GB2559162A

GB2559162A - Compound for use in medicine

Info

Publication number: GB2559162A
Application number: GB1701400.2A
Authority: GB
Inventors: El-Tanani Mohamed
Original assignee: University of Bradford
Current assignee: University of Bradford
Priority date: 2017-01-27
Filing date: 2017-01-27
Publication date: 2018-08-01
Also published as: GB201701400D0; WO2018138510A1

Abstract

Disclosed is; the use of mebendazole (MBZ, Vermox) as a Ran inhibitor and as a treatment for cancer than overexpresses RAN (e.g. triple negative breast cancer). Preferably, MBZ is used in combination with a tyrosine kinase inhibitor (TKI); more preferably; the TKI is an inhibitor of EGFR or Bcr-Abl; most preferably, the TKI is gefitinib (Iressa) or one of (afatinib, axitinib, bosutinib, crizotinib, dasatinib, erlonib, osimertinib, imatinib, lapatinib, nilotinib, pazopanib, regorafenib, sorafenib, sunitinib, ibrutinib). The MBZ and TKI may be administered sequentially, simultaneously, or separately. More broadly, use of any Ran inhibitor in combination with any tyrosine kinase inhibitor is claimed; apparently, use of mebendazole serves to overcome resistance to the TKI. Also claimed is the use of mebendazole (monotherapy) as an immunotherapy, most preferably an immunotherapy used to downregulate C5a.

Description

(71) Applicant(s):

University of Bradford (Incorporated in the United Kingdom) Richmond Road, BRADFORD, West Yorkshire, BD1 7DP, United Kingdom (72) Inventor(s):

Mohamed El-Tanani (74) Agent and/or Address for Service:

TLIP Ltd

Leeds Innovation Centre, 103 Clarendon Road, LEEDS, LS2 9DF, United Kingdom (56) Documents Cited:

WO 2016/127168 A2 US 20110142923 A1

US 20150065526 A1 US 20050038022 A1

Proceedings ofthe 107th Annual Meeting ofthe American Association for Cancer Research, Vol. 76. No. 14, July 2016, C. M. Simbulan-Rosenthal, Abstract 3860: The repurposed anthelmintic mebendazole in combination with trametinib suppresses refractory NRASQ61K melanoma Oncotarget, Vol. 8, No. 8, 24 January 2017, J. E. Barrett, The purpose of repurposing, Available at http://www.impactjournals.com/oncotarget/ index.php?

journal=oncotarget8ipage=article&op=view&path%5b %5d=14806, pages 12534-12535 (58) Field of Search:

INT CL A61K, A61P

Other: BIOSIS, EPODOC, Patent Full Text, MEDLINE, WPI, XPESP, XPSPRNG (54) Title ofthe Invention: Compound for use in medicine

Abstract Title: Use of mebendazole and a tyrosine kinase inhibitor (gefitinib) as a treatment for cancer that overexpresses RAN, Ran or Ran mRNA (57) Disclosed is; the use of mebendazole (MBZ, Vermox) as a Ran inhibitor and as a treatment for cancer than overexpresses RAN (e.g. triple negative breast cancer). Preferably, MBZ is used in combination with a tyrosine kinase inhibitor (TKI); more preferably; the TKI is an inhibitor of EGFR or Bcr-Abl; most preferably, the TKI is gefitinib (Iressa) or one of (afatinib, axitinib, bosutinib, crizotinib, dasatinib, erlonib, osimertinib, imatinib, lapatinib, nilotinib, pazopanib, regorafenib, sorafenib, sunitinib, ibrutinib). The MBZ and TKI may be administered sequentially, simultaneously, or separately. More broadly, use of any Ran inhibitor in combination with any tyrosine kinase inhibitor is claimed; apparently, use of mebendazole serves to overcome resistance to the TKI. Also claimed is the use of mebendazole (monotherapy) as an immunotherapy, most preferably an immunotherapy used to downregulate C5a.

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COMPOUND FOR USE IN MEDICINE

Field of Invention

The present invention relates to mebendazole or a pharmaceutically acceptable form thereof for use as an inhibitor of RAN, for example for use in the treatment of a cancer that overexpresses RAN or its protein product Ran. Overexpression of RAN may be characteristic of the cancer type or may be stimulated by prior cancer therapy.

Background of the Invention

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

The term cancer relates to a family of diseases characterised by abnormal cell growth. The abnormal new growth of cells is referred to as a neoplasm. The cells in a neoplasm usually grow more rapidly than normal cells and will continue to grow if left untreated. As they grow, neoplasms can impinge upon and damage adjacent structures, and can eventually compromise the survival of the host organism. Neoplasms can be benign or malignant (cancerous) and are often referred to as tumours. As used herein the term tumour is used to refer to malignant (cancerous) neoplasms. As a result, reference to the treatment of tumours and the treatment of cancer have the same meaning herein, while reference to breast cancer or breast tumour, and treatment thereof, refers to a cancer or tumour that originates in the breast, or treatment thereof, rather than a tumour that originates in another organ and that has spread to the breast through the process of metastasis. Tumours or cancers that originate in other organs or sites are referred to in a similar manner. Tumours that are derived from the process of metastasis are referred to as metastases or secondary tumours.

Hanahan and Weinburg (The Hallmarks of Cancer. Cell 100 (1): 57-70) postulated that there are a number of traits or hallmarks that are common to the diseases known as cancer, namely (i) cancer cells stimulate their own growth; (ii) cancer ceils resist inhibitory signals that might otherwise stop their growth; (iii) cancer cells resist their programmed cell death (they evade apoptosis); (iv) cancer cells can multiply indefinitely; (v) cancer ceils stimulate the growth of blood vessels to supply nutrients to tumours (angiogenesis); and (vi) cancer cells invade local tissue and spread to distant sites (metastasis). Whether or not the full set of hallmarks, that have subsequently been supplemented (Hallmarks of Cancer: The Next Generation Ceil 144 (5): 646-674), are exhibited by all cancers, it is apparent that targeting one or more of these hallmarks affords an opportunity to exert a selective killing effect on cancer cells.

Cancer cells are addicted to oncogenes or oncogenic pathways (Weinstein, Science 2002;297:63-4), which are usually uniquely present or hyperactivated, and are essential for tumour ceil growth and survival. The two most frequently dysregulated signaling pathways in cancers are the phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 and Ras/MEK/ERK [mitogenactivated protein/extraceilular signal-regulated kinase (ERK; MEK)] pathways (Grant, J Clin invest 2008; 113:3003-6). These growth signaling pathways usually exert their ultimate effect by regulating the translocation of transcription factors into, or out of, the nucleus, therebyaltering the transcriptome and consequently, the expressome (Kau & Silver, Drug Discov Today 2003;8:78-85). The activity of several oncogenic or tumour suppressive transcription factors, such as NF-κΒ, FOXO1, p53, and beta-catenln, and non-transcription factors, such as survivin, are regulated by their subcellular localization. Therefore, the uncoupling of nucleocytoplasmic transport from growth and survival signaling pathways has been suggested to be a potential target for cancer therapy.

Tumours or cancers are commonly referred to by the primary site of their occurrence, i.e. where the tumour first develops, and often also by reference to further characteristics, for example whether their growth is stimulated by hormones or whether they express certain receptors. Thus, primary tumours located in the breast are referred to breast cancers while tumours whose growth is stimulated by hormones such as estrogen, progesterone and testosterone are referred to as hormone dependent cancers. Breast cancers whose growth is stimulated by the presence of estrogen are known as estrogen receptor positive (ER+ve) breast cancers. Knowledge of tumour type can be exploited to select the best mode of therapy. For example, treatment of ER+ve tumours can involve estrogen deprivation, e.g. through blockage of estrogen biosynthesis with an aromatase inhibitor, or blocking of the signal derived from the interaction of an estrogen ligand with its receptor with a selective estrogen receptor modulator such as tamoxifen. These therapeutic interventions for the treatment of ER+ve tumours arrest the normal transcriptional activation of the estrogen receptor thereby blocking the estrogen stimulated growth of the tumour. Often, after presenting in an initial ER+ve state, breast cancer tumours progress to a hormone refactory state where their growth is independent of estrogen as, for example, through mutation, the estrogen receptor adopts a form in which it is transcriptionally activated even in the absence of estrogen, such tumours are also referred to as estrogen receptor negative (ER-ve) tumours. Similarly, breast cancers that express progesterone receptors and whose growth is sensitive to the presence of progesterone are referred to as progesterone positive (PR+ve) breast cancers whilst those whose growth is independent of the expression of progesterone receptor are progesterone receptor negative (PR-ve) breast cancers.

Another common sub-type of breast cancer is HER2-positive breast cancer, a subset of breast cancers that over express human epidermal growth factor 2. A number of HER2 targeted therapies have been developed, examples include the anti-HER2 antibodies trastuzumab and pertuzumab and the antibody-drug-conjugate trastuzumab emtansine.

Breast cancers that are negative for ER, PR and HER2, i.e. those tumour types whose growth is independent of ER, PR or HER2 status, are commonly referred to as triple negative breast cancers. Patients suffering from triple negative breast cancers presently have the worst overall and disease free survival rates. Treatment options for patients with triple negative breast tumours are relatively limited (Ontilo et al, Clin Med Res. 2009 Jun; 7(1-2): 4-13) with the present treatment paradigms usually involving a combination of surgery and radiation as appropriate with cytotoxic chemotherapy with agents such as anthracyclines, taxanes or platinum chemotherapeutic agents, such as carboplatin, unless the patient has a tumour with a BRCA1 mutation. There is a clear clinical need to deliver new therapies for use in treating of such conditions to improve patient prognosis (Andre & Zielinski, Ann Oncol (2012) 23 (suppl 6): vi46-vi51). It is an object of the present invention to provide new therapies that are effective and are well tolerated for cancers such as triple negative breast cancer.

Tyrosine kinases are enzymes responsible for the activation, via phosphorylation, of many proteins by signal transduction cascades. Inhibition of tyrosine kinases has proven to be a successful therapeutic approach from the treatment of a number of cancer types and drugs that selectively inhibit specific tyrosine kinases have been developed. For example, the BcrAbl tyrosine kinases inhibitor nilotinib is used to treat chronic myelogenous leukaemia, while the epidermal growth factor receptor tyrosine kinase (EGFR/ ErbB-1) inhibitor gefitinib is used for the treatment of certain breast and lung cancers, especially for patient with EGFR mutations. Problems with development of drug resistance are however common for patients treated with tyrosine kinase inhibitors such as gefitinib despite initial and sometimes dramatic responses in EGFR-addicted tumours. In response to this resistance problem new kinase inhibitors such as osimertinib have been developed to treat tumours that have acquired resistance to gefitinib treatment through T790 mutation. Nonetheless, there is a significant and pressing need for new agents that can overcome acquired resistance to tyrosine kinase inhibitor therapy.

RAN, a member of the RAS Oncogene family, is a gene that encodes the GTP-binding nuclear protein Ran. Overexpression of RAN gene is observed in a number of cancers and this overexpression has been linked to poor patient prognosis. For example, RAN overexpression has been shown to correlate with increased aggressiveness of cancer cells in vitro and in vivo (Kurisetty et al, Oncogene 2008, 27, 7139-49), i.e. RAN overexpressing cancer cells are seen to grow rapidly and exhibit high metastatic potential. In contrast, silencing RAN by siRNA or shRNA reduced cell adhesion, migration and invasion in vitro and metastasis in vivo. Furthermore, in vitro studies have demonstrated that silencing the RAN gene with SiRNA or shRNA induces a greater degree of apoptosis in cancer cells relative to that induced in normal cells and in activated KRas-mutant cells relative to their isogenic KRas wild-type counterparts. Cancer cells are observed to be more sensitive to changes in RAN status than their normal counterparts. RAN silencing has also been observed to promote apoptosis in cancer cells with mutations that correlate with activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK signalling pathways (Yuen et al, Clin Cancer Res 2011; 18(2); 1-12). As a result, RAN has been suggested as a potential therapeutic target for cancer phenotypes in which the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways are activated.

Ran (Ras-related nuclear) protein, a 25-kDa protein product encoded by RAN gene, is a Gprotein GTPase that cycles between a GDP-bound (RanGDP) and a GTP-bound (RanGTP) state that regulates nucleocytoplasmic transport, mitotic spindle fibre assembly and postmitotic nuclear envelope dynamics.

Ran exists in a different conformation depending on whether it is bound to GTP or GDP. In its GTP bound state, Ran is capable of binding karyopherins (importins and exportins) a set of proteins that are involved in importing and exporting molecules between the nucleus and the cytoplasm of a eukaryotic ceil. Importins release a molecular cargo upon binding to RanGTP in the nucleus, while exportins must bind RanGTP in the nucleus to form a ternary complex with their export cargo in order to transport the cargo to the cytoplasm.

The dominant nucleotide binding state of Ran depends on whether it is located in the nucleus (RanGTP) or the cytoplasm (RanGDP), with RanGTP being formed inside the nucleus through interaction of Ran with its specific guanine nucleotide exchange factor (GEF), regulator of chromosome condensation 1 referred to herein as RCC1, which catalyses the exchange of GDP for GTP on the nucleotide binding pocket of Ran. Hydrolysis of RanGTP to RanGDP in the cytoplasm by RanGAP and RanBPI releases energy and causes the ternary complex of Ran, exportin and cargo to dissociate thus releasing the cargo exported from the nucleus. Cytoplasmic RanGDP is in turn imported into the nucleus by the small protein NTF2 (Nuclear Transport Factor 2), where RCC1 can then catalyse exchange of GDP for GTP on Ran and complete the Ran cycle.

During mitosis, the Ran cycle is involved in mitotic spindle assembly and nuclear envelope reassembly after the chromosomes have been separated. During prophase, the steep gradient in RanGTP-RanGDP ratio at the nuclear pores breaks down as the nuclear envelope becomes leaky and disassembles. RanGTP concentration stays high around the chromosomes as RCC1 stays attached to chromatin as the nucleoporin RanBP2 (Nup358) and Ran GTPase activating protein (RanGAP) move to the kinetochores where they facilitate the attachment of spindle fibres to chromosomes. Moreover, RanGTP promotes spindle assembly by mechanisms similar to mechanisms of nuclear transport: the activity of spindle assembly factors such as NuMA and TPX2 is inhibited by the binding to importins. By releasing importins, RanGTP activates these factors and therefore promotes the assembly of the mitotic spindle. In telophase, RanGTP hydrolysis and nucleotide exchange are required for vesicle fusion at the reforming nuclear envelopes of the daughter nuclei.

There is a growing awareness that RAN is a potential target for cancer therapy, in particular breast and lung cancer therapy. Studies have shown that breast cancer patients whose primary tumours have a higher percentage of malignant cell nuclei that stain for Ran have a shorter median survival time than those with less than 1% of cell nuclei that stain for Ran (P <0.001). When analyzed alongside tumour size, grade, and lymph node involvement using Cox-regression analysis, Ran nuclear staining was independently associated with patient survival time [relative risk (RR) 12.53, 95% Cl: 3.95-39.758, P < 0.001] (Yuen HF, El-Tanani M, etal. J Natl Cancer Inst. 2013;105(7):475-88 & Yuen HF, El-Tanani M, etal. Clin Cancer Res. 2012; 18(2):380-91).

Analysis of the GEO breast cancer data set f (200 patients (GSE2034)) indicated that a high level of Ran significantly correlates to shorter survival time in patients with PIK3CA mutation gene signature (P = 0.018), but not in those with PIK3CA wild-type gene signature (P = 0.186) (Yuen HF, El-Tanani M, etal. J Natl Cancer Inst. 2013; 105(7):475-88 & Yuen HF, El-Tanani M, etal. Clin Cancer Res. 2012;18(2):380-91).

The present inventors have previously established that higher levels of Ran are significantly correlated with a shorter survival time in lung cancer patients. In addition, it has also been established that there is a much greater difference in survival time between patients with high and low levels of Ran expression in c-Met positive tumours (median survival 58.1 months, final proportion surviving 18% vs >216 months, 81%, respectively; Wilcoxon-Gehan χ2 = 16.5, p < 0.001) than in c-Met negative tumours (median survival > 216 months, 78% vs > 216 months, 100%, respectively; Wilcoxon-Gehan χ2 = 9, p = 0.003). When analysed with other prognostic factors such as clinical stage, lymph node involvement, and histology, Ran expression was independently associated with patient survival times in Cox-regression analyses.

Stable transfection of non-invasive mammary cells (R37 and MCF-10A) with an expression vector for Ran (R37-Ran and MCF-10A-Ran) was found to induce an invasive/metastatic phenotype in vitro and the development of metastases in vivo (Kurisetty VV, El-Tanani MK et al. Oncogene. 2008:27(57):7139-49). Stable transfection of invasive cells (MDA-MB-231, R37-OPN and R37-RAN) with siRNA directed at Ran specifically inhibits the invasive/metastatic phenotype in vitro and in vivo (Kurisetty VV, El-Tanani MK et al. Oncogene. 2008;27(57):7139-49, Yuen HF, El-Tanani M, et al. J Natl Cancer Inst. 2013; 105(7):475-88 & Yuen HF, El-Tanani M, etal. Clin Cancer Res. 2012; 18(2):380-91. In addition, inhibition of Ran expression in tumour cell lines was found to cause abnormal mitotic spindle formation, mitochondrial dysfunction, and apoptosis. Use of siRNA/shRNAi or expression vector for Ran dominant negative (G19V/Q69L) mutant was found to lead to an anti-proliferative and/or anti-invasive phenotype in multiple models including breast and lung (Kurisetty W, El-Tanani MKeia/. Oncogene. 2008;27(57):7139-49).

Ran silencing was found to induce a significant apoptotic response in GR5 c-Met amplified cells in the presence of gefitinib, but not in HCC827 parental cells (p <0.01), suggesting that Ran silencing may be used as a modulator of chemosensitivity (Yuen HF, El-Tanani M, et al. J Natl Cancer Inst. 2013; 105(7):475-88).

Silencing Ran expression induced higher apoptosis in breast cancer MDA-MB-231 than immortalized MCF10a cells, and c-Met amplified cells compared to their isogenic c-Met wildtype counterparts, respectively (Yuen HF, El-Tanani M, et al. J Natl Cancer Inst. 2013; 105(7):475-88).

Gefitinib, erlotinib and osimertinib are a small molecule tyrosine kinase inhibitors (TKIs) of epidermal growth factor receptor (EGFR) used in the treatment of non-small-cell lung cancer (NSCLC) patients. Since 2004, a substantial proportion of patients with NSCLC harbouring activating mutations in the EGFR gene have obtained an objective response to TKIs, with longer progression-free survival, a more favourable toxicity profile and better quality-of-life in randomized clinical trials. Unfortunately, despite good initial responses to TKI therapy, acquired resistance is a significant issue. 50% of acquired resistance to EGFR tyrosine kinase inhibitors (TKI) is caused by a mutation in the ATP binding pocket of the EGFR kinase domain involving substitution of a small polar threonine residue with a large nonpolar methionine residue, T790M. Osimertinib was developed to treat T790M mutation-positive NSCLCs. Exploration of novel approaches to further enhance the therapeutic efficacy of TKIs in such patient cohorts aimed at improving progression-free and overall survival times remains an important challenge.

It is an object of the present invention to provide small molecules that inhibit RAN at the transcriptional level and their use in the treatment of cancers that overexpress RAN or its protein product Ran, for instance in patients who have been identified as having a cancer that overexpresses RAN or its protein product Ran or that are receiving or have progressed on tyrosine kinase inhibitor therapy. Diseases overexpressing RAN include but are not limited to certain breast, lung, prostate, ovarian, blood, brain and renal cancers and include those cancers currently associated with poor patient prognosis such as triple negative breast cancer.

Summary of the Invention

In a first aspect of the invention there is provided mebendazole, or a pharmaceutically acceptable form thereof, for use as an inhibitor of RAN at a transcriptional level. In preferred embodiments, the mebendazole for use as an inhibitor of RAN at a transcriptional level, is for use in the treatment of cancer, for example a cancer that overexpresses RAN or its protein product Ran. In most preferred cases the mebendazole for use as an inhibitor of RAN at a transcriptional level in the treatment of cancer, is for use in patients identified as having a cancer that overexpresses RAN, for example a cancer that overexpresses Ran mRNA, or its protein product Ran. In embodiments the mebendazole for use is used in the treatment of breast, lung, prostate, ovarian, blood, brain and renal cancers including those cancers currently associated with poor patient prognosis such as triple negative breast cancer. As used herein and above, a cancer that overexpresses RAN, may overexpress Ran mRNA and/or Ran protein and may be identified, for example, by establishing that elevated levels of Ran mRNA and/or Ran protein are present in the cancer cells.

The invention also provides a Ran inhibitor, i.e. a compound capable of inhibiting RAN, for example at a transcriptional level, or its protein product Ran, for example a small molecule inhibitor of RAN at a transcriptional level such as mebendazole or pimozide (as described in UK patent application No. GB1616880.9) or a protein inhibitor of the Ran I RCC-1 interaction (as described in UK patent application No. GB1607593.9), for use in combination with a tyrosine kinase inhibitor, such as a small molecule or an antibody tyrosine kinase inhibitor, for the treatment of cancer. Examples of tyrosine kinase inhibitors that may be used in combination with a RAN inhibitor for the treatment of cancer include afatinib (Giotrif™), axitinib (Inlyta™), bosutinib (Bosulif™), crizotinib (Xalkori™), dasatinib (Sprycel™), erlotinib (Tarceva), gefitinib (Iressa™), osimertinib (Tagrisso™), imatinib (Glivec™), lapatinib (Tyverb™), nilotinib (Tasigna™), pazopanib (Votrient™), regorafenib (Stivarga™), sorafenib (Nexavar™), sunitinib (Sutent™) and ibrutinib (Imbruvica™). In embodiments, the inhibitor of RAN for use with a tyrosine kinase inhibitor is an inhibitor of RAN at a transcriptional level such as mebendazole.

In embodiments, mebendazole is used as an inhibitor of RAN in combination with an inhibitor of an epidermal growth factor receptor (EGFR) tyrosine kinase such as the small molecules gefitinib, erlotinib, afatinib, brigatinib, icotinib, or osimertinib or the antibodies cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab. Molecules and antibodies that inhibit epidermal growth factor receptor (EGFR) tyrosine kinase are generally referred to herein as EGFR inhibitors. In such cases the co-administration of mebendazole and an EGFR inhibitor may be simultaneous, sequential or separate. It has been demonstrated that the combination of mebendazole and an EGFR inhibitor has a synergistic effect against cancer cell proliferation. It has also been shown that mebendazole can restore sensitivity of gefitinib resistant cancer cells to gefitinib treatment therefore providing a new therapeutic approach for the treatment of patients who have progressed on EGFR inhibitor therapy and, potentially, a new mechanism to prevent development of EGFR inhibitor resistance.

Identification of the patients indicated for mebendazole treatment, i.e. those having a cancer that overexpresses RAN, its protein product Ran or its target pathways including c-myc, cMet, osteopontin, PI3K/AKT/mTORC and KRas/MEK/ERK1/2 may be achieved by obtaining a sample from the candidate patient, for example a tumour biopsy or a blood sample, and analysing the sample for high expression of Ran mRNA or protein or a biomarker for Ran expression. Alternatively, or additionally, the patient identification may involve testing for cMet amplification, PI3K/AKT/mTORC and KRas/MEK/ERK1/2 or mutation status of a tyrosine kinase such as EGFR.

Analysis of a tumour biopsy for identifying a patient having a RAN overexpressing tumour (i.e. a tumour that overexpresses Ran mRNA or Ran protein) can, for example, be carried out by immunohistochemical means. Thus, a tumour biopsy sample can be stained with a stain comprising a RAN selective antibody linked to a visualising means such as an enzyme or fluorophore. The stained tumour biopsy can then be categorised as a RAN overexpressing tumour or a non-RA/V overexpressing tumour. This categorisation can be done by a visual or automated scoring. Testing for tyrosine kinase mutation status can be by the methods available in the art, for example the commercially available testing kits produced by Genzyme, QIAGEN and Argenomics S.A. for predicting response to the various tyrosine kinase inhibitors.

Analysis of a patient blood sample to determine whether a patient has a RAN overexpressing tumour (i.e. a tumour that overexpresses Ran mRNA or Ran protein) may be performed by isolating the serum exosome and then carrying out an ELISA (to assess Ran protein expression) or quantitative real time PCR (q RT-PCR, to detect RNA expression) analysis to provide the necessary tumour/patient classification.

In preferred embodiments, the patient is a human patient.

In some embodiments, the mebendazole for use is administered in a dosage of from 1 to 5000 mg/day.

The invention also relates to a method of treatment of a cancer that overexpresses RAN (or Ran mRNA) or its protein product Ran, comprising a step of administering to a patient in need thereof an effective amount of mebendazole or a pharmaceutically acceptable form thereof. This method of treatment may further comprise a step of testing a sample obtained from said patient to determine whether said patient has a RAN overexpressing cancer prior to said administration step.

The invention also provides a method of treatment of a cancer that overexpresses RAN (or Ran mRNA), or its protein product Ran, comprising the step of administering to a patient who has been identified as having a cancer that overexpresses RAN, or its protein product Ran, an effective amount of mebendazole or a pharmaceutically acceptable form thereof. This method of treatment may further comprise a step of testing a sample obtained from said patient to determine whether said patient has a RAN overexpressing cancer prior to said administration step.

The invention also provides a method of treatment of a cancer that overexpresses RAN, (or Ran mRNA) or its protein product Ran, comprising the steps of selecting a patient who has a cancer that overexpresses RAN or its protein product Ran, and administering an effective amount of mebendazole or a pharmaceutically acceptable form thereof to that patient. This method of treatment may further comprise a step of testing a sample obtained from said patient to determine whether said patient has a RAN overexpressing cancer prior to said administration step.

The invention also provides a method of treatment for cancer in a patient diagnosed as having a cancer characterised by an overexpression of RAN or its protein product Ran involving administration to a patient in need thereof an effective amount of mebendazole. The cancer characterised by an overexpression of RAN may be triple negative breast cancer. The cancer characterised by an overexpression of RAN may be lung cancer, and in particular lung cancers that have previously been treated with a tyrosine kinase inhibitor, for example wherein the previous treatment involved administration of an tyrosine kinase inhibitor such as an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor such as gefitinib, erlotinib, afatinib, brigatinib, icotinib or osimertinib to a lung cancer patient and wherein the disease no longer responds to the treatment with the EGFR inhibitor. Such methods may involve coadministration of the previously used tyrosine kinase inhibitor with a Ran inhibitor, in particular wherein the Ran inhibitor is mebendazole.

The invention also provides a method of treatment for cancer in a patient diagnosed as having a cancer characterised by an overexpression of a tyrosine kinase, such as a mutated tyrosine kinase, such as epidermal growth factor receptor (EGFR) tyrosine kinase, involving administering mebendazole in combination with an inhibitor of the tyrosine kinase that is overexpressed, for example in a cancer with a mutation in the EGFR tyrosine kinase domain, an EGFR inhibitor may be used. In such methods the administration of mebendazole and an EGFR inhibitor may be simultaneous, sequential or separate. Advantageously the combination of mebendazole and an EGFR inhibitor may prevent or substantially delay the emergence of resistance to the EGFR inhibitor associated with EGFR inhibitor monotherapy. Advantageously the combination of mebendazole and an EGFR inhibitor may resensitize a tumour to treatment with an EGFR inhibitor to which the patient no longer responds as a monotherapy.

The invention also provides mebendazole for use as an immunotherapy, for example for the treatment of therapy, for instance by down regulating C5a.

In a further aspect, the invention relates to the use of mebendazole, or a pharmaceutically acceptable form thereof, for the manufacture of a medicament for the treatment of cancer, optionally wherein the cancer overexpresses RAN or its protein product Ran. The resultant medicament may be provided in a package with instruction for use in the treatment of a cancer that overexpresses RAN or its protein product Ran. The resultant medicament may be provided in a package with instruction for use in combination with an EGFR inhibitor such as gefitinib.

In a further aspect, the invention provides a package comprising mebendazole or a pharmaceutically acceptable form thereof and instructions for its use in the treatment of a cancer that overexpresses RAN or its protein product Ran or instructions for its use in the treatment of a cancer that overexpresses epidermal growth factor receptor (EGFR) tyrosine kinase.

Description of Figures

Figure 1 shows the effects of drug treatment on A) cell morphology and B) survival of HCC827 lung cancer cells on exposure to control (DMSO), mebendazole (0.1 μΜ, 0.5 μΜ, 1 μΜ), gefitinib (1 μΜ) and dasatinib (1 μΜ) and combinations of gefitinib and dasatinib with mebendazole.

Figure 2 shows the effects of drug treatment on A) cell morphology and B) survival of HCC827 GR5 (gefitinib resistant) lung cancer cells on exposure to control (DMSO), mebendazole (0.1 μΜ, 0.5 μΜ, 1 μΜ), gefitinib (1 μΜ) and dasatinib (1 μΜ) and combinations of gefitinib and dasatinib with mebendazole.

Figure 3 shows the relative gene expression (fold change) in HCC827 cells treated with mebendazole (0.1 μΜ, 0.5 μΜ) for 24h relative to control.

Figure 4 shows the relative gene expression (fold change) in HCC827 GR5 (gefitinib resistant) cells treated with mebendazole (0.1 μΜ, 0.5 μΜ) for 24h relative to control.

Figure 5 show the results of immunoblotting for A) Ran (28 kDa, rabbit, 1:3000 dil in 5% milk powder), B) pBad (27 kDa, mouse, 1:3000 dil in 5% BSA), C) Actin (40 kDa, mouse, 1:10 000 dil in 5% milk powder) in the lysate obtained from HCC827 GR5 cells treated with 1) Control (DMSO), 2) 1 μΜ Gefitinib, 3) 300 nM osimertinib, 4) 100 nM mebendazole, 5) 100 nM mebendazole + 1 μΜ Gefitinib, 6) 100 nM mebendazole + 300 nM osimertinib, 7) 500 nM mebendazole, 8) 500 nM mebendazole + 1 μΜ Gefitinib and 9) 500 nM mebendazole + 300 nM osimertinib.

Figure 6 shows the effect on HCC827 human cancer cell survival following treatment with A) gefitinib, B) pimozide and C) various concentrations of pimozide in combination with 300 nM gefitinib.

Figure 7 shows the effect on HCC827 GR5 (gefitinib resistant) human cancer cell survival following treatment with A) gefitinib B) pimozide and C) various concentrations of pimozide in combination with 300 nM gefitinib.

Detailed description of the invention

The present invention relates to mebendazole, or a pharmaceutically acceptable form thereof, for use as a Ran inhibitor for the treatment of cancer, for example in patients identified as having a cancer that overexpresses RAN or Ran, for example a cancer in which atypically high elevated levels of Ran mRNA and/or Ran protein are present in the cancerous cells.

Ran, a protein product of the gene RAN, is a member of the Ras superfamily that regulates nucleocytoplasmic transport, mitotic spindle fibre assembly and post-mitotic nuclear envelope dynamics. Ran acts as a molecular switch through a GTP-GDP cycle in which the conversion between GTP-bound and GDP-bound conformations controls its interaction with different effectors. RanGTP is formed inside the nucleus by interaction of Ran with its specific guanine nucleotide exchange factor (GEF), referred to herein as RCC1, which catalyses the exchange of GDPfor GTP (and vice versa) on the nucleotide binding pocket of Ran. RAN, and its protein product Ran are known to be overexpressed in a number of cancers including those in which the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways are activated. Diseases overexpressing Ran include but are not limited to certain breast, lung, prostate, ovarian, blood, brain and renal cancers and include those cancers currently associated with poor patient prognosis such as triple negative breast cancer. Inhibiting Ran, either through blockage of its interactions with other proteins or by downregulation of Ran synthesis (e.g. by blocking RAN transcription) therefore provides a potential approach for the treatment of cancer.

Mebendazole, methyl (5-benzoyl-1H-benzimidazol-2-yl)carbamate (Formula I below), is a broad spectrum antihelmintic drug sold under trade names such as Vermox™ and Emverm™ indicated for the treatment of nematode infestations, including roundworm, hookworm, whipworm, threadworm, pinworm, and the intestinal form of trichinosis prior to its spread into the tissues beyond the digestive tract. The clinical effect of mebendazole as an antihelmintic agent is thought to stem from selective inhibition of the synthesis of microtubules in parasitic worms through binding at the colchicine site of tubulin. Destruction of extant cytoplasmic microtubes in the worm intestinal cells, blocks the uptake of glucose and other nutrients, resulting in the gradual immobilization and eventual death of the helminths.

Mebendazole, Formula (I)

A number of reports have also indicated that mebendazole has activity against the proliferation of cancers cells in in vivo and in vitro models of cancer (see e.g. Mukhopadhyay et al, Clin Cancer Res 8(9) 2963-9).

Prior to the present invention, it was not known that mebendazole could inhibit RAN or production of its protein product Ran, nor was it known that mebendazole inhibits RAN at a transcriptional level. As described herein, the identification of mebendazole as a Ran inhibitor that acts by inhibiting RAN at a transcriptional level, thereby suppressing the downstream effects of the RAN gene, for example Ran mRNA expression, provides a new and advantageous option for therapy of patients with cancers that overexpress RAN or its protein product Ran. The present invention advantageously allows identification of groups of cancer patients that are indicated for mebendazole treatment and advantageously allows a therapeutic intervention for such patients that is targeted to the genetic profile of their cancer. The present invention thus potentially provides for improved therapeutic outcomes for cancer patients with cancers including solid and blood cancers (including solid and blood cancers, e.g. acute lymphoblastic leukaemia, acute myeloid leukaemia, chronic lymphocytic leukaemia, chronic myeloid leukaemia, lymphoma and myeloma) that overexpress RAN and its protein product Ran.

The invention provides mebendazole, or a pharmaceutically acceptable form thereof, for use as an inhibitor of RAN at a transcriptional level. In preferred embodiments, the mebendazole for use as an inhibitor of RAN at a transcriptional level, is for use in the treatment of cancer, for example a cancer that overexpresses RAN or its protein product Ran. In most preferred cases the mebendazole for use as an inhibitor of RAN at a transcriptional level in the treatment of cancer, is for use in patients identified as having a cancer that overexpresses RAN or its protein product Ran. Examples of cancer types that are known to overexpress RAN or its protein product Ran, and for which mebendazole as a Ran inhibitor is indicated for use include, but are not limited to certain breast, lung, prostate, ovarian, bladder, colorectal, blood, brain and renal cancers and include certain cancers currently associated with poor patient prognosis such as triple negative breast cancer.

The selection of patients to be treated with mebendazole for use according to the invention is advantageously performed by establishing that they have a cancer that overexpresses RAN or its protein product Ran. This selection may be performed by obtaining a sample from the candidate patient, for example a tumour biopsy or a blood sample, and analysing the sample for the presence of RAN or a biomarker for RAN expression.

Analysis of a tumour biopsy for identifying a patient having a RAN overexpressing tumour (i.e. a tumour that overexpresses Ran mRNA or Ran protein) can, for example, be carried out by immunohistochemical means. Thus, a tumour biopsy sample can be stained with a stain comprising a RAN selective antibody linked to a visualising means such as an enzyme or fluorophore. The stained tumour biopsy can then be categorised as a RAN overexpressing tumour or a non-RA/V overexpressing tumour. This categorisation can be done by a visual or automated scoring.

Analysis of a patient blood sample to determine whether a patient has a RAN overexpressing tumour and is therefore indicated for treatment with mebendazole may be performed by isolating the serum exosome and then carrying out an ELISA or quantitative real time PCR (q RT-PCR) analysis to provide the necessary tumour/patient classification.

The present invention also relates to a Ran inhibitor, for example mebendazole, for use in combination with a tyrosine kinase inhibitor, for example a receptor tyrosine kinase inhibitor such as an inhibitor of EGFR tyrosine kinase or a non-receptor tyrosine kinase inhibitor such as a Bcr-Abl kinase inhibitor, for the treatment of cancer. Other receptor tyrosine kinases inhibitors that may be used in combination with a Ran inhibitor such as mebendazole include inhibitors of PDGFR and FGFR kinases. Other non-receptor tyrosine kinases inhibitors that may be used in combination with a Ran inhibitor such as mebendazole include inhibitors of SRC, FAKand JAK. Other Ran inhibitors, such as small molecule, peptide, protein or antibody inhibitors of Ran, may be used in conjunction with a tyrosine kinase inhibitor. As can be seen from the data provided in Figures 1 and 2, co-administration of a Ran inhibitor with the receptor tyrosine kinase inhibitor gefitinib or a non-receptor tyrosine kinase inhibitor dasatinib provides a synergistic, i.e. better than additive, effect against the survival of human lung cancer cells. The present inventors have also determined that a further small molecule Ran inhibitor, pimozide, that, like mebendazole, inhibits RAN at a transcriptional level, also works in a synergistic manner with the tyrosine kinase inhibitor gefitinib against survival of HCC827 cancer cells and the gefitinib resistant HCC827-GR5 cell line. This data suggests that the combination of Ran inhibitors in general in combination with tyrosine kinase inhibitors is useful in the treatment of cancer. In particular, inhibitors of RAN at a transcriptional level have proven to act in a synergistic manner with inhibitors of EGFR and Bcr-Abl tyrosine kinases.

A new approach for the treatment of cancers that are indicated for tyrosine kinase inhibitor therapy is thus provided that involves co-administration of the appropriate tyrosine kinase inhibitor with a Ran inhibitor. The Ran inhibitor may be a small molecule inhibitor of RAN at a transcriptional level such as mebendazole or pimozide, a peptide inhibitor of the Ran I RCC1 interaction or an antibody. The tyrosine kinase inhibitor may be as a small molecule or an antibody tyrosine kinase inhibitor. Examples of tyrosine kinase inhibitors that may be used in combination with a Ran inhibitor for the treatment of cancer include afatinib (Giotrif™), axitinib (Inlyta™), bosutinib (Bosulif™), crizotinib (Xalkori™), dasatinib (Sprycel™), erlotinib (Tarceva), gefitinib (Iressa™), osimertinib (Tagrisso™), imatinib (Glivec™), lapatinib (Tyverb™), nilotinib (Tasigna™), pazopanib (Votrient™), regorafenib (Stivarga™), sorafenib (Nexavar™), sunitinib (Sutent™) and ibrutinib (Imbruvica™). In preferred embodiments, the Ran inhibitor for use with a tyrosine kinase inhibitor is an inhibitor of RAN at a transcriptional level such as mebendazole. The co-administration of a Ran inhibitor and tyrosine kinase inhibitor may be simultaneous, sequential or separate.

In earlier studies Ran RNAi was shown to silence Ran protein by 96%. The studies presented herein indicate that a combination of EGFR TKI and Ran inhibitor therapy may profoundly improve the outcome for resistant EGFR TKI cancer patients (see e.g. Figure 2). Gefitinib suppresses AKT and ERK activation in HCC827 NSCLC parental cells but has no effect on these signals in HCC827 GR5 cells that are resistant to gefitinib in vitro and show MET amplification. Interestingly, it was also found that Ran silencing induced a significant apoptotic response in GR5 c-Met amplified cells in the presence of gefitinib, but not in HCC827 parental cells (p < 0.01), suggesting that Ran silencing may be used as a modulator of chemosensitivity. The present inventors have established that Ran silencing induced significantly higher cell death and activation of apoptotic pathways in the A549 NSCLC ceil line (P < 0.01) (results not shown). Treatment of A549 cells by the mTORCl inhibitor, rapamycin, the class I P!3K/mTORC1 dual inhibitor, Pi 103, and the MEK1/2 inhibitor, PD184352, inhibited phosphorylation of the corresponding downstream targets and reduced cell proliferation. These inhibitors of the PI3K/Akt/mTORC1 or MEK/ERK pathways significantly reduced Ran siiencing-induced apoptosis in A549 cells (P < 0.05). Similar results were obtained in a MTT cell survival assay. These results suggest that Ran may be preferentially required for survival in NSCLC ceils with higher activities of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Ran silencing resulted in a significantly higher apoptotic response in the mutant K-Ras transformed ceils compared with their wild-type (WT) K-Ras counterparts in the DLD-1/DKO-3 colon cancer isogenic pair (P < 0.01). This result suggests that cancer cells with Ras activation are more susceptible to Ran siiencing-induced apoptosis. In another isogenic cell pair, the HCT116 WT and Pten-nuli colon cancer cell lines, the activation of the Pi3K/Akt pathway through deletion in Pten sensitized the cancer ceils to Ran siiencing-induced apoptosis, which was also potentially in a PI3K/Akt-'mTORC1 pathway-dependent manner.

c-MET amplification, which leads to activation of the PI3K/AKT and MEK/ERK pathway promotes gefitinib resistance in lung cancer cells. Gefitinib-resistant cells, with c-Met amplification, have been shown to have an increased apoptotic response to Ran silencing compared to their wild-type counterparts, indicating that cells with c-MET amplification are more susceptible to apoptosis in the presence of Ran silencing. These results were confirmed using a MTT cell survival assay. Furthermore, inhibition of the PI3K/AKT and RAS/MEK/ERK pathways significantly reduced c-MET-mediated sensitization to Ran knock-down (p < 0.01).

The combination of Ran inhibitor (Ran silencing) and TKIs as gefitinib (as a first line EGFR TKI) would enhance anti-tumour activity in NSCLC. To date, the therapeutic effect of the combination therapy of Ran inhibitor with EGFR TKI’s on human cancers has not been reported.

In embodiments mebendazole is used as an inhibitor of RAN in combination with an inhibitor of an epidermal growth factor receptor (EGFR) tyrosine kinase such as the small molecules gefitinib, erlotinib, afatinib, brigatinib, icotinib, or osimertinib or the antibodies cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab. Molecules and antibodies that inhibit epidermal growth factor receptor (EGFR) tyrosine kinase are generally referred to herein as EGFR inhibitors. In such cases the co-administration of mebendazole and an EGFR inhibitor may be simultaneous, sequential or separate. It has been demonstrated that the combination of mebendazole and an EGFR inhibitor, namely gefitinib, has a synergistic effect against cancer cell proliferation (see Figure 1). It has also been shown that mebendazole can restore sensitivity of gefitinib resistant cancer cells to gefitinib treatment therefore providing a new therapeutic approach for the treatment of patients who have progressed on EGFR inhibitor therapy and, potentially, a new mechanism to prevent development of EGFR inhibitor resistance (see Figure 2). Furthermore, it has been shown that the Ran inhibitor mebendazole can eradicate the induction of Ran stimulated by the EGFR inhibitors gefitinib and osimertinib thus indicating that co-administration of a Ran inhibitor with a tyrosine kinase inhibitor may overcome or substantially delay acquired resistance to tyrosine kinase inhibitor therapy.

Identification of the patients indicated for combination therapy with a Ran inhibitor and a tyrosine kinase inhibitor may involve testing for mutation status of a tyrosine kinase such as EGFR. Testing for tyrosine kinase expression/mutation status can be by the methods available in the art, for example the commercially available testing kits produced by Genzyme, QIAGEN and Argenomics S.A. for predicting response to the various tyrosine kinase inhibitors.

The present inventors have also identified that mebendazole can down regulate C5a (Figures 3 and 4). This finding indicates that mebendazole may be used to potentiate or promote an innate immune response to cancer cells/tumours. The invention thus provides mebendazole for use to potentiate or promote any innate immune response to cancer cells/tumours (i.e. as an immunotherapy) for the treatment of cancer via to its ability to down regulate C5a.

In preferred embodiments, the patient is a mammal and in most preferred cases the patient is a human patient.

The invention also relates to a method of treatment of a cancer that overexpresses RAN, or its protein product Ran or Ran mRNA, comprising a step of administering to a patient in need thereof an effective amount of mebendazole or a pharmaceutically acceptable form thereof. This method of treatment may further comprise a step of testing a sample obtained from said patient to determine whether said patient has a RAN overexpressing cancer prior to said administration step.

The invention also provides a method of treatment of a cancer that overexpresses RAN, or its protein product Ran or Ran mRNA, comprising the step of administering to a patient who has been identified as having a cancer that overexpresses RAN, or its protein product Ran, an effective amount of mebendazole or a pharmaceutically acceptable form thereof. This method of treatment may further comprise a step of testing a sample obtained from said patient to determine whether said patient has a RAN overexpressing cancer prior to said administration step.

The invention also provides a method of treatment of a cancer that overexpresses RAN, or its protein product Ran or Ran mRNA, comprising the steps of selecting a patient who has a cancer that overexpresses RAN or its protein product Ran or Ran mRNA, and administering an effective amount of mebendazole or a pharmaceutically acceptable form thereof to that patient. This method of treatment may further comprise a step of testing a sample obtained from said patient to determine whether said patient has a RAN overexpressing cancer prior to said administration step.

The invention also provides a method of treatment for cancer in a patient diagnosed as having a cancer characterised by an overexpression of RAN or its protein product Ran or Ran mRNA involving administration to a patient in need thereof an effective amount of mebendazole. The cancer characterised by an overexpression of RAN may be triple negative breast cancer. The cancer characterised by an overexpression of RAN may be lung cancer, and in particular lung cancers that have previously been treated with a tyrosine kinase inhibitor, for example wherein the previous treatment involved administration of an tyrosine kinase inhibitor such as an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor such as gefitinib, erlotinib, afatinib, brigatinib, icotinib or osimertinib to a lung cancer patient and wherein the disease no longer responds to the treatment with the EGFR inhibitor. Such methods may involve coadministration of the previously used tyrosine kinase inhibitor with a Ran inhibitor, preferably wherein the Ran inhibitor is mebendazole.

The invention also provides a method of treatment for cancer in a patient diagnosed as having a cancer characterised indicated for tyrosine kinase therapy, by an overexpression of an tyrosine kinase, such as a mutated epidermal growth factor receptor (EGFR) tyrosine kinase, involving administering mebendazole in combination with an inhibitor of the tyrosine kinase that is overexpressed, for example in a cancer with a mutation in the EGFR tyrosine kinase domain, an EGFR inhibitor may be used. In such methods, the administration of mebendazole and an EGFR inhibitor may be simultaneous, sequential or separate. Advantageously the combination of mebendazole and an EGFR inhibitor may prevent or substantially delay the emergence of resistance to the EGFR inhibitor associated with EGFR inhibitor monotherapy. Advantageously the combination of mebendazole and an EGFR inhibitor may resensitize a tumour to treatment with an EGFR inhibitor to which the patient no longer responds as a monotherapy.

The invention relates to the use of mebendazole, or a pharmaceutically acceptable form thereof, for the manufacture of a medicament for the treatment of cancer, wherein the cancer overexpresses RAN or its protein product Ran or wherein the cancer is indicated for tyrosine kinase inhibitor therapy. The resultant medicament may be provided in a package with instruction for use in the treatment of a cancer that overexpresses RAN or its protein product Ran or for use in combination with a tyrosine kinase inhibitor for the treatment of cancer. Where the medicament is to be used in combination with a second pharmacologically active agent the administration of mebendazole and the second pharmacologically active agent may be simultaneous, sequential or separate. The resultant, mebendazole containing medicament may be provided in a package with instruction for use in combination with an EGFR inhibitor such as gefitinib.

The invention provides a package comprising mebendazole or a pharmaceutically acceptable form thereof and instructions for its use in the treatment of a cancer, wherein the cancer overexpresses RAN or its protein product Ran or Rna mRNA or instructions for its use in the treatment of a cancer that overexpresses a tyrosine kinase such as epidermal growth factor receptor (EGFR) tyrosine kinase.

The combination of mebendazole with a EGFR tyrosine kinase inhibitor, e.g. an EGFR inhibitor such as gefitinib, can be used for the treatment of a cancer that is naive to EGFR treatment or that has previously been treated with an EGFR inhibitor, for example a cancer that has previously responded to treatment with a first EGFR inhibitor and that has subsequently developed resistance to that first EGFR inhibitor. The realisation of the potential to use a Ran inhibitor in combination with an EGFR inhibitor derives from the synergistic effect observed in combinations of mebendazole with gefitinib shown in Figures 1 and 2.

Reference to mebendazole herein and above relates to mebendazole itself or a pharmaceutically acceptable form of mebendazole. Pharmaceutically acceptable forms of mebendazole include prodrugs of mebendazole, that is to say compounds which break down and/or are metabolised in vivo to provide an active compound of formula (I). General examples of prodrugs include simple esters, and other esters such as mixed carbonate esters, carbamates, glycosides, ethers, acetals and ketals. Prodrug motifs may, for example, be directly introduced at the benzimidazole NH position of mebendazole.

Reference to pharmaceutically acceptable forms of mebendazole expressly include reference to known prodrugs of mebendazole, for example /V-alkoxycarbonyl prodrugs as described in Ini J Pharmaceutics 1994 (10) p 231-239 that advantageously improve aqueous solubility and, as result, oral bioavailability of mebendazole. /V-methoxycarbonyl, /V-ethoxy carbonyl and Npropoxycarbonyl forms of mebendazole (at the benzimidazole NH position of mebendazole), in their respective isomeric forms, are examples of prodrugs that are pharmaceutically acceptable forms of mebendazole.

Pharmaceutically acceptable forms of mebendazole include metabolites of mebendazole, in particular metabolites that retain an inhibitory effect on RAN transcription. A metabolite, as employed herein, is a compound that is produced in vivo from the metabolism of mebendazole, such as, without limitation, oxidative metabolites.

Pharmaceutically acceptable forms of mebendazole also include deuterated forms of mebendazole. Deuterated forms of mebendazole are derivatives of pimozide in which one or more aliphatic or aromatic hydrogen atom is substituted for a deuterium atom. For example, a hydrogen atom at a site susceptible to metabolism may be replaced by a deuterium atom to reduce metabolism at that site.

Pharmaceutically acceptable forms of mebendazole include formulations of mebendazole, for example formulations that exhibit a pharmacological profile that is different to the clinical formulations presently in use for treatment of neurological conditions.

Methods of treatment

As noted above, it is an object of the present invention to provide mebendazole, or a pharmaceutically acceptable form thereof, as an inhibitor of RAN transcription for use as a medicament.

In one aspect, the present invention provides methods for treatment or alleviation of a cancer over expressing RAN, or its protein product Ran or Ran mRNA, in the tissue of one or more organs as mentioned herein, comprising the step of administering mebendazole or a pharmaceutically acceptable form thereof to a patient in need thereof. In embodiments the method treatment is for patients with a cancer that have been diagnosed as having a cancer that overexpresses RAN or Ran or Ran mRNA.

Such methods according to the present invention may comprise one or more steps of administration or release of an effective amount of mebendazole or a pharmaceutically acceptable form thereof, or a pharmaceutical composition comprising mebendazole or a pharmaceutically acceptable form thereof, to an individual in need thereof. In one embodiment, such steps of administration or release according to the present invention is simultaneous, sequential or separate.

An individual in need as referred to herein may be an individual that benefits from the administration of a mebendazole or a pharmaceutically acceptable form thereof, in particular an individual with a cancer that overexpresses RAN or its protein product Ran or Ran mRNA. Such an individual in one embodiment suffers from a malignant neoplasm (tumour) in the tissue of one or more organs. In preferred examples the tumour(s) overexpresses Ran to that is subject to overexpressing Ran on treatment with a second pharmacologically agent that induces RAN or its protein product Ran. The cancer may be selected from cancers of the breast, lung, ovary or kidney or a leukaemia. The cancer may be one in which the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways are activated. The cancer may be triple negative breast cancer.

The individual may be any human being, male or female, infant, middle-aged or old. The individual may be a non-human mammal, for example a mammal from the members of the Canidae, Felinae, Bovidae, Equidae, Suidae, Camelini and Cervidae families.

Identification of the patient to be treated in the methods of the invention can be achieved in a number of ways. For example, the identification may involve analysis of a sample obtained from a patient, for example a blood sample or a tumour biopsy sample. Analysis of a patient blood sample may be performed by isolating the serum exosome and then carrying out an ELISA or quantitative real time PCR (q RT-PCR) analysis to provide the necessary tumour/patient classification.

Analysis of a tumour biopsy for identifying a patient having a RAN overexpressing tumour can, for example, be carried out by immunohistochemical means. Thus, a tumour biopsy sample can be stained with a stain comprising a RAN selective antibody linked to a visualising means such as an enzyme or fluorophore. The stained tumour biopsy can then be categorised as a

RAN overexpressing tumour or a non-RA/V overexpressing tumour. This categorisation may be performed by a visual or automated scoring. Studies have demonstrated that scoring of Ran immunohistochemically stained tumour biopsy sample slides can be performed under a light microscope and that an assessment of the % of tumour cells staining over the whole section provides a method to identify tumours that overexpress RAN.

The terms treatment and treating as used herein refer to the management and care of a patient for the purpose of combating a condition, disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of pimozide or a pharmaceutically acceptable form thereof for the purpose of: alleviating or relieving symptoms or complications; delaying the progression of the condition, partially arresting the clinical manifestations, disease or disorder; curing or eliminating the condition, disease or disorder; and/or preventing or reducing the risk of acquiring the condition, disease or disorder, wherein preventing or prevention is to be understood to refer to the management and care of a patient for the purpose of hindering the development of the condition, disease or disorder, and includes the administration of the active compounds to prevent or reduce the risk of the onset of symptoms or complications. The patient to be treated is preferably a mammal, in particular a human being. Treatment of animals, such as mice, rats, dogs, cats, cows, horses, sheep and pigs, is, however, also within the scope of the present invention. The patients to be treated according to the present invention can be of various ages, for example, adults, children, children under 16, children age 6-16, children age 2-16, children age 2 months to 6 years or children age 2 months to 5 years.

The invention is thus, in one embodiment, directed to mebendazole or a pharmaceutically acceptable form thereof for use in the treatment of cancer in the tissue of one or more organs of a mammal. In one embodiment said treatment is ameliorative and/or curative. In one embodiment, said mammal is a human (homo sapiens).

When referring to the tissue of one or more organs, said organ is in one embodiment selected from the group consisting of breast, lung, ovarian, prostate, blood, brain and renal cancers

Formulations

Mebendazole or a pharmaceutically acceptable form thereof for use as described herein, for example in the treatment of cancer that overexpresses RAN, or its protein product Ran, may be provided as a pharmaceutical composition comprising mebendazole or a pharmaceutically acceptable form thereof in combination with one or more pharmaceutically acceptable diluents or carriers. The formulation may be a formulation currently used in the clinic for mebendazole or may be a new formulation specifically adapted to the purpose of treating RAN overexpressing cancers or cancers indicated for tyrosine kinase inhibitor therapy.

Diluents and carriers may include those suitable for parenteral, oral, topical, mucosal and rectal administration.

The present invention also provides a process for preparing such a pharmaceutical composition (for example a pharmaceutical composition for parenteral, oral, topical, mucosal or rectal administration), said process comprising mixing the ingredients.

As mentioned above, such compositions may be prepared e.g. for parenteral, subcutaneous, intramuscular or intravenous administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; for topical e.g. pulmonary or intranasal administration, particularly in the form of powders, nasal drops or aerosols and transdermal administration; for mucosal administration e.g. to buccal, sublingual or vaginal mucosa, and for rectal administration e.g. in the form of a suppository.

The compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well-known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA., (1985). Formulations for parenteral administration may contain as excipients sterile water or saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. Formulations for nasal administration may be solid and may contain excipients, for example, lactose or dextran, or may be aqueous or oily solutions for use in the form of nasal drops or metered sprays. For buccal administration, typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinated starch, and the like.

Compositions suitable for oral administration may comprise one or more physiologically compatible carriers and/or excipients and may be in solid or liquid form. Tablets and capsules may be prepared with binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrollidone (PVP); fillers, such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, or glycine; lubricants, such as magnesium stearate, talc, polyethylene glycol, or silica; and surfactants, such as sodium lauryl sulfate. Liquid compositions may contain conventional additives such as suspending agents, for example sorbitol syrup, methyl cellulose, sugar syrup, gelatin, carboxymethyl-cellulose, or edible fats; emulsifying agents such as lecithin, or acacia; vegetable oils such as almond oil, coconut oil, cod liver oil, or peanut oil; preservatives such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Liquid compositions may be encapsulated in, for example, gelatin to provide a unit dosage form.

Solid oral dosage forms include tablets, two-piece hard shell capsules and soft elastic gelatin (SEG) capsules.

A dry shell formulation typically comprises of about 40% to 60% w/w concentration of gelatin, about a 20% to 30% concentration of plasticizer (such as glycerin, sorbitol or propylene glycol) and about a 30% to 40% concentration of water. Other materials such as preservatives, dyes, opacifiers and flavours also may be present. The liquid fill material comprises a solid drug that has been dissolved, solubilized or dispersed (with suspending agents such as beeswax, hydrogenated castor oil or polyethylene glycol 4000) or a liquid drug in vehicles or combinations of vehicles such as mineral oil, vegetable oils, triglycerides, glycols, polyols and surface-active agents.

Second active ingredients

In some embodiments, mebendazole, or a pharmaceutically acceptable form thereof, for use or for use in a method of treatment as described herein, can be combined with or comprise one or more second active ingredients which are understood as other therapeutic compounds or pharmaceutically acceptable derivatives thereof.

Methods for treatment according to the present invention may comprise one or more steps of administration of one or more second active ingredients, either concomitantly or sequentially, and in any suitable ratios. In embodiments, such second active ingredients are, for example, selected from compounds used to treat or prevent cancer in the tissue of one or more organs or symptoms or used to treat or prevent complications associated with treatment of cancer in the tissue of one or more organs. In embodiments, such a second active ingredients may be intended for the treatment of the side effects associated with the primary treatment or may be aimed at overcoming or suppressing resistance that may arise in the cancer. Thus, the one or more second active ingredients may be directed to the treatment of cancer, the treatment or suppression of emesis, the blockage of drug efflux pumps or potentiation of the anti-cancer effect of mebendazole.

A preferred class of second active ingredient for use in combination with mebendazole are tyrosine kinase inhibitors. The tyrosine kinase inhibitor may be as a small molecule or an antibody tyrosine kinase inhibitor. Examples of tyrosine kinase inhibitors that may be used in combination with a Ran inhibitor, for example mebendazole, for the treatment of cancer include afatinib (Giotrif™), axitinib (Inlyta™), bosutinib (Bosulif™), crizotinib (Xalkori™), dasatinib (Sprycel™), erlotinib (Tarceva), gefitinib (Iressa™), osimertinib (Tagrisso™), imatinib (Glivec™), lapatinib (Tyverb™), nilotinib (Tasigna™), pazopanib (Votrient™), regorafenib (Stivarga™), sorafenib (Nexavar™), sunitinib (Sutent™) and ibrutinib (Imbruvica™). Examples of EGFR tyrosine kinase inhibitors that may be used in combination with a Ran inhibitor, for example mebendazole, for the treatment of cancer include the small molecules gefitinib, erlotinib, afatinib, brigatinib, icotinib, and osimertinib and the antibodies cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab.

Methods of treatment according to the present invention in embodiments include a step wherein mebendazole is administered simultaneously, sequentially or separately in combination with one or more second active ingredients. In preferred embodiments, the action of the second therapeutic agent and mebenazole is synergistic.

Kit of parts

The present invention also provides a kit of parts. A kit of parts according to the present invention in one embodiment comprises mebendazole or a pharmaceutically acceptable form thereof or a composition thereof as defined herein for treatment of cancer in the tissue of one or more organs in combination with a further active ingredient as described herein, for example a tyrosine kinase inhibitor such as an inhibitor of EGFR. Kits according to the present invention in one embodiment allows for simultaneous, sequential or separate administration of mebendazole and one or more second active ingredients as described herein and are optionally provided with instructions therefore.

The invention provides a package containing mebendazole or a pharmaceutically acceptable form thereof and instructions for its use for the treatment of cancer, for example for patients with a cancer that overexpresses RAN or its protein product Ran or that is indicated for tyrosine kinase inhibitor therapy.

Administration and dosage

According to the present invention, a composition comprising mebendazole or a pharmaceutically acceptable form thereof is in embodiments administered to a cancer patient having a cancer that overexpresses RAN or its protein product Ran in a pharmaceutically effective dose or a therapeutically effective amount. A composition comprising mebendazole or a pharmaceutically acceptable form thereof may be administered to a cancer patient that is receiving or that has already received tyrosine kinase inhibitor therapy.

A therapeutically effective amount of mebendazole or a pharmaceutically acceptable form thereof according to the present invention is an amount sufficient to cure, prevent, reduce the risk of, alleviate or partially arrest the clinical manifestations of a cancer that overexpresses RAN or its protein product Ran or that has been or is being treated with tyrosine kinase inhibitor therapy. The amount that is effective will depend on the severity of the cancer as well as on the weight and general state of the subject. An amount adequate to accomplish this is defined as a therapeutically effective amount.

In one embodiment of the present invention, the composition is administered in doses of from 1 mg/day to 5000 mg/day.

Routes of administration

It will be appreciated that the preferred route of administration will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated, the location of the tissue to be treated in the body and the formulation of the active ingredient chosen. Mebendazole, or a pharmaceutically acceptable form thereof, may be administered as an oral formulation, for instance in the form of a tablet. In some cases, the administration may be in the form a local infusion or injection, or an intravenous or subcutaneous injection depending on the location of the cancer to be treated.

Systemic treatment

In one embodiment, the route of administration allows for introducing mebendazole, or a pharmaceutically acceptable form thereof, for use into the blood stream to ultimately target the desired site(s) of action.

In embodiments, the routes of administration is any suitable route, such as an enteral route (including the oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal and intraperitoneal administration), and/or a parenteral route (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal administration).

Appropriate dosage forms for such administration may be prepared by conventional techniques.

Parenteral administration

Parenteral administration is any administration route not being the oral/enteral route whereby the medicament avoids first-pass degradation in the liver. Accordingly, parenteral administration includes any injections and infusions, for example bolus injection or continuous infusion, such as intravenous administration, intramuscular administration or subcutaneous administration. Furthermore, parenteral administration includes inhalations and topical administration.

Accordingly, the composition may in embodiments be administered topically to cross any mucosal membrane of an animal to which the substance or peptide is to be given, e.g. in the nose, vagina, eye, mouth, genital tract, lungs, gastrointestinal tract, or rectum, for example the mucosa of the nose, or mouth, and accordingly, parenteral administration may also include buccal, sublingual, nasal, rectal, vaginal and intraperitoneal administration as well as pulmonal and bronchial administration by inhalation or installation. In some embodiments, the composition is administered topically to cross the skin.

In embodiments, intravenous, subcutaneous and intramuscular forms of parenteral administration may be employed.

In one embodiment, the mebendazole composition according to the invention is used as a local treatment, i.e. is introduced directly to the site(s) of action. Accordingly, mebendazole may be applied to the skin or mucosa directly, or may be injected into the site of action, for example into the diseased tissue or to an end artery leading directly to the diseased tissue.

Alternative pharmaceutical formulations

In one embodiment, mebendazole or a pharmaceutically acceptable form thereof according to the present invention or pharmaceutically acceptable derivatives thereof are administered alone or in combination with pharmaceutically acceptable carriers or excipients, in either single or multiple doses. The pharmaceutical compositions or compounds according to the invention may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 2000.

The term pharmaceutically acceptable form in present context includes pharmaceutically acceptable salts, which indicate a salt which is not harmful to the patient. Such salts include pharmaceutically acceptable basic or acid addition salts as well as pharmaceutically acceptable metal salts, ammonium salts and alkylated ammonium salts. A pharmaceutically acceptable derivative further includes esters and prodrugs, or other precursors of a compound which may be biologically metabolized into the active compound, or crystal forms of a compound.

The pharmaceutical composition or pharmaceutically acceptable composition may be specifically formulated for administration by any suitable route, such as an enteral route, the oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal, intraperitoneal, and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route.

In an embodiment of the present invention, the pharmaceutical compositions or compounds of the present invention are formulated for crossing the blood-brain-barrier.

Pharmaceutical compositions for oral administration include solid dosage forms such as hard or soft capsules, tablets, troches, dragees, pills, lozenges, powders and granules. Where appropriate, they can be prepared with coatings such as enteric coatings, or they can be formulated so as to provide controlled release of the active ingredient, such as sustained or prolonged release, according to methods well known in the art. In the same solid dosage form two active ingredients may be combined so as to provide controlled release of one active ingredient and immediate release of another active ingredient.

Liquid dosage forms for oral administration include solutions, emulsions, aqueous or oily suspensions, syrups and elixirs.

Pharmaceutical compositions for parenteral administration include sterile aqueous and nonaqueous injectable solutions, dispersions, suspensions or emulsions, as well as sterile powders to be reconstituted in sterile injectable solutions or dispersions prior to use. Depot injectable formulations are also regarded as being within the scope of the present invention.

Other suitable administration forms include suppositories, sprays, ointments, creams/lotions, gels, inhalants, dermal patches, implants, etc.

Mebendazole for use according to the present invention is generally utilized as the free substance or as a pharmaceutically derivative such as a pharmaceutically acceptable salt thereof

The term prodrug refers to derivatives of mebendazole that are rapidly transformed in vivo to yield mebendazole, for example, by hydrolysis in blood or by metabolism in cells, such as for example the cells of the basal ganglia. A thorough discussion is provided in T. Higuchi and V Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are hereby incorporated by reference.

Examples of prodrugs include pharmaceutically acceptable, non-toxic esters of the compounds of the present invention.

Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid and lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water. Moreover, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax. The pharmaceutical compositions formed by combining the compounds for use according to the present invention and the pharmaceutically acceptable carriers are then readily administered in a variety of dosage forms suitable for the disclosed routes of administration. The formulations may conveniently be presented in unit dosage form by methods known in the art of pharmacy.

Formulations of the present invention suitable for oral administration may be presented as discrete units, such as capsules or tablets, which each contain a predetermined amount of the active ingredient, and which may include a suitable excipient.

Furthermore, the orally available formulations may be in the form of a powder or granules, a solution or suspension in an aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsion.

Compositions intended for oral use may be prepared according to any known method, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient(s) in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may, for example, be: inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch or alginic acid; binding agents, for example, starch, gelatine or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in U.S. Patent Nos. 4,356,108; 4,166,452; and 4,265,874, the contents of which are incorporated herein by reference, to form osmotic therapeutic tablets for controlled release.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as a liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active compound in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavouring, and colouring agents may also be present.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavouring and colouring agent. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known methods using suitable dispersing or wetting agents and suspending agents described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conveniently employed as solvent or suspending medium. For this purpose, any bland fixed oil may be employed using synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compositions may also be in the form of suppositories for rectal administration of the compounds of the invention. These compositions can be prepared by mixing the compound with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will thus melt in the rectum to release the drug. Such materials include, for example, cocoa butter and polyethylene glycols.

Examples

The present invention is illustrated by the following examples, which are not intended to limit the scope of the invention.

Examples

Cell Morphology and Survival of HCC827 during treatment with mebendazole, gefitinib, dasatinib and combinations of mebendazole with gefitinib or dasatinib after 24 Hours

The effects of mebendazole treatment on the morphology of, and survival of, HCC827 epithelial lung adenocarcinoma cells (ATCC® CRL-2868™) with mebendazole, gefitinib (an EGFR inhibitor) and dasatinib (a Bcr-Abl Tyr Kinase inhibitor) alone or with combinations of mebendazole with gefitinib or dasatinib are shown in Figures 1A and 1B, respectively. As can be seen in Figure 1A, a significant change in the morphology of all the cells in all of the treated cohorts relative to control, DMSO treated, cells was produced.

The histogram of Figure 1B shows the survival of HCC827 cells 24h after treatment with mebendazole, gefitinib and dasatinib, or with a combination of the Ran inhibitor mebendazole and a tyrosine kinase inhibitor with 100% survival being evaluated relative to the control (DMSO) treated cells. As can be seen in Figure 1B treatment with mebendazole at concentrations of 0.1 μΜ and 0.5 μΜ causes a ca 30% and 65% reduction in cell survival. Treatment with 1μΜ concentrations of gefitinib and dasatinib caused a ca 40 and 70 % reduction in survival, respectively, relative to control. When mebendazole, at a concentration of 0.1 μΜ, was used in combination with gefitinib and dasatinib (each at 1 μΜ), a 70% and 80% reduction in cell survival was observed thus indicating a synergistic effect between the Ran inhibitor (mebendazole) and the tyrosine kinase inhibitors against the survival of human cancer cells.

Cell Morphology and Survival of HCC827 GR5 (gefitinib resistant) during treatment with mebendazole, gefitinib, dasatinib and combinations of mebendazole with gefitinib or dasatinib after 24 Hours

The experiments performed in wild type HCC827 cells were replicated with the HCC827 derived gefitinib resistant cell line HCC827 GR5. As can be seen in figure 2A the effects on morphology mirrored that observed with HCC827 with the exception of the gefitinib treated cohort that were largely unaffected by treatment. The lack of activity of gefitinib on the survival of these resistant cells at a concentration of 1 μΜ can be clearly seen in figure 2B and this contrasts strongly to the 65% reduction in cell survival when gefitinib was administered with 0.1 μΜ mebendazole. When the effect of 0.1 μΜ mebendazole (20% reduction in cell survival) is considered alongside the result obtained for the combination of mebendazole and gefitinb 65% reduction in cell survival then the synergy obtained by use of a RAN inhibitor in combination with an EGFR inhibitor is even more evident than in the HCC827 wild type cell line (see slide 1). The potential to use a Ran inhibitor, in particular mebendazole, as an adjunct to tyrosine kinase inhibitor therapy, such as EGFR inhibitor therapy, is therefore evident and may circumvent the emergence of acquired (e.g. to EGFR inhibitor therapy) resistance in patients.

The enhancement in activity of dasatinib treatment when the drug was given in combination with mebendazole demonstrates once more the synergy that can be derived from dosing of a Ran inhibitor with a second type of tyrosine kinase inhibitor (a Bcr-Abl tyrosine kinase).

Effect of Ran inhibitor treatment on the expression of mRNA

The direct effects of mebendazole on RAN at a transcriptional level is evident from Figures 3 and 4. In these slides the mRNA levels for various proteins in wild type HCC827 cells (Figures 3) and in the gefitinib resistant HCC827-GR5 cells (Figures 4) can be seen. The dose dependent reduction in Ran mRNA following mebendazole treatment demonstrates that mebendazole inhibits mebendazole at a transcriptional level.

Mebendazole treatment was also found to inhibit the mRNAs that code for a number of other proteins that are significant in cancer, for example the Akt1, Akt2, p53 and Bcl2 that are all markers of apoptosis. Akt1 and Akt2 are members of the PI3K/mTOR pathway that is found to be hyperactive in most of common cancers and in 90% of tyrosine kinase resistant cancers. c-Met, also called tyrosine-protein kinase Met or hepatocyte growth factor receptor (HGFR), is a protein that in humans is encoded by the MET gene. Abnormal c-Met activation/amplification is found to correlate with poor patient prognosis in cancer patients and overexpression of Met/HGFR have been implicated in oncogenesis and drug resistance in non small cell lung carcinoma (NSCLC).

A significant finding in this study that is evident from Figures 3 and 4 is the effect of mebendazole treatment on the mRNA for C5a (C5a is a protein fragment released from cleavage of complement component C5 by protease CS-convertase into C5a and C5b fragments).

Previous studies have shown that silencing Ran by shRNA dysregulated the immune response target genes. In more detail gene expression profiling to comprehensively delineate expression programs associated with Ran knock down in MDA MB 231 breast cell line. Total RNA from MDA MB 231-transfect with scrambled shRNA (control) or with specific Ran shRNA, MDA MB 231-shRNA (Ran knock down) cells was purified using the RNeasy Mini kit (Qiagen). The labeled cDNA samples were hybridized to NimbleGen human gene expression 12 x 135 K microarrays (Roche NimbleGen) that represent 44, 049 transcripts. Hybridized arrays were scanned (Molecular Devices). The data was extracted from scanned images using NimbleScan and the Robust Multichip Average (RMA) algorithm was used to generate gene expression values. Microarray data was normalised using Robust Multichip Average (RMA) method and differentially expressed genes between RAN knock-down and Control were identified by ANOVA. A total of 707 probes passing the filter of p-value <0.05 and fold change > |2| were selected for pathway analysis using GeneGo MetaCore. Important canonical pathways enriched in the hypergeometric statistical analysis included Regulation of epithelialto-mesenchymal transition (EMT) (FDR corrected p-value - 0.000008) and TGF-beta dependent induction of EMT via MAPK (FDR corrected p-value = 0.00000005).

The microarray data showed that

1. Ran silencing downregulates immune response - CD40 signalling (p= 8.17012E-10

Including STAT3, Acid sphingomyelinase, STAT5A, PI3K cat class IA, TRAF3, ATF-2, TRAF1, TRAF2, IKK-alpha, IL-8, Lyn, MEK3(MAP2K3), c-Jun, NF-kB, AKT(PKB), p53, PDK(PDPKI), P27KIP1, PLC-gamma 2, A20

2. Ran silencing downregulates immune response - Gastrin in inflammatory response (p= 2.6462E-09) including GRO-2, MEF2D, PI3K cat class IA, c-Fos, ATF-2, IKK-alpha, IL-8, PLC-gamma 1, EGFR, c-Jun, FAK1, G-protein alpha-q, SOS, MEF2, AKT(PKB), G-protein aipha-q/11, PDK (PDPK1), Stromelysin-1, Elk-1, ATF-2/c-Jun

3. Ran silencing downregulates immune response Immune response J L-S signaling pathway (p= 4.87983E-08) including STATS, gp130, c-Fos, JAK1, SHP-2, c-Jun, SOS, NF-kB, ADAM17, Eik-1, IL-6 receptor, IL6RA

4. Ran silencing downregulates immune responseJL-2 activation and signaling pathway (p= 1.18371 E-07) including STAT5A, PI3K cat class ΙΑ, AP-1, CISH, c-Fos, JAK1, IKKalpha, SHP-2, STATS, c-Jun/c-Fos, SOS, NF-kB, AKT(PKB), PDK(PDPKI), Elk-1

5. Ran silencing downregulates immune response_JL-5 signalling (=1.82897E-07) including STATS, RISK cat class IA, c-Fos, JAK1, cPLA2, Lyn, SHP-2, c-Jun, STAT5, SOS, AKT(PKB), C3G, PDK (PDPK1), STAT1

6. Ran silencing downregulates immune response__BCR pathway (p= 7.59585E-08) including ATF-2/c-Jun, NF-kB, c-Jun, AP-1, NF-kB p65/c-Rel, NF-kB p65/p65, NF-kB p50/p65

7, Ran silencing downregulates immune response_JL~1 (p= 4,9421 E-08) including COX2 (PTGS2), IL-6, Heme oxygenase 1, IL-8, AP-1, IL-1 alpha, c-Jun/c-Jun, PAI1, c-Jun

8, Ran silencing downregulates immune response Histamine H1 receptor signaling (p= 1.7861E-07) including gp130, SHP-2, OSM receptor, AP-1, OSMR, c-Jun, LIFR, UF receptor and CD16, Human NKG2D,

9, Ran silencing downregulates immune responsejDncostatin M signaling via MARK in human cells (p= 1.7861E-07).

The data in Figures 3 and 4 demonstrates that mebendazole, a small molecule inhibitor Ran, down regulates G5a. The significance of this evident when the following are considered. Although the role of the complement system in innate immunity is well characterized, recent studies suggest that the complement anaphylatoxins C3a and C5a are insidious propagators of tumour growth and progression. In more detail, it is now recognized that certain tumours elaborate C3a and C5a and that complement, as a mediator of chronic inflammation and regulator of immune function, and this may in fact foster rather than defend against tumour growth.

The tumour microenvironment is crucial for understanding cancer, and is subject to varying levels of immunosurveiliance and immunosuppression. It is believed that the complement anaphylatoxins C3a and C5a enhance tumour growth by shifting the balance toward immunosuppression, thus challenging longstanding dogma that complement activation is advantageous in cancer patients. Furthermore, the ability of neoplastic ceils to evade attack by complement proteins while simultaneously activating complement undermines traditional concepts of complement in tumour control. Numerous studies have investigated the relationship between local and/or systemic complement signalling, the host immune response, and tumour progression in experimental models of lymphoma and ovarian, mammary, breast, lung, and cervical cancer. Their findings collectively support the paradigm that C3a and/or C5a signalling modifies the immune infiltrate within the tumour microenvironment and/or the peripheral blood and lymphoid organs, with consequential effects on tumour growth. In addition, complement activation modulates the function or efficiency of several types of immune effector cells. Findings from in vivo cancer models clearly indicate that tumour progression can be halted and tumour regression achieved through the restoration of effective antitumor immunity.

The ability of mebendazole to down regulate C5a thus provide the potential to promote or potentiate any innate immune response to cancer cells in addition to direct anti-tumour effects derived from its ability to inhibit Ran, Mebendazole may therefore be used as an immunotherapy due to its ability to down regulate C5a. Without being bound by theory, it is believed that downregulation of C5a causes upregulation of T ceils and NK natural killer) cells therefore promoting/potentiatsng the immune response.

Effect of Ran inhibitor treatment and tyrosine kinases inhibitor treatment on the expression of Ran and pBad in HCC827 GR5

Further data on the effects of treatment of HCG827 cancer ceils with mebendazole and the EGFR inhibitors gefitinib and osimertinib are presented in Figures 5A, 5B and 5C. In these experiments HCC827-GR5 cells (in a quantity of 0.65 - 0.8 χ 10⁶) were seeded in a 10 cm petri dish and allowed to adhere overnight. The ceils were then treated as with (number correspond to the lane number of the Western Blots): 1) Control (DMSO), 2) 1 μΜ Gefitinib,

3) 300 nM Osimertinib, 4) 100 nM mebendazole, 5) 100 nM mebendazole + 1 μΜ Gefitinib, 6) 100 nM mebendazole 5 + 300 nM Osimertinib, 7) 500 nM mebendazole, 8) 500 nM mebendazole + 1 μΜ Gefitinib, 9) 500 nM mebendazole + 300 nM Osimertinib. After 24b of exposure to the drugs the cells from each cohort were lysed and 40 pg of each resultant lysate was separated on 12% PAG and blotted to nitrocellulose.

As can be seen in Figure 5A, blotting for Ran with 28 kDa, rabbit anti-Ran (1:3000 dilution in 5% milk powder) revealed the strong induction of Ran in the HCC827-GR5 ceils treated with gefitinib and osimertinib (lanes 2 and 3 respectively), a hitherto unknown effect. The induction of Ran by gefitinib was completely abolished by co-administration of 1G0nM mebendazole (lane 5). in contrast, the use of 100 nM mebendazole with 300 nM osimertinib (lane 8) was not sufficient to knock down all of the Ran, for this purpose 500 nM of mebendazole was required (lane 9).

These results suggest that a mechanism by which TKI inhibitors develop resistance proceeds via Ran upregulation and that it is this Ran upregulation that in turn delivers an increase c-Met and upregulation anti-apoptotic signaling pathways, it is thus apparent that a Ran inhibitor, acting at the protein or gene transcription level, can potentially be used to reverse TKI inhibitor resistance and restore resistance to TKis. Based on these results, a new therapeutic strategy for treatment of tumours having acquired tyrosine kinase inhibitor resistance involving treatment with a combination of a Ran inhibitor, such as mebendazole, and a tyrosine kinase inhibitor is foreshadowed. The results presented in Figures 1 to 5 demonstrate that the sensitivity to gefitinib is restored in the gefitinib resistant HCC827-GR5 cell line (see Figure 2) as discussed above. Furthermore, based on these results it appears that using a Ran inhibitor in conjunction with a tyrosine kinase inhibitor in patients indicated for tyrosine kinase inhibitor therapy may prevent or delay the development of acquired resistance to tyrosine kinase inhibitors in cancer patients. A new combination therapy for first line cancer therapy is therefore possible, especially when the synergistic effects of Ran inhibitor and tyrosine kinase therapy shown in Figures 1 and 2 is considered.

Figure 5B shows the results of a Western blot for pBad. When Bad is phosphoryiated it cannot interact with Bel and therefore cannot engage with the pro-apoptotic machinery of the cell. Figure 5C demonstrates that actin levels remain stable under drug treatment.

BAD is a member of the BCL-2 family. BCL-2 family members are regulators of the programmed cell death pathways. BAD induces apoptosis by inhibiting antiapoptotic BCL-2family members - BCL-x, Bcl~2, thereby allowing two other pro-apoptotic proteins, BAK and BAX, to aggregate and induce release of cytochrome. Proapoptotic activity of this protein is regulated through its phosphorylation. Protein kinases AKT and MAP kinase, as well as protein phosphatase caicineurin were found to be involved in the regulation of this protein. The BAD protein is a pro-apoptotic member of the Bci-2 family whose ability to heterodimerize with survival proteins such as Bci-X(L) and to promote ceil death is inhibited by phosphorylation.

Gefitinib treatment results in a reduction in pBad whereas osimertinib appears to have no effect, compared to control, on pBad in GR5 cells. Mebendazole at 500 nM alone and in combination increases the dephosphorylation of pBad, and thus increased apoptosis, whereas the effect of 100 nM mebendazole is small after 24 hr.

Activity of the Ran inhibitor pimozide and gefitinib, as single agents or in combination, against the survival of HCC827 human lung cancer cells and HCC827 GR5 (gefitinib resistant) human lung cancer cells

As can be seen in Figure 6a, after 24 h of treatment with the epidermal growth factor receptor (EGFR) inhibitor gefitinib at all of the evaluated concentrations above 15 nM the survival of the HCC827 cells ranges from ca 50 to 60 %. Treatment with 5 μΜ pimozide causes a reduction in cell survival relative to control of approximately 40%. Treatment of HCC827 cells with gefitinib (at 300 nM) with a 5 μΜ concentrations of pimozide causes a reduction in cell survival of ca 80%, thus illustrating a synergistic effect against cancer cell survival that is realised from the combination of a Ran inhibitor and a EGFR inhibitor (gefitinib).

Figure 7 shows the effects of drug treatment for 24 h on the survival of gefitinib resistant HCC827 GR5 cells, a cell line that is derived from normal HCC827 human lung cancer cells (cf Figure 6). As can be seen in Figure YA, treatment with gefitinib in this instance at all of the concentrations evaluated causes no more than a 15% reduction in cell survival. Pimozide treatment (Figure 7B) causes a ca 30% reduction in cell survival at a concentration of 5 μΜ. The combination of various concentrations of pimozide and gefitinib (300 nM) meanwhile proved superior to either agent when they were presented as single agents. As can be seen in Figure 7C, a ca 40% reduction in cell survival at a pimozide concentration was observed at 5 μΜ pimozide, while higher concentrations of pimozide saw cell survival fall to 20% at 7.5 μΜ and to less than 10% at 12 μΜ. It is thus evident that pimozide restores sensitivity to the EGFR inhibitor gefitinib in these resistant cells. Furthermore, as with the data obtained in the wild type HCC827 as presented in Figure 6, the combination of the EGFR gefitinib inhibitor (gefitinib) and the Ran inhibitor (pimozide) proves to be synergistic since the reduction in cell survival observed is greater than additive.

Claims

1) Mebendazole, or a pharmaceutically acceptable form thereof, for use as a Ran inhibitor.

2) Mebendazole for use according to claim 1 for use in medicine.

3) Mebendazole for use according to claim 1 or claim 2, for the treatment of cancer.

4) Mebendazole for use according to claim 3, wherein the cancer is a cancer that overexpresses RAN or its protein product Ran or Ran mRNA.

5) Mebendazole for use according to claim 3 or claim 4, wherein the use is in a patient identified as having a cancer that overexpresses RAN or its protein product Ran or Ran mRNA.

6) Mebendazole for use according to any preceding claim in combination with a tyrosine kinase inhibitor.

7) Mebendazole for use according to claim 6, wherein the tyrosine kinase inhibitor is an inhibitor of EGFR or Bcr-Abl.

8) Mebendazole for use according to claim 6 or 7 wherein the mebendazole for use and the tyrosine kinase inhibitor is administered simultaneously, sequentially or separately.

9) Mebendazole for use according to any of claim 6 to 8 wherein the tyrosine kinase inhibitor is gefitinib.

10) A Ran inhibitor for use in the treatment of cancer, wherein the use is in combination with a tyrosine kinase inhibitor.

11) A Ran inhibitor for use according to claim 10, wherein the use is in a patient that has previously been previously treated with the tyrosine kinase inhibitor.

12) A Ran inhibitor for use according to claim 10, wherein the use is in a patient that has not previously been treated with the tyrosine kinase inhibitor.

13) Use according to any of claims 10 to 12, wherein the Ran inhibitor is mebendazole or a pharmaceutically acceptable form thereof.

14) Use according to any of claims 10 to 13, wherein the tyrosine kinase inhibitor is afatinib, axitinib, bosutinib, crizotinib, dasatinib, erlotinib, gefitinib, osimertinib, imatinib, lapatinib, nilotinib, pazopanib, regorafenib, sorafenib, sunitinib and ibrutinib.

15) Use according to any of claims 10 to 14, wherein the tyrosine kinase inhibitor is an inhibitor of EGFR.

16) Use according to any of claims 10 to 15, wherein the mebendazole for use serves to overcome the resistance to the tyrosine kinase inhibitor with which it is administered.

17) Use according to claim 16, wherein the mebendazole for use prevents c-Met upregulation in a tumour.

18) A method of treatment of a cancer that overexpresses RAN, or its protein product Ran, or Ran mRNA, comprising a step of administering to a patient in need thereof an effective amount of mebendazole or a pharmaceutically acceptable form thereof.

19) Method according to claim 18, further comprising testing a sample obtained from said patient to determine whether said patient has a RAN, Ran or Ran mRNA overexpressing cancer prior to said administration step.

20) A method of treatment of a cancer that overexpresses RAN, or its protein product Ran, or Ran mRNA, comprising the step of administering to a patient who has been identified as having a cancer that overexpresses RAN, or its protein product Ran, an effective amount of mebendazole or a pharmaceutically acceptable form thereof.

21) Method according to claim 20, further comprising testing a sample obtained from said patient to determine whether said patient has a cancer that overexpresses RAN, Ran or Ran mRNA prior to said administration step.

22) A method of treatment for cancer in a patient diagnosed as having a cancer characterised by an overexpression of RAN, or its protein product Ran or Ran mRNA involving administration to a patient in need thereof an effective amount of mebendazole or a pharmaceutically acceptable form thereof.

23) Method according to any of claims 18 to 22, wherein the cancer is triple negative breast cancer.

24) Method according to any of claims 18 to 22, wherein the cancer is lung cancer, optionally wherein the lung cancer has previously been treated with a tyrosine kinase inhibitor.

25) Method according to claim 24 wherein the previous treatment involved administration of an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor such as, or selected from, gefitinib, erlotinib, afatinib, brigatinib, icotinib or osimertinib.

26) Method according to any of claims 18 to 25, wherein the cancer has progressed following prior treatment with EGFR inhibitor therapy.

27) A method of treatment for cancer in a patient diagnosed as having a cancer characterised by an overexpression of c-Met or a tyrosine kinase or mutation of a tyrosine kinase, involving administering a Ran inhibitor in combination with an inhibitor of the overexpressed or mutated tyrosine kinase.

28) Method according to claim 27, wherein the tyrosine kinase is EGFR.

29) Method according to claim 27 or 28, wherein the Ran inhibitor is mebendazole or a pharmaceutically acceptable form thereof.

30) Method according to any of claims 27, 28 or 29, wherein the cancer is resistant to the tyrosine kinase inhibitor when that tyrosine kinase inhibitor is administered as a monotherapy.

31) Use of mebendazole, or a pharmaceutically acceptable form thereof, for the manufacture of a medicament for the treatment of cancer, wherein the cancer i) overexpresses RAN or its protein product Ran or Ran mRNA; or ii) is characterised by an overexpression or mutation of a tyrosine kinase.

32) Use according to claim 31, wherein the tyrosine kinase is EGFR.

33) A kit containing mebendazole, or a pharmaceutically acceptable form thereof, in a package with instruction for use in the treatment of a cancer that i) overexpresses RAN or its protein product Ran or Ran mRNA or ii) is characterised by an overexpression or mutation of a tyrosine kinase.

34) Kit according to claim 33, wherein the tyrosine kinase is EGFR.

35) Mebendazole, or a pharmaceutically acceptable form thereof, for use as an immunotherapy.

36) Mebendazole, or a pharmaceutically acceptable form thereof, for use according to claim 35, wherein the use is for the treatment of cancer.

37) Mebendazole, or a pharmaceutically acceptable form thereof, for use according to claim 35 or 36, wherein the mebendazole is used to downregulate C5a.

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Application No: Claims searched:

GB1701400.2

1-37