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

BACKGROUND
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%. Despite several improvements in the treatment of patients with unresectable CRC prognosis is poor and there is the need of developing new treatment strategies for patients with metastatic chemorefractory disease.

The S100 calcium binding protein A4 (S100A4) predicts metastasis formation and reduced CRC patient survival. S100A4 was previously identified as transcriptional target of the Wnt/β-catenin signaling pathway. The Food and Drug Administration (FDA)-approved anti-helminthic drug niclosamide is known to intervene in the Wnt/β-catenin pathway signaling, leading to reduced expression of S100A4 linked to restricted in vivo metastasis formation. Thus, we aim at translation of our findings on restricting S100A4-driven metastasis into clinical practice for treating metastasized CRC patients progressing after standard therapy.


METHODS/DESIGN
NIKOLO is a phase II, single center, one-arm open-label clinical trial to investigate the safety and efficacy of niclosamide tablets in patients with metastasized CRC progressing under standard therapy. Eligible patients will receive 2 g of orally applied niclosamide once a day and will continue with the treatment once daily till disease progression or toxicity. Toxicities will be graded according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v4.03. The primary objective of this trial is to assess the progression free survival after 4 months, secondary objectives are overall survival, time to progression, disease control rate (remission + partial remission + stable disease), and safety. Furthermore, pharmacokinetic analysis will be conducted to evaluate niclosamide plasma concentration.


DISCUSSION
This study is expected to provide evidence of the feasibility, toxicity and efficacy of niclosamide in the treatment of patients with metastasized CRC and could help to establish a new treatment option.


TRIAL REGISTRATION
The study is registered with ClinicalTrials.gov (NCT02519582) and the European Clinical Trials Database (EudraCT 2014-005151-20).


Background
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.
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 therapyfree 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 (FOL-FOX 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].
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].
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.

Study aims
This study evaluates the safety and efficacy of orally applied niclosamide in patients with metachronous or synchronous metastases of CRC origin, who progressed under previous therapy.

Study objectives
Primary objective is PFS after 4 months. PFS after 4 months will be evaluated according to the Response Evaluation Criteria In Solid Tumors (RECIST) Version 1.1.
Secondary objectives are OS, time to progression, disease control rate (remission + partial remission + stable disease), and safety.
Pharmacokinetic analysis will be done to investigate niclosamide plasma concentration and a potential correlation with efficacy.

Study design
The study is a phase II single center, one-arm open-label clinical trial for patients with metastatic CRC.

Study schedule
The trial has started on August 2015. The estimated accrual duration is 30 months. The estimated study completion date is 2018 (final data collection, date for primary outcome measure). Survival status will be collected for 2 years after registration.

Trial organization
The trial is organised by the Charité Comprehensive Cancer Center (CCCC) in collaboration with the Max-Delbrück-Center for Molecular Medicine (MDC), Berlin, Germany.

Statistics
The study is a one-arm open-label clinical trial with interim analyses for futility of patients with metastatic CRC.
A Simon two-stage design will be used to allow early termination due to futility. The null hypothesis is that the probability of being free of progression (having stable disease, partial or complete response) at 4 months is p 0 = 0.25 (H 0 : p < =p 0 = 0.25) estimated according to the recent literature [50,51]. This null hypothesis will be tested with the Binomial test one-sided against the alternative H 1 (estimated progression-free probability at 4 months of the experimental therapy) p1 = 0.5. Samples size for both stages were calculated according to the optimal design after Simon [52].
In the first stage 17 patients will be recruited. If less than 5 of the first 17 patients are progression-free at 4 months the trial will be terminated prematurely. Otherwise 20 additional patients will be enrolled (up to a total of 37 patients).
The null hypothesis will be rejected if at least 13 patients out of 37 will be free of progression at 4 months. This design (optimal design after Simon) yields a type I error rate of 0.046 and a power of 0.902.
Analyses of PFS and OS survival will performed with Kaplan-Meier-curves, hazard ration will be reported with 95% confidence intervals. For safety analysis the worst toxicities in each cycle will be reported according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) Version 4.03. A descriptive analysis of adverse events (AEs) and serious adverse events (SAEs) will be provided.

Recruitment process
The trial is expected to take up to 30 months to complete recruitment. Participants will be recruited from the Charité Universitätsmedizin Berlin. Following verbal and written study information in form of the patient information sheet and extensive discussion with a clinical trial physician, patients will get at least 24 h time to consider participation.
Patients who meet all the inclusion criteria and none of the exclusion criteria listed in Table 1 will be invited to provide written informed consent.

Study intervention and general considerations for dose modifications
Patients will start the treatment with the oral intake of 2 g niclosamide on day 1 and will continue with the medication once daily at the same time after a meal till disease progression or toxicity. Toxicities will be graded according to NCI CTCAE v4.03.
In case of an allergic reaction or vomiting ≥ grade II or diarrhoea or nausea or any other toxicity ≥ grade 3 the study medication will be paused until recovery and consequently reduced by one dose level (500 mg). If dose reductions to levels less than 1 g or discontinuation of the study treatment longer than 3 weeks are necessary, the study treatment will be terminated.

Assessments
Patients will be followed up closely for side effects. This includes physical examinations as well as blood tests. Additional intensive analyses of niclosamide levels in the blood will be conducted in the first patient cohort (patients 1-5) in the first week to examine the absorption of niclosamide from the intestine and systemic circulation. To assess efficacy imaging will be performed every 2 months to evaluate the disease status.

Translational research
Patients will be asked to consent for optional translational research including analysis of patients' tumor tissue and additional blood analysis as described hereunder.

Analysis of S100A4 expression in patient primary tumors and/or metastases or recurrent tumors
If available, tumor tissue of the primary tumor and/or metastasis or recurrent tumor will be collected for patients who consent for optional translational research to do further analyses in specific subgroups (responder/ non-responder). These tissues will be analyzed for S100A4 expression. These initial analyses characterizing the molecular starting point of the patient, depend on the availability of the amount of tissue procured for research purposes.

Analysis of potential niclosamide-induced effects on circulating S100A4 transcripts in patient plasma
The following initial analyses for characterization of the molecular starting point of the patient will be performed by using patients' plasma for determination of the basal levels of circulating S100A4 transcripts.
Peripheral blood samples will be collected at baseline and at end of treatment (due to progression, toxicity or other reasons) for all patients. For patients who consented for the optional translational research additional blood samples will be collected during routine blood taking at week 1, week 4 and thereafter monthly until progression. Blood samples will be used to discover and validate the prognostic and predictive value of S100A4 as well as for response to niclosamide and to evaluate relationship between other clinical outcome parameters.

Plasma generation
Immediately after every blood taking, plasma will be generated. Blood initially collected in EDTA tubes will be processed in the lab for generation of plasma aliquots (400 μl volume) which will be stored frozen at − 80°C. Analysis will be performed after completion of collection of all samples.
Additional analyses of further circulating transcripts such as beta-catenin might follow in patients who consented for the optional translational research.

Discussion
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 antihelminthic 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 At least one metastasis has to be measurable according to RECIST V 1.1 in X-ray computed tomography or magnetic resonance imaging (not older than 2 weeks before inclusion into the trial)  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.

Availability of data and materials
Not applicable-this is a protocol paper outline of the study being conducted. All data supporting the development of the study are outlined in the manuscript.
Authors' contributions SB, US and WW wrote the manuscript. KN provided statistical counseling in clinical trial design. SB, US, UK and SD designed the study. All authors have read, critically reviewed and approved the final manuscript for publication.

Ethics approval and consent to participate
The final protocol was approved by the ethics committee of the Landesamt für Gesundheit und Soziales (LAGeSo), Berlin, Germany and the Bundesinstitut für Arzneimittel und Medizinprodukte (BfArM) and is registered at EudraCT database (EudraCT 2014-005151-20) and at Clinicaltrials.gov (NCT02519582). This study complies with the Declaration of Helsinki in its recent version, the Medical Association's professional code of conduct, the principles of the International Conference on Harmonisation about Good Clinical Practice guidelines (ICH-GCP) and the Federal Data Protection Act. The trial will also be carried out in keeping with local legal and regulatory requirements. The medical secrecy and the Federal Data Protection Act will be followed. Written informed consent is obtained from each patient in oral and written form before inclusion in the trial and the nature, scope, and possible consequences of the trial have been explained by a physician. The investigator will not undertake any measures specifically required only for the clinical trial until valid consent has been obtained.