2015-773