Phase II trial to investigate the safety and efficacy of orally applied niclosamide in patients with metachronous or sychronous metastases of a colorectal cancer progressing after therapy: the NIKOLO trial

Colorectal cancer (CRC) is the second most common cause of all cancer deaths in Europe and the Western world with a lifetime risk of approximately 5% [1]. Despite the implementation of early detection programs about 20–25% of all patients have already synchronous metastases at the time of diagnosis and approximately 25–50% will develop metastases in the course of disease mainly in liver and lung [2, 3]. Liver metastases are the main cause of death and the only currently available curative therapy is the surgical R0 resection of metastases. Without any further treatment the median survival after metastases detection is poor with about 7.5 month [4].

For patients with unresectable disease several systemic treatment options are available. In recent years prognosis has been improved with median survival rates in clinical trials of up to 30 months [5]. The optimal treatment strategy is highly depending on the aim of the treatment, e.g. prolongation of survival, downsizing with secondary (potentially curative) resection, improving tumor-related symptoms, stopping tumor progression and/or maintaining quality of life, the side effect profile of the regimen, tumor -specific factors and comorbidities of the patient.

Combination chemotherapy regimens with 5-fluorouracil/leucovorin/oxaliplatin (FOLFOX) or 5-fluorouracil/leucovorin/irinotecan (FOLFIRI) provide higher response rates, longer progression-free survival (PFS) and better overall survival (OS) in first-line treatment compared to monotherapy regimens with 5-fluorouracil/leucovorin alone.

For patients with a good performance status and adequate organ function second-line chemotherapy should be offered. The chemotherapy regime in this situation is highly depending on the previous therapies, the therapy-free interval and pre-existing toxicities.

For patients who received an irinotecan-based regimen as first-line treatment the second-line treatment should consist of an oxaliplatin-containing combination (FOLFOX or capecitabine/oxaliplatin). For patients who received an oxaliplatin-based regimen as first-line treatment, the second-line treatment should be irinotecan-based (FOLFIRI or irinotecan alone) [6, 7].

The combination of conventional chemotherapy with molecular targeted therapies such as the anti-vascular endothelial growth factor (anti-VEGF) agents bevacizumab, ramucirumab and aflibercept or the anti-epidermal growth factor receptor (anti-EGFR) agents cetuximab and panitumumab should be considered depending on the tumor biology. After second-line chemotherapy many patients are still in a good condition to receive third-line treatment depending on the organ function and pre-existing toxicities.

Despite the recent improvements in CRC therapy there is still the need of developing new treatment strategies for patients with metastatic chemorefractory disease. One important pathway which is constitutively active in 90% of CRC is the canonical Wnt/β-catenin pathway [8].

The S100 calcium binding protein A4 (S100A4) was identified as a Wnt/β-catenin target gene [9]. S100A4, originally named metastasin 1 (MTS1), is associated with many physiological processes (e.g. migration, invasion, wound healing, neovascularisation). S100A4 is able to induce metastases in different experimental in vitro and animal models [10‐13]. S100A4 is overexpressed in many different types of cancer [14, 15]. Its overexpression correlates with poor patients’ prognosis; high S100A4 levels are prognostic for metachronous metastases formation and correlate with reduced patient survival in several cancer types [16‐22]. However, S100A4 itself is not tumorigenic as shown by Ambartsumian et al. because transgenic mice with an overexpression of S100A4 do not develop tumors per se [23].

In our own group we identified niclosamide as a transcriptional inhibitor of S100A4 expression by performing a high throughput screen for the human S100A4 promoter–driven reporter gene expression. Niclosamide is used since mid of the 1960s as an anti-helminthic drug which inhibits glucose uptake, oxidative phosphorylation and anaerobic metabolism [24]. Niclosamide is approved by the Food and Drug Administration (FDA) and the European medicines agency (EMA) for treatment of tapeworm infections.

Consequently, we demonstrated the efficacy of niclosamide in cell culture by inhibition of S100A4-induced cell migration and invasion. We identified niclosamide as anti-metastatic drug in colon cancer xenografted mice by restriction of liver metastases linked to shorter survival. In this experimental setting, niclosamide interfered in the Wnt signaling pathway by disrupting the complexation of β-catenin/TCF with the S100A4 promoter leading to reduced expression of S100A4 [13].

Other studies reported that niclosamide also interfered with the Wnt signaling pathways by inhibiting Wnt/Frizzled 1 signaling or by inducing the degradation of the Wnt co-receptor low density lipoprotein receptor-related protein 6 (LRP6) [25, 26]. Osada et al. demonstrated a niclosamide-induced decrease in Dvl2 expression, leading to reduced downstream β-catenin signaling [27]. In hepatocellular cancer cells, niclosamide reduced cell proliferation, induced apoptosis, decreased TOP activity, and lowered β-catenin, Dvl2 and cyclin D1 levels [28]. Ono et al. reported niclosamide-induced reduction of proliferation of primary human leiomyoma cells by inhibiting Wnt/β-catenin pathway activation, such as inhibited TOP activity, down-regulated pathway target genes e.g. Axin2, and reduced nuclear β-catenin translocation [29]. Londoño-Joshi et al. demonstrated niclosamide-reduced levels of LRP6 and β-catenin for basal-like breast cancers [30]. For ovarian cancer, niclosamide treatment resulted in increased cytotoxicity, reduction of Wnt/β-catenin signaling by TOP assay, and decreased Wnt pathway proteins e.g. Axin2 and cyclin D1 [31]. King et al. demonstrated niclosamide-induced abrogation of Wnt7A/β-catenin signaling, inhibition of β-catenin transcriptional activity, cell viability, and decrease in cell migration [32]. Satoh et al. showed that niclosamide inhibited cell proliferation, induced caspase-dependent apoptosis and G1 cell cycle arrest, and decreased cell migration, reduced the level of mediators of epithelial-to-mesenchymal transition, and decreased β-catenin expression, [33]. Functionally, niclosamide is able to block further pathways besides Wnt/β-catenin signaling, known to be decisive for cancer initiation, progression and metastases [34, 35]. For instance, it inhibits the mTORC1 activity by lysosomal dysfunction in different cancer types [36‐38], blocks the STAT3 phosphorylation and its translocation to the nucleus in prostate cancer cells [39], and inhibits the NF-κB and Notch pathways [40, 41]. Thus, niclosamide has been reported as an anti-cancer molecule in numerous cancer types like CRC [13], osteosarcoma [42], breast cancer [30, 37], ovarian cancer [31, 43], prostate cancer [44], non-small cell lung cancer (NSCLC) [45, 46], glioblastoma [40], head and neck cancer [47], multiple myeloma, and leukemia [41, 48].

Niclosamide has only little side effects and seems to be well tolerated even when applied over a long period [49]. The oral dose of niclosamide for adults in anti-helminthic treatments is 2 g on day one followed by 1 g daily for 6 consecutive days. The serum concentration of niclosamide after a single dose of 2 g leads to maximal serum concentrations of 0.25–6.0 μg/ml which corresponds to the concentration used on the above mentioned models [34, 49].

As there is strong evidence from in vitro and in vivo experiments that niclosamide might be beneficial as anti-tumorigenic and anti-metastatic drug we designed the hereafter described clinical trial to investigate the safety and efficacy of orally applied niclosamide in patients with metachronous or sychronous metastases of a CRC who progressed after therapy and for whom no other standard therapy is available.

Several research groups and our own studies could show that niclosamide inhibits crucial pathways associated with proliferation, migration and invasion like the Wnt/β-catenin pathway, leading to anti-cancer activity of the drug. Niclosamide is initially EMA- and FDA-approved for anti-helminthic treatment and has a good safety profile, which creates a solid basis for repositioning this drug for alternate uses. Our preclinical studies supported the strong potential of niclosamide as anti-cancer drug, which interferes with tumor progression and metastases formation via S100A4 inhibition. However, its application in cancer patients has not been investigated in patients yet. Therefore, the use of niclosamide represents a novel approach of particularly metastases intervention via inhibition of a defined molecular target, such as S100A4.

To better explore the therapeutic potential of niclosamide there is the need to conduct a clinical trial to investigate the safety, efficacy and pharmacokinetics of orally applied niclosamide in cancer patients, and most importantly, to investigate if niclosamide treatment will result in prolongation of the PFS of the patients. The attractiveness of this study is based on the fact that the drug activity in patients will be correlated with the molecular analysis of the target molecule S100A4 in patient’s blood as the easiest accessible liquid biopsy for the timely monitoring. Furthermore, potential limiting factors, such as possible intra-individual difference in the drug absorption rate, will provide important additional information to what extend this will lead to variations of its anti-cancer efficacy.

As there is currently no predictive biomarker for response to niclosamide available we included an intense translational research program in our study to gain deeper insights in the mechanisms of niclosamide mediated anti-cancer activity. Thus, exploring the therapeutic potential of niclosamide in colon cancer patients in correlation to its target molecule S100A4 will enable us to evaluate this approach for broader uses in cancer therapy, where Wnt-signaling interference represents a rationale.