Despite the recent advances in the treatment of multiple myeloma (MM), MM patients with high-risk cytogenetic changes such as t(4;14) translocation or deletion of chromosome 17 still have extremely poor prognoses. With the goal of helping these high-risk MM patients, we previously developed a novel phthalimide derivative, TC11. Here we report the further characterization of TC11 including anti-myeloma effects in vitro and in vivo, a pharmacokinetic study in mice, and anti-osteoclastogenic activity. Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous tumors in human myeloma-bearing SCID mice. Immunohistochemical analyses showed that TC11 induced apoptosis of MM cells in vivo. In the pharmacokinetic analyses, the Cmax was 2.1 μM at 1 h after the injection of TC11, with 1.2 h as the half-life. TC11 significantly inhibited the differentiation and function of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts in mouse osteoclast cultures using M-CSF and RANKL. We also revealed that TC11 induced the apoptosis of myeloma cells accompanied by α-tubulin fragmentation. In addition, TC11 and lenalidomide, another phthalimide derivative, directly bound to nucleophosmin 1 (NPM1), whose role in MM is unknown. Thus, through multiple molecular interactions, TC11 is a potentially effective drug for high-risk MM patients with bone lesions. The present results suggest the possibility of the further development of novel thalidomide derivatives by drug designing.

Multiple myeloma (MM) is one of the hematological malignancy that is characterized by proliferation of malignant plasma cells. Recent advance in the treatment of MM using newly developed drugs, prognosis of the MM patients have been significantly improved. For example, immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide and pomalidomide have been developed for treatment of MM. However, IMiDs have only limited effects against MM patients with high risk chromosomal abnormalities such as t(4;14) and del17p (high-risk MM). In 2010, it was reported that IMiDs directly bind to cereblon (CRBN), a component of ubiquitin ligase 3 complex, and induced teratogenicity as well as anti-tumor effects. We have previously reported that a novel phthalimide derivative, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione (TC11) induced apoptosis against high-risk MM cells in vivo and in vitro, and inhibited differentiation of osteoclasts. We also reported that TC11 directly bound to α-tubulin and nucleophosmin-1 (NPM1), but did not bind to CRBN. However, TC11 was not well dissolved in water with only 0.02 mg/mL solubility. Therefore, TC11 showed poor absorption into blood and limited anti-tumor activity when it was intraperitoneally administered in tumor-bearing mice. To resolve these problems, we synthesized PEG(E)-TC11, in which TC11 is linked to polyethylene glycol through an ester bond, and consequently enhanced water solubility of PEG(E)-TC11 to 88.9 mg/mL. PEG(E)-TC11 revealed as potent growth inhibitory effect on high-risk MM cells as TC11 in vitro. In pharmacokinetic study, PEG-modification of TC11 improved the peak blood concentration (Cmax) from 2.6 to 24.4 μM and extended elimination half-life (t1/2) from 1.4 to 2.2 hr when 186 μM/kg of these compounds were intraperitoneally injected. More importantly, these pharmacokinetic improvement led to more potent growth inhibition of MM cells in vivo than TC11. We also explored mechanisms of anti-myeloma effect of PEG(E)-TC11 and found that PEG(E)-TC11 induced apoptosis via G2/M cell cycle arrest. However, unlike IMiDs family, BIACORE assay revealed that PEG(E)-TC11 didn’t directly bind to CRBN, indicating that growth inhibitory effect of PEG(E)-TC11 against MM cells was independent of binding to CRBN. In conclusion, PEGylation of TC11 significantly increased water solubility, resulted in potentiated anti-myeloma activity in vivo. Furthermore, PEG(E)-TC11 inhibited cell growth via G2/M arrest in a CRBN-independent manner. Thus, PEG(E)-TC11 is considered as a candidate compound for overcoming high-risk MM. Citation Format: Shuji Aida, Daiju Ichikawa, Kazuki Iida, Masashi Hozumi, Misa Nakamura, Ryo Uozaki, Nahoko Hashimoto, Mikio Okayama, Yuko Yonemura, Noriko Tabata, Taketo Yamada, Maiko Matsushita, Takeshi Sugai, Hiroshi Yanagawa, Yutaka Hattori. PEG(E)-TC11, a novel polyethylene glycol-linked phthalimide derivative, inhibited high-risk MM cell growth in vivo and in vitro via cell cycle G2/M arrest in a CRBN-independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5121. doi:10.1158/1538-7445.AM2017-5121

PURPOSE: Recent progress of the treatment of multiple myeloma (MM) has significantly improved prognosis. However, the MM patients with high-risk cytogenetic abnormalities still showed significantly shorter survival. The purpose of this study is to find compounds effective for high-risk myeloma with lower toxicity by developing chemical structure of the phthalimide. We also tried to isolate their binding molecules to understand molecular pharmacology of the compounds. RESULTS: (1) Screening and optimization of phthalimide derivatives: We screened library of synthetic phthalimide derivatives by their ability to induce apoptosis of MM cell line, KMS34, which has high-risk cytogenetic abnormalities such as t(4;14) and TP53 gene deletion. We finally found TC11, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione, which showed the most potent tumor growth inhibition. Based on the results of screening of phthalimide library, we found the two important points of chemical structure: (i) A 4-amino group lacked growth inhibitory effect, and thus, 5-amino branch is necessary for strong anti-tumor activity. (ii) Modification of 2,6-diisopropylphenyl in phenyl ring of TC11 significantly decreased inhibitory effects of tumor cell growth. (2) Anti-myeloma effects of phthalimide derivatives: TC11 showed significant growth inhibition of all myeloma cell lines (IC50=3-8μM) including those with high-risk cytogenetic changes. Lenalidomide also significantly inhibited growth of myeloma cell lines. However, 30μM of lenalidomide failed to inhibit growth of KMS34 and KMS28 cell lines that have high-risk cytogenetic alterations. TC11 also inhibited growth of bone marrow myeloma cells obtained from the MM patients. (3) Anti-myeloma effects of TC11 in vivo: Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous plasmacytoma in human myeloma cell (KMS34 and KMS11)-bearing SCID xenografts. In the pharmacokinetic analyses, the Cmax was 2.1μM at 1 h after the injection of TC11, with 1.2 h as the half-life. Cmax was 18.1μM at 1.5hr (Tmax), and T1/2 was 4.5hr, when 100mg/kg of TC11 was injected. Cmax was 2.1μM at 1.0hr (Tmax), and T1/2 was 1.2hr, if 20mg/kg was injected. In oral administration of 200mg/kg of TC11 in lcr mice, Cmax was 3.81μM at 8.0hr (Tmax), and T1/2 was 2.77hr. (4) Toxicity of TC11 and lenalidomide: When 20mg/kg of TC11 was injected to SCID-mice for two weeks, weight loss compared with control mice was not observed. Clonological assay using mice bone marrow cells showed that 10μM TC11 did not suppress the colony formation while 5μM lenalidomide significantly decreased the colony out put. Therefore, TC11 in the concentrations that induced apoptosis of myeloma cells unlikely caused systemic and hematological toxicity. (5) Binding molecules: To understand the molecular mechanism, we employed our unique in vitro screening system using mRNA display, in vitro virus (IVV) method. It was found that TC11 directly bound to nucleophosmin 1 (NPM1) and α-tubulin. Knock-down of NPM1 gene in myeloma cells significantly delayed tumor cell growth. However, cereblon (CRBN), which was considered to be associated with teratogenicity of thalidomide, was not included in TC11-binding molecules in IVV assay. TC11 lacked glutarimide moiety to which CRBN was reported to bind. Thus, TC11 revealed anti-myeloma activity in a CRBN-independent pathway and is conceptionally expected as a non-teratogenic thalidomide-related compound. Interestingly, lenalidomide also directly bound to NPM1. Further analyses were needed to elucidate biological significance of interaction of IMiDs with NPM1 and α-tubulin. CONCLUSION: Our results suggested the possibility that phthalimide derivatives induce tumor cell death independent of CRBN pathway. It was also suggested that drug design and modification of chemical structure of phthalimide enable us to develop further new thalidomide derivatives which have more potent antitumor activity and less toxicity. Disclosures Ichikawa: Takeda Pharmaceutical Company: Research Funding. Matsushita:Takeda Pharmaceutical Company: Research Funding. Hattori:MSD company: Research Funding; Ono Pharmaceutical company: Research Funding; Takeda Pharmaceutical Company: Consultancy; Novo Nordisk company: Research Funding; Mitsubisi Tanabe Pharm: Research Funding; Cosmic Corporation: Research Funding.

BACKGROUND: Despite recent advances in the use of newly developed drugs including immune-modulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide and proteasome inhibitors such as bortezomib, carfilzomib, and MLN9708, MM is still an incurable disease. In particular, MM patients harboring 17p deletion, t(14;16), t(14;20), or t(4;14) are classified as a high-risk group and have shown significantly shorter survival. With the goal of helping prolong the survival of these high-risk MM patients, we screened 29 synthetic phthalimide derivatives and found a novel compound, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione (TC11), which induced the apoptosis of KMS34 cells with t(4;14) and del17p13. PURPOSE:The purpose of this project is to clarify preclinical effects of the synthetic phthalimide derivative, TC11, on high-risk MM cell lines and osteoclasts. Namely, anti-myeloma and anti-osteoclastogenic activities and pharmacokinetic study in mice were shown. We also try to isolate directly binding molecules. Safety issues including hematological toxicities and teratogenicity were also discussed. METHODS AND RESULTS: TC11 significantly inhibited growth of MM cell lines (IC50 4-8μM) including KMS34 and KMS11 cells which have high-risk chromosomal abnormalities. TC11 also suppressed the proliferation of all of the bone marrow cells obtained from the MM patients, in a dose-dependent manner. TC11 increased annexin V-positive fraction and induced apoptosis. TC11 was injected intraperitonealy into myeloma (KMS34 and KMS11 cells)-bearing lcr/SCID mice, and anti-myeloma activity was evaluated in vivo. Twenty mg/kg of TC11 significantly inhibited growth of KMS34 or KMS11-derived plasmacytomas. Apoptosis of MM cells was observed by histopathological examination. In order to evaluate hematological toxicity of TC11, growth of colony-forming cells was examined. In the presence of 5μM of TC11, formation of CFCs was not significantly suppressed, suggesting low hematopoietic toxicity. In the pharmacokinetic analyses using lcr mice, the plasma concentrations of TC11 was examined; Cmaxwas 18.1μM at 1.5hr (Tmax), and T1/2 was 2.5hr, when 100mg/kg of TC11 was injected. If 20mg/kg was injected, Cmaxwas 2.1μM at 1.0hr (Tmax), and T1/2 was 1.2hr. Oral administration of TC11 to Icr mice was safely carried out, and results of pharmacokinetic study will be shown. Aiming at the therapeutic use of TC11 to bone disease, anti-osteoclastogenic activity was examined. Mouse bone marrow mononuclear cells were incubated in the presence of M-CSF and RANK-ligand. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts was reduced in number in the presence of 1μM of TC11. It was also found that 1μM of TC11 inhibited bone resorption by pit assay. We have identified nucleophosmin 1 (NPM1) and α-tubulin as TC11-binding molecules using our unique in vitro selection system using mRNA display, in vitro virus (IVV) method. However, cereblon (CRBN) was not detected as a TC11-binding protein by this method. The immunofluorescent analysis showed that TC11-treated cells exhibited elevated levels of α-tubulin fragmentation. Together with our previous observation of induction of centrosomal disruption of HeLa cells by NPM1-knock down, TC11 may cause anti-myeloma effects via mitotic catastrophe. CONCLUSION: We have demonstrated that TC11, a novel phthalimide derivative, has anti-tumor activity against MM cells with high-risk genetic abnormality including del 17p and t(4;14), in vitro and in vivo. This novel compound also down-regulates the differentiation and function of osteoclasts. Our data provide a strong preclinical rationale for TC11 as a safe and effective drug for the treatment of high-risk MM patients with bone disease. The actions of this drug relating to α-tubulin and NPM1 remain to be further investigated. TC11 exerts its anti-myeloma effect via molecular interactions which do not involve CRBN. In addition, TC11 does not form racemate and is expected to lack teratogenicity. The results of our present study suggest that new phthalimide derivatives other than thalidomide, lenalidomide and pomalidomide could be developed by drug designing for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

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