Repositioning of the anthelmintic drug mebendazole for the treatment for colon cancer

In the past decades, target-based approaches with the aim to modulate specific diagnosis-related molecules and pathways have dominated the cancer drug discovery process and resulted in several successful novel therapies, best exemplified by the bcr–abl inhibitor imatinib for the treatment for chronic myelocytic leukemia (CML) (Swinney and Anthony 2011). However, this approach is slow, has a high failure rate and it has become obvious that the rate of new drug approvals is decreasing (Hurle et al. 2013; Sukhai et al. 2011). Furthermore, the compounds that reach the market produce modest benefit in solid tumors (Hurle et al. 2013). Thus, new strategies for drug discovery are needed. One such strategy is drug repositioning (or repurposing) in which a new indication for an existing drug is identified. In this approach, known on-patent, off-patent, discontinued and withdrawn drugs with unrecognized cancer activity can be rapidly advanced into clinical trials for this new indication as much or all of the required documentation to support clinical trials can rely on previously published and readily available data (Hurle et al. 2013).

Genomics-based target identification and screening using cell-free systems have been the dominating principle in cancer drug discovery during the recent decade (Mayr and Bojanic 2009). As an alternative to this approach, the use of phenotype-cell-based screening using human tumor cell lines may provide some distinct advantages (van Staveren et al. 2009). Cellular phenotype results from many interconnecting signaling and feedback pathways. Compound screens based on isolated targets or cell components do not reflect this complexity. Indeed, it has recently been shown that the contribution of phenotypic screening to the discovery of first-in-class small molecule drugs exceeded that of target-based approaches (Swinney and Anthony 2011). Furthermore, recent findings from large panels of cell lines indicate that they retain genomic features of the primary tumor and can recapitulate clinical findings with regard to response to targeted inhibitors (Sharma et al. 2010).

In the present study, we used an annotated library containing 1,600 compounds with documented clinical use for screening in colon cancer cell lines with the aim to identify drugs with repositioning potential in this cancer type.

The cytotoxic/antiproliferative activities in response to the 1,600 annotated compounds with documented clinical history at a concentration of 10 μM in the colon cancer cell lines RKO and HCT116 are shown in Fig. 1a. Sixty-eight hits, as defined above, were retrieved (4.25 % hit rate). Among the hits retrieved, the highest frequency was obtained for known antineoplastic agents followed by anti-parasitic drugs, anti-infectives and cardiovascular drugs (Fig. 1b). Clustering the compounds according to chemical structure using Vortex revealed clusters with similar chemical structure including anthracyclines, vinca alkaloids and cardiac glycosides (Fig. 1c). Interestingly, we also observed a distinct cluster containing the antihelmintic benzimidazole compounds albendazole (ABZ), mebendazole (MBZ), oxibendazole and fenbendazole (Fig. 1d). This group of compounds were within 2 standard deviations of the library with respect to log P, molecular weight, hydrogen donors and hydrogen acceptor, and were thus well in agreement with the Lipinski’s rule of 5 criteria (not shown). Oxibendazole and MBZ showed the lowest SIs in the two cell lines screened closely followed by ABZ and fenbendazole (Fig. 2a). From a repositioning perspective, MBZ and ABZ are registered pharmaceuticals for clinical use in humans, thus easily accessible for clinical testing, and were therefore prioritized for further analysis.

ABZ has previously demonstrated activity in several cancer types including colon cancer (Králová et al. 2013; Pourgholami et al. 2001, 2005). MBZ on the other hand has not previously been reported to be active in this disease and may have advantages both with respect to toxicity and efficacy. Thus, ABZ appears to have a more severe toxicity profile, including neutropenia and anemia (http://​bioinformatics.​charite.​de/​promiscuous/​, von Eichborn et al. 2011). In a clinical study of patients with advanced cancers, ABZ-induced neutropenia (Morris et al. 2001) and neutropenia was also the dose-limiting toxicity in a subsequent formal dose-escalating phase-I trial (Pourgholami et al. 2010). ABZ has been reported to be redox active leading to strong reactive oxygen species (ROS) generation (Locatelli et al. 2004) potentially adding to the reported drug-induced toxicity. ROS generation could lead to several toxicological consequences as a result of oxidative injury to important biomolecules such as DNA, proteins and lipids (Kalinowski and Richardson 2007). In contrast, MBZ, although belonging to the benzimidazole group of compounds, showed only a low and transient effect on ROS generation (Locatelli et al. 2004). This suggests that MBZ could be the first-choice drug since it causes only a mild oxidative stress and thus is expected to better tolerate in the clinical setting.

Also from the standpoint of efficacy, MBZ may have advantages as demonstrated in a study of preclinical glioma models (Bai et al. 2011). In this study, MBZ, but not ABZ, was able to significantly increase survival in two orthotopic mouse models of glioma (Bai et al. 2011). In this context, it is worth noting that ABZ and MBZ activity patterns show only moderate correlations in the NCI60 panel (Pearson’s correlation coefficient 0.64, Fig. 2b). Furthermore, when the ABZ- and MBZ-induced gene expression profiles were compared using the CMAP database, ABZ was only ranked number 222 using MBZ as the query signature (Fig. 2c). These results indicate differences in mechanism of action between ABZ and MBZ.

We subsequently tested the binding affinities of MBZ and ABZ at 10 µM against a panel of 97 kinases. Whereas no kinase binding was observed for ABZ, MBZ appeared to be a potent inhibitor of several kinases with high affinity (POC <22, Fig. 3). Binding constants (Kds) in response to MBZ were also determined for selected kinases (Table 1) Interestingly, MBZ showed strong activity with Kds in the nanomolar range against ABL kinases including the T315I gatekeeper binding site point mutation which remain a major clinical challenge in treatment for CML. Cells that harbor this mutation are insensitive to most of the clinically available ABL-targeted drugs. Furthermore, MBZ was also shown to potently bind to BRAF and especially to mutant BRAF(V600E), which is of clinical relevance in melanoma and colon cancer (Fig. 3; Table 1). In CMAP, the MBZ-induced gene expression profile correlated strongly with nocodazole a well-known tubulin inhibitor with chemical structure similarity to MBZ. Also nocodazole has recently been shown to inhibit several protein kinases including Bcr–Abl (Park et al. 2012). These results thus suggest additional potential targets of importance for MBZ efficacy.

Analysis of the diagnosis-specific activity of MBZ using NCI 60 z score data revealed 80 % of the colon cancer cell lines to be sensitive, second only to the leukemia panel (Fig. 4a). The lung and renal cancer subpanels showed the lowest frequency of sensitive cell lines, 25 and 28 %, respectively (Fig. 4b). The average GI 50 over the panel was 0.46 μM.

Five colon cancer cell lines, including those used in the primary screen, and three cell lines with non-malignant phenotypes were subsequently tested for response to MBZ (Fig. 4c). All 5 colon cancer cell lines showed a MBZ IC₅₀ of <5 µM whereas the drug was largely inactive in the non-malignant cell line models, thus indicating a potentially good therapeutic index in colon cancer (Fig. 4c). Indeed as a first indication of this possibility, a patient with refractory metastatic colon cancer was treated with MBZ at the standard anthelmintic dose of 100 mg twice daily. The patient experienced no subjective adverse effects at all from the drug and computerized tomography evaluation after six weeks of therapy showed near complete remission of the metastases in the lungs and lymph nodes and a good partial remission in the liver (case report accepted for publication in Acta Oncologica).

In conclusion, the overall results indicate that MBZ have repositioning potential for treatment of advanced colon cancer and clinical trials seem warranted.