Antitumor Activity of IMC-038525, a Novel Oral Tubulin Polymerization Inhibitor

Abstract

Microtubules are a well-validated target for anticancer therapy. Molecules that bind tubulin affect dynamic instability of microtubules causing mitotic arrest of proliferating cells, leading to cell death and tumor growth inhibition. Natural antitubulin agents such as taxanes and Vinca alkaloids have been successful in the treatment of cancer; however, several limitations have encouraged the development of synthetic small molecule inhibitors of tubulin function. We have previously reported the discovery of two novel chemical series of tubulin polymerization inhibitors, triazoles (Ouyang et al. Synthesis and structure-activity relationships of 1,2,4-triazoles as a novel class of potent tubulin polymerization inhibitors. Bioorg Med Chem Lett. 2005; 15:5154-5159) and oxadiazole derivatives (Ouyang et al. Oxadiazole derivatives as a novel class of antimitotic agents: synthesis, inhibition of tubulin polymerization, and activity in tumor cell lines. Bioorg Med Chem Lett. 2006; 16:1191-1196). Here, we report on the anticancer effects of a lead oxadiazole derivative in vitro and in vivo. In vitro, IMC-038525 caused mitotic arrest at nanomolar concentrations in epidermoid carcinoma and breast tumor cells, including multidrug-resistant cells. In vivo, IMC-038525 had a desirable pharmacokinetic profile with sustained plasma levels after oral dosing. IMC-038525 reduced subcutaneous xenograft tumor growth with significantly greater efficacy than the taxane paclitaxel. At efficacious doses, IMC-038525 did not cause substantial myelosuppression or peripheral neurotoxicity, as evaluated by neutrophil counts and changes in myelination of the sciatic nerve, respectively. These data indicate that IMC-038525 is a promising candidate for further development as a chemotherapeutic agent.

Figure 1

Chemical structures of IMC-094332 and IMC-038525.

Figure 2

IMC-038525 inhibits polymerization in vitro, competes for the colchicine-binding site on tubulin, and disrupts microtubules in tumor cells. (A) Inhibition of tubulin polymerization in vitro. Purified tubulin in a GTP-containing buffer was incubated in presence or absence of increasing concentrations of IMC-038525 or colchicine at 10 µM, as a positive control. Assembly of tubulin into microtubules is measured by increasing absorbance at 340 nm. IMC-038525 inhibits tubulin polymerization in a dose-dependent manner. (B) Competition assay for the colchicine-binding site. Tubulin was incubated with radiolabeled (³H) colchicine at 37°C, and the amount of bound ³H colchicine was determined by scintillation of proximity beads. Dose-response curves were plotted showing inhibition of colchicine binding by IMC-094332, IMC-038525, and combretastatin A-4 (positive control). (C) Immunofluorescence staining of microtubules in tumor cells. A431 (a and b) and NCI-ADR-RES cells (c and d) were treated with DMSO (a and c) or IMC-038525 at 100 nM (b and d), for 30 minutes. After treatment, cells were fixed in methanol, and microtubules stained with an antitubulin antibody and a secondary Alexa-488 secondary antibody. Epifluorescence micrographs show an intricate microtubule network in control cells (a and c), absent in cells treated with IMC-038525 (b and d).

Figure 3

Antimitotic effect of IMC-038525. IMC-038525 causes mitotic arrest in tumor cell lines. (A) NCI/ADR-RES cells were treated with 0.1% DMSO (control), 10 and 0.5 nM IMC-038525 for 24 hours, DNA stained with propidium iodide, and cell cycle distribution determined by flow cytometry. (B) Dose-response curves were plotted for the effect of IMC-038525 in NCI/ADR-RES cells.

Figure 4

PK properties and in vivo antitumor activity of IMC-038525. (A) Rapid assessment of compound exposure analysis of IMC- 038525 dosed PO at 300 mg/kg and paclitaxel dosed IP at 20 mg/kg. Plasma samples were collected at 30, 60, and 240 minutes after injection into mice, and levels of compounds analyzed by LCMS. Plasma levels graph points represent mean values ± SEM (n = 3). (B) Growth of MDA-MB-435-LM2 xenografts in nude mice treated with vehicle, paclitaxel, or IMC-038525 (dosage regimens indicated on graph legend). (C) Body weight measurements of nude mice used in the previously described xenograft studies described. Tumor volume and body weight graph points represent mean values ± SEM (n = 10). Q7D indicates every 7 days.

Figure 5

Evaluation of toxicity caused by treatment with IMC-038525. (A) Assessment of neurotoxicity by morphologic evaluation of myelin sheaths in the sciatic nerve. Sciatic nerves of nude mice treated with vehicle (a), paclitaxel (b), or IMC-038525 (c) were thin-sectioned and stained with osmium tetroxide. No abnormalities were detected in any of the treatments, as shown in representative micrographs (n = 4–8). (B) Evaluation of bone marrow toxicity. White blood cells were drawn and counted from nude mice in all treatment groups (indicated on the graph). (C) Evaluation of treatment effects on neutropenia. Absolute neutrophil counts from blood collected from treated mice. Bars represent mean values (n = 10) ± SEM.