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Transl Oncol. 2010 October; 3(5): 318–325.
Published online 2010 October 1.
PMCID: PMC2935635

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.

Introduction

Chemotherapeutics are among the most widely used therapies for cancer treatment. As cytotoxic agents targeting dividing cells, they are broadly efficacious across multiple cancers. Commonly used cytotoxics include DNA-targeting agents, such as anthracyclines and platinum compounds, and microtubule-targeting agents. Microtubules are a major component of the cellular cytoskeleton and are involved in a variety of cellular functions such as cell motility, intracellular transport, and cell division. Microtubules are dynamic polymeric structures consisting of α- and β-tubulin heterodimers that, together with associated motor and structural proteins, form the mitotic spindle, a self-organizing machine responsible for proper chromosome segregation during cell division. Polymerization and depolymerization of microtubules occur in a dynamic equilibrium disrupted by tubulin-targeting chemotherapeutics [1,2]. Molecules that bind to tubulin and impair/enhance polymerization or depolymerization cause mitotic arrest of dividing cells, inhibiting cell division and leading to cell death. The first natural tubulin-targeting agents were approved as cancer chemotherapeutics more than 40 years ago, and like these, most antitubulin/ antimicrotubule agents currently available are structurally complex naturally occurring compounds.

Several classes of natural antitubulin products have been effectively used as antitumoral agents. The most successful in the clinic to date are the taxanes (paclitaxel [Taxol] and the semisynthetic analog docetaxel [Taxotere]) and the Vinca alkaloids (vincristine, vinblastine, and vinorelbine). Whereas taxanes are widely used in several types of cancers including breast, ovarian, and non-small cell lung cancer [3], Vinca alkaloids are used primarily for the treatment of certain leukemias, such as childhood acute lymphoblastic leukemia [4]. Despite their wide use and clinical success, however, these approved therapies have several limitations that have driven significant efforts toward the discovery of novel antitubulin agents.

Among the most critical limitations of antitubulin natural products are their sensitivity to multidrug resistance pumps and the development of drug resistance [5]. Resistance to taxanes and other microtubule-targeting agents is often related to expression of transport proteins such as the drug efflux pump P-glycoprotein/multidrug resistance (MDR) protein and/or additional mechanisms such as mutations and alterations in tubulin isotype levels as well as alterations in signaling pathways associated with cell death [5,6]. In addition to drug resistance, toxicity is a major limitation of commonly used chemotherapeutic agents, including microtubule-targeting therapies. Bone marrow toxicity, especially neutropenia (reduction in neutrophils), is a dose-limiting adverse effect for classic and novel Vinca alkaloids and is also a significant toxicity associated with taxanes. Peripheral neuropathy is another common toxicity associated with taxanes [7]. Both toxicities pose a major clinical problem for cancer patients. Thus, although the clinical and commercial success of natural antitubulin compounds and their semisynthetic analogs validate tubulin as a target for cancer therapeutics, difficulties in chemical synthesis, poor solubility, and oral availability, along with drug resistance and toxicities (both discussed above), have driven the pursuit of new and improved antitubulin agents [8].

Notable efforts have been made toward the development of natural compounds, mainly novel semisynthetic taxane derivatives aiming for a wider spectrum of activity and/or a greater therapeutic window. In addition to novel taxanes, Vinca alkaloids [9] and newer classes of compounds such as epothilones [10,11] and dolastatins [12] are currently in clinical development. An additional promising strategy is the development of small molecules, known for their simpler synthetic processes and potential for oral availability. In the past 10 years, there have been numerous reports of small molecule inhibitors of tubulin function, identified through screening of compound libraries or derivatization of natural compounds. Several of these inhibitors are currently in preclinical or clinical development as antimitotics or vascular-disrupting agents [7,8]. Yet, to date, no antitubulin small molecule has been approved clinically.

We have previously described the discovery and synthesis of two series of small molecule tubulin inhibitors with potent antimitotic activity in tumor cells, triazoles [13] and oxadiazoles [14]. Here, we report the biologic activity of a novel lead compound from the oxadiazole series, IMC-038525. IMC-038525 has potent antiproliferative activity against tumor cells, including those with MDR and resistance to paclitaxel. In vivo, orally administered IMC-038525 had significant antitumor effects at doses that did not cause myelosuppression or peripheral neuropathy. This profile supports the further development of IMC-038525 as a novel cytotoxic agent, with the benefit of being applicable for therapy for MDR and paclitaxel-resistant cancers.

Materials and Methods

Compounds

IMC-038525 ((2,3-dihydro-benzo[1,4]dioxin-6-yl)-(5-{2-[(pyridin-4-ylmethyl)-amino]-phenyl}-[1,3,4]oxadiazol-2-yl)-amine) and IMC-094332 (N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-(2-(pyridin-4-ylmethylamino)pyridin-3-yl)-1,3,4-oxadiazol-2-amine) were synthesized according to previously described protocols [14]. Combretastatin A-4 was synthesized according to published protocols [15]. Colchicine was purchased from Sigma-Aldrich (St Louis, MO), and paclitaxel was purchased from Tocris Bioscience (Ellisville, MO).

Cell Line

A431 cells (human epidermoid cancer cell line) were obtained from American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle medium with 10% FBS and 1% glutaMAX. NCI/ADR-RES cells were obtained from the National Cancer Institute (Bethesda, MD) [16] and maintained in RPMI medium supplemented with 10% FBS. MDA-MB-435-LM2 cells were derived at ImClone Systems (New York, NY) from lung metastases of MDA-MB-435 cells originally obtained from American Type Culture Collection.

In Vitro Tubulin Polymerization Assay

Treatment effects on tubulin polymerization in vitro were determined in a turbidity assay (measured at 340 nm), a modified version of the screening assay developed by Cytoskeleton, Inc (Cytodynamix 12; Denver, CO), optimized for maximum throughput and sensitivity. Lyophilized bovine tubulin (HTS02; Cytoskeleton, Inc) was resuspended in G-PEM buffer (80 mM 1,4-piperazinediethane sulfonic acid pH 7, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP, 5% glycerol) to a final concentration of 3 mg/ml and kept at 4°C. Compounds in 100x DMSO stock solutions were dotted onto prewarmed 96-well plates (product no. 3696; Corning Costar, Lowell, MA), and the plates were immediately transferred to a 37°C plate reader (Spectramax Plus; Molecular Devices, Sunnyvale, CA). Cold tubulin was added to the wells, plate contents were mixed by shaking, and absorbance at 340 nm was read every minute for 30 minutes. Kinetic curves with 30 points each were collected for every compound, with a dynamic range between 0 and 0.4 OD units. Percentage inhibition values were calculated using the 30-minute data point based on control samples (1% DMSO). IC50 (concentration that causes 50% inhibition of polymerization) values were determined by sigmoidal curve fitting using Excel-based software (Microsoft Corporation, Redmond, WA).

Cell Cycle Analysis - G2/M Arrest

A431, MDA-MB-435-LM2, or NCI/ADR cells were plated onto six-well plates at a final density of 500,000 cells per well, treated with compounds at 0.01 to 1 µM final concentrations (final 0.1% DMSO) for 24 hours, trypsinized, collected, rinsed in PBS, and fixed in 70% cold ethanol overnight at 4°C. Cells were rinsed with PBS, resuspended in PBS with 0.2% Tween, incubated with RNAse (final concentration of 1 µg/ml) at 37°C for 15 minutes, added with propidium iodide (final 50 µg/ml), and incubated for 30 minutes at room temperature. DNA ploidy was analyzed using cell sorters (Epics Excel [Beckman Coulter, Brea, CA] or Guava PCA-96 [Guava Technologies/Millipore, Hayward, CA]), and mitotic arrest was characterized by accumulation of cells in the G2/M phase of the cell cycle. EC50 (effective concentration that causes 50% mitotic arrest) values were determined by sigmoidal curve fitting using Excel-based software.

Colchicine-Binding Site Competition Assay

To determine whether compounds compete for the colchicine-binding site on tubulin, we used a modified competition scintillation proximity assay (SPA) [17]. Briefly, lyophilized biotinylated tubulin (Cytodynamix screen 15; Cytoskeleton, Inc) was dissolved in G-PEM with 5% glycerol and was incubated with streptavidin-conjugated yttrium SPA beads (Amersham/GE Healthcare, Piscataway, NJ) for 45 minutes at 4°C, to a final concentration of 0.88 mg/ml beads and 40 nM tubulin. 3H colchicine (American Radiolabeled Chemicals, St Louis, MO) was added to a final concentration of 65 nM. Compounds at 10 µM for single point screen or 0.01 to 100 µM for dose-response curves and the tubulin bead mixture were added to flat-bottom black 96-well plates. Plates were incubated for an additional 45 minutes at 37°C and read with a scintillation plate reader (TopCount; Perkin Elmer, Wellesley, MA), with no need for a separation step. Counts were normalized to control wells without SPA beads, and inhibition was calculated from DMSO controls. IC50 values were calculated based on sigmoidal curve fitting using Prizm software (GraphPad Software, La Jolla, CA).

Immunofluorescence

A431 or NCI/ADR-RES cells were treated with PBS containing 0.1% DMSO or IMC-038525 (in 0.1% DMSO) for 60 minutes at 37°C, then fixed with ice-cold methanol, further permeabilized with PBS-Tween (2%), and stained with a primary monoclonal antitubulin antibody (Sigma) followed by a secondary Alexa-488 antimouse antibody (Molecular Probes/Invitrogen, Carlsbad, CA). Images were acquired with an epifluorescence microscope (Nikon, Melville, NY).

Pharmacokinetics (PK)

All animal studies were conducted in accordance with current regulations and standards of the US Department of Agriculture and the National Institutes of Health. Non-tumor-bearing female nu/nu athymic mice (7–8 weeks) were dosed intraperitoneally (IP) or orally (PO) at a 10-ml dosing solution/kg of body weight. IMC-038525 was prepared in ETP vehicle (5% ethanol/5% Tween-80/5% Peg400/85% PBS), and paclitaxel was dissolved in 5% EtOH/CREM vehicle (5% DMSO, 5% cremophor, and 90% sodium carboxymethylcellulose). Plasma samples from blood collected by cardiac puncture in CO2-asphyxiated mice were run on appropriate solid phase sorbent, samples were eluted under steady vacuum with an organic solvent, eluted volumes were dried and resuspended in applicable liquid chromatography mass spectrometry (LCMS) mobile phase (0.2% formic acid in water or methanol) and processed for LCMS analysis (Agilent 1100 LC-MSD, Santa Clara, CA) of compound concentration.

In Vivo Efficacy and Toxicity

nu/nu mice (female, 7–8 weeks) were injected subcutaneously with 5 x 106 MDA-MB-435-LM2 cells/mouse. When tumors reached approximately 270 mm3, mice were randomized by tumor size into one of three groups (n = 10) as follows: E/T/P vehicle (5% ethanol/5% Tween-80/5% Peg400/85% PBS) at 10 µl/g, 300 mg/kg IMC-038525 in E/T/P, or 20 mg/kg paclitaxel in 5% EtOH/CREM vehicle every 7 days. Mice were dosed PO for IMC-038525 and IP for paclitaxel. Mice received treatments once a week for 28 days in the paclitaxel group and for 36 days in the IMC-038525 group. Caliper measurements were used to calculate tumor volume (mm3) using the formula V = π/6 x (length x width x height). At the end of the study period (day 36 of treatment), 10 mice in the E/T/P group, 9 mice in the IMC-038525 group, and 4 mice in the paclitaxel group were killed, and whole blood and sciatic nerves were collected for the measurement of white blood count, absolute neutrophil count, and sciatic nerve myelination.

For counting of white blood cells and neutrophils, blood was drawn in the presence of an anticoagulant, and measurements were obtained with a CDC Mascot (CDC Technologies, Oxford, CT). For evaluation of sciatic nerve myelination, sciatic nerves were fixed overnight in 10% neutral-buffered formalin followed by overnight fixation in 2.5% glutaraldehyde at 4°C, rinsed in PBS 3 x 5 minutes, and stained with 2% osmium tetroxide for 2 hours. Nerves were then protected in 25% sucrose overnight at 4°C, washed in 30% alcohol, paraffin-embedded, and sectioned (4 µm) for microscopic analysis.

In addition, five mice per group had their tumors resected and frozen. Heart, lungs, liver, stomach, duodenum, and brains were removed and sent for pathologic analysis (Experimental Pathology Laboratories, Inc, Sterling, VA). The % T/C for each treatment group was calculated as follows: % T/C = 100 x (treatment volume / initial volume) / (control volume / initial volume). Tumor volumes were analyzed using repeated-measures analysis of variance (RM ANOVA) using JMP version 5 (SAS Institute, Inc, Cary, NC).

Results

IMC-038525 Disrupts Tubulin Polymerization In Vitro, Binds to the Colchicine-Binding Site, and Disrupts Microtubules in Intact Cells

We have previously identified oxadiazoles as a novel scaffold of tubulin small molecule inhibitors [14]. Compound 13 (here referred to as IMC-094332; Figure 1) was reported to demonstrate acceptable oral availability and pharmacokinetic (PK) profile. Through further structure-activity relationship (SAR) efforts around in vitro potency, oral availability, and PK profile, IMC-038525 (Figure 1) was identified. IMC-038525 caused dose-dependent inhibition of microtubule formation in an in vitro tubulin polymerization assay (Figure 2A). To determine the binding site of our compounds onto the tubulin molecule, we used radioactive competition and determined that IMC-038525 competes with colchicine in a dose-dependent manner (Figure 2B). This binding competition was comparable to that of combretastatin A-4, an established colchicine site binder with a higher affinity for tubulin than colchicine. Immunofluorescence was used to evaluate the effects of treatment on microtubule distribution and cell morphology. IMC-038525 at concentrations of 100 nM (Figure 2C) and 10 nM (data not shown) caused major disruption of the microtubule cytoskeleton in NCI/ADR-RESMDR-expressing human breast carcinoma cells and in A431 human epidermoid cancer cells, in both cases leading to dramatic changes in cell shape (cells became less spread out and more rounded). Taken together, these data indicate that IMC-038525 binds to tubulin at or near the colchicine-binding site and depolymerizes microtubules in tumor cells.

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 ...

IMC-038525 Causes Cell Cycle Arrest in MDR Tumor Cells

A hallmark of tubulin-targeting agents, whether depolymerizers or polymerizers/stabilizers, is the alteration of microtubule dynamics that causes mitotic arrest. We evaluated the effects of IMC-038525 on the cell cycle, side by side with paclitaxel (microtubule stabilizer) and combretastatin A-4 (microtubule depolymerizer). IMC-038525 caused a significant mitotic arrest of A431 and NCI/ADR-RES cells (Figure 3 and Table 1). In comparison with other oxadiazoles previously tested in SAR studies [14], IMC-038525 is one of the most potent compounds in this class. The EC50 values of IMC-038525 and paclitaxel for causing mitotic arrest of A431 epidermoid cells were very similar (Table 1). In contrast, 0.5 nM IMC-038525 caused a G2/M arrest of greater than 50% in NCI/ADR-RES cells (Figure 3A), whereas paclitaxel was completely ineffective in these MDR-expressing cells (Table 1). Thus, IMC-038525 exerts antimitotic activity in cells that are multidrug resistant and do not respond to paclitaxel.

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 ...
Table 1
EC50 Values for G2/M Arrest in Tumor Cell Lines.

IMC-038525 Dosed Orally Inhibits Tumor Growth In Vivo

IMC-038525 had an encouraging exposure on oral dosing and a superior PK profile compared with IMC-094332, a previously described oxadiazole [14]. When dosed PO side by side, the levels of IMC-038525 reached within the first hour were twofold higher than those of IMC-094332 (data not shown). This difference in achieved maximum concentration favored the choice of IMC-038525 as a lead compound. Furthermore, when evaluated in parallel with paclitaxel, IMC-038525 dosed PO showed an acceptable PK profile compared with that of paclitaxel administered IP. IMC-038525 reached 6.6 µM plasma levels 30 minutes after dosing, decreasing considerably by 4 hours (Figure 4A); this was maintained to more than 790 nM through 24 hours after dosing, a concentration 50-fold above the concentration determined in vitro to cause 50% G2/M arrest in tumor cells. Paclitaxel levels, however, were no longer detectable at 24 hours after dosing.

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 ...

IMC-038525 was tested in parallel with paclitaxel in two xenograft models for an evaluation of antitumor effects in vivo. In an A431 xenograft tumor model, oral administration of IMC-038525 resulted in a dose-dependent inhibition of tumor growth, with the greatest efficacy reached at the highest dose tested, 300 mg/kg (% T/C = 56, RM ANOVA P = .08). This efficacy was greater than that of paclitaxel dosed IP at 20 mg/kg (% T/C = 74, P = .226). In the MDA-MB-435-LM2 model, likely of melanoma origin [18], animals were treated with paclitaxel at 20 mg/kg (IP) or with IMC-038525 at 300 mg/kg (PO), and both doses were established to be the maximum tolerated dose (MTD) for each compound (not shown). IMC-038525 was well tolerated when dosed PO, once a week, at 300-mg/kg treatment. In contrast, treatment with paclitaxel, dosed IP, once a week, at 20 mg/kg, was toxic after 3 weeks of treatment in this study. In the paclitaxel group, several mice died or had to be killed because of morbidity, so mice in this group did not receive treatment beyond day 28. Necropsy of remaining mice showed enlarged, blood-filled, inflamed intestines, indicating that the MTD for paclitaxel was clearly reached; nevertheless, no significant antitumor efficacy was achieved (Figure 4B). In contrast, treatment with IMC-038525 resulted in a statistically significant (RM ANOVA, P = .004) tumor growth inhibition through day 36 (% T/C = 39; Table 2). In this group, only one animal died before the end of the study, with no abnormalities observed during necropsy or in histopathologic evaluation of tissues. Body weight measurements taken during the study period are reported in Figure 4C. Whereas at the beginning of the study IMC-038525 caused a transient body weight loss (RM ANOVA, P = .03) compared with the control group, final body weights were unaffected (Table 2). There were no treatment-related body weight changes observed in the paclitaxel group through day 28 (Figure 4C). Thus, although both drugs have similar antimitotic activity against MDA-MB- 435-LM2 in vitro (Table 1), our data indicate that IMC-038525 has greater efficacy than paclitaxel in inhibiting tumor growth in vivo in this xenograft model, when both drugs were dosed at their MTD values.

Table 2
Antitumor Activity and Body Weight Changes on Treatment with IMC-038525 in MDA-MB-435-LM2 Xenografts.

IMC-038525 Does Not Cause Peripheral Neuropathy

Peripheral neuropathy is a toxicity associated with a number of chemotherapeutic agents, including antitubulin compounds. To assess potential neurotoxic effects of IMC-038525, we evaluated the morphology of myelin sheaths in the sciatic nerve. Histology of the sciatic nerve revealed that there was no treatment-related nerve degeneration or myelin fragmentation. In all treatment groups, myelin sheaths were generally intact and open, although infrequent whirl-like or collapsed myelin sheaths were observed (Figure 5A). No histologic differences were observed between groups that could be attributed to treatment with either paclitaxel or IMC-038525. As the MTD for paclitaxel was reached, the lack of detectable myelin sheath anomalies is likely related to the dosage regimen used. According to Mimura et al. [19], paclitaxel-induced neuropathy in mice is dependent on treatment schedule. In that study, drug related degeneration was evaluated in mice treated with paclitaxel at 30 mg/kg in regimens varying from every 3 hours for 3 days to once a week for 4 weeks, and the severity of nerve degeneration was dependent on the treatment schedule.

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 ...

IMC-038525 Does Not Cause Myelotoxicity

Another commonly reported toxic effect of inhibitors of tubulin polymerization is bone marrow toxicity or myelotoxicity. To evaluate myelotoxicity in IMC-038525-treated mice, white blood cell and absolute neutrophil counts were performed (Figure 5, B and C). Treatment with paclitaxel resulted in a decrease in white blood cells and absolute neutrophil counts in blood compared with control, although the difference was not statistically significant (one-way ANOVA, P = .136 and P = .176, white blood cells and absolute neutrophils, respectively). The lack of statistical significance for the effect of paclitaxel is attributed to the low number of surviving mice in the paclitaxel group (only four mice survived to the end of the study). In the IMC-038525 group, however, there were no treatment-related changes in white blood cells and neutrophil counts, indicating that this compound does not cause significant myelotoxicity.

Discussion

Taxanes have demonstrated great utility in the treatment of human malignancies, including cancers of the lung, ovary, and breast. Paclitaxel, for example, is widely used for the treatment of metastatic and early stage breast cancer, with a response rate of 25% to 69% in the first-line setting and significant benefits on overall and disease-free survival [20]. Paramount among the limitations to the effectiveness of taxane therapies is drug resistance, intrinsic or acquired during treatment. In addition, taxane treatment is associated with two major toxicities: neurotoxicity commonly manifested as peripheral neuropathy and bone marrow toxicity leading to neutropenia. Furthermore, currently approved taxanes cannot be administered PO, and formulation requirements for intravenous delivery often lead to hypersensitivity reactions. Given the established therapeutic success of taxanes, significant efforts are underway to search for novel tubulin-targeted agents with efficacy equivalent to or greater than taxanes, especially in taxane-resistant models. Novel tubulin-targeted agents may also be selected for further development based on an improved toxicological profile or oral availability, potentially allowing for greater ease of administration and patient convenience, besides minimizing the risk of hypersensitivity reactions.

While several new taxanes are being developed, most of them seem to offer only marginal advantages over approved drugs [21]. On account of their size and simpler synthetic processes, small molecules have the potential to offer advantages over natural or semisynthetic products. Through SAR processes, small molecules can be selected with improved solubility, oral availability, as well as lower susceptibility to drug efflux mechanisms. Here, we report a novel small molecule inhibitor of tubulin polymerization that is PO available, has antimitotic activity against MDR expressing tumor cells that do not respond to paclitaxel, is efficacious in vivo against a taxane-resistant breast tumor xenograft, and has an acceptable therapeutic window.

IMC-038525 dosed PO resulted in robust plasma exposure, with rapid achievement of micromolar levels lasting for at least 1 hour. The plasma concentrations of paclitaxel dosed IP in mice declined in a biphasic manner, consistent with the pattern seen in patients after intravenous administration of TAXOL. The initial rapid decline likely represents distribution of the drug to the peripheral compartment and drug elimination, and the later and longer phase corresponds to a slow efflux of paclitaxel from the peripheral compartment [22]. Importantly, despite a large decline in the levels of IMC-038525 during the first 4 hours, the concentrations remained much higher than those necessary for antimitotic activity in vitro, up to 24 hours. Interestingly, whereas IMC-038525 and paclitaxel showed similar potency in causing mitotic arrest in MDA-MB-435-LM2 cells in vitro, only IMC-038525 demonstrated antitumor efficacy in vivo, potentially related to the PK differences. The known wide tissue distribution and avid tissue binding of taxanes suggests that the most effective way to prolong exposure of this taxane in the peripheral compartment is by administering it at more frequent schedules [23]. Yet, IMC-038525 showed antitumor efficacy when given PO just once a week. Hence, our data indicate that a more frequent administration is unlikely to be a requirement for IMC-038525 dosing.

Resistance to taxanes is multifaceted, involving defects in tubulin and its microtubule-associated proteins, as well as misregulation of signaling pathways, in addition to expression of drug efflux pumps [20]. Our results show that IMC-038525 is not a substrate for the Pgp-1/MDR transporter, a prevalent mechanism for drug resistance to chemotherapeutics. More importantly, antitumor efficacy of IMC-038525 was demonstrated against breast tumor cells that do not respond to paclitaxel, suggesting that this compound might overcome more than one resistance mechanism and could be a viable alternative for taxane-resistant tumors. Thus, IMC-038525 may be active in cancer indications and patients who do not respond to paclitaxel.

Two major toxicities are associated with tubulin targeting agents: neurotoxicity and bone marrow toxicity. Peripheral neuropathy is a major dose limiting side effect of commonly used chemotherapeutic agents including tubulin-targeting drugs, platinum agents, and the proteasome inhibitor bortezomib [24]. The incidence and severity of taxane-associated neuropathy are dose- and schedule-dependent [25]. In a published study, paclitaxel has been reported to cause detectable neurotoxicity in mice only when administered at a frequency greater than that used in the present study [19]. At the dose regimen used in this study, IMC-038525 at its MTD showed antitumor activity without causing detectable neuropathy, whereas paclitaxel did not affect tumor growth or nerve histology, despite its overall toxic effects (morbidity and death of several mice). Although the precise mechanisms of neurotoxicity associated with tubulin inhibitors are not fully understood, it is believed that the neuropathies are a consequence of interruption of axonal transport within neurons. Despite the fact that all antitubulin drugs alter microtubule dynamics, other differences in the mechanism of action, not yet completely defined, might explain the range of toxicity levels seen among different tubulin targeting drugs. Interestingly, other novel tubulin-targeted agents, such as the small molecule D-24851 [26] and the semisynthetic compound EM015 [27], have also been reported to lack neurotoxicity.

Bone marrow toxicity is also associated with taxane and Vinca alkaloid therapy [28]. Unlike paclitaxel, IMC-038525 did not cause myelotoxicity at an efficacious dose. The clinical implications of these results are that treatment with IMC-038525 might not require cytokine or stem cell support, usually given with paclitaxel [29], or IMC-038525 may have a wider therapeutic window with regard to this toxicity. Additional studies are necessary to clarify the potential toxicities associated with IMC-038525 and more accurately define the therapeutic window.

One gauge for the potential application of antitubulin agents is the number of investigations of novel tubulin-targeted agents, microtubule depolymerizers, or stabilizers at preclinical and clinical levels. Although most of the novel small molecule antitubulin agents are being explored in clinical development as vascular-disrupting agents, there are also some promising antimitotic small molecules in preclinical and clinical development (see [7] for a review). Of note are the semisynthetic compound EM015 [27] and the small molecule MPC-6827 [30], both PO available, active against MDR-expressing cells in vitro, and efficacious in vivo. No detectable toxicities have been reported for EM015 or MPC-6827. In an analogy to what is expected of novel taxane analogs, for a novel antitubulin agent to be considered truly superior to paclitaxel, robust data of activity in clearly taxane refractory malignancies are crucial [21]. Antitumor efficacy against taxane-resistant tumors is yet to be demonstrated for the above mentioned novel tubulin agents. In this regard, it is important that IMC-038525, which is also a PO available antimitotic small molecule active against MDR-expressing cells in vitro, was efficacious in vivo in a tumor model that does not respond to paclitaxel, without causing detectable neutropenia or peripheral neuropathy.

In conclusion, our data suggest that IMC-038525 has desirable efficacy, PK properties, and therapeutic window. These data support further development of IMC-038525 with screening for activity in a broad range of cancer indications, including those who do not respond to or develop resistance to paclitaxel.

Acknowledgments

The authors thank Peter Bohlen and Daniel Hicklin for their valuable input and Sue Lee, Kai Zhou, and Erik Corcoran for technical assistance.

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