Development of a Novel Phthalimide Derivative, Preclinical Effects on High-Risk Myeloma Cells and Osteoclasts

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; C_maxwas 18.1μM at 1.5hr (T_max), and T_1/2 was 2.5hr, when 100mg/kg of TC11 was injected. If 20mg/kg was injected, C_maxwas 2.1μM at 1.0hr (T_max), and T_1/2 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.