Nuclear transport inhibitors for Anti-cancer combination therapy

Abstract

A combination for use in treating cancer is provided. The combination comprises a therapeutically effective amount of an inhibitor of nuclear import protein Karyopherin Beta 1 (Kpnβ1) such as the substituted pyrrolo[2,3-b]quinoxalines of Formula I and a therapeutically effective amount of an inhibitor of nuclear export protein Chromosome Maintenance 1 (Crm1) such as the hydrazide- containing compounds of Formula II. The combination therapy provides an enhanced anti-cancer therapeutic effect compared to the effect of each of the nuclear transport inhibitors administered alone.

Description

[0001] NUCLEAR TRANSPORT INHIBITORS FOR ANTI-CANCER COMBINATION THERAPY

[0002] CROSS-REFERENCE(S) TO RELATED APPLICATIONS

[0003] This application claims priority from United Kingdom patent application number 2411999.2 filed on 14 August 2024, which is incorporated by reference herein.

[0004] FIELD

[0005] This disclosure relates to small molecule nuclear transport inhibitors for anti-cancer combination therapy. In particular, it relates to the use of a combination of small molecules that modulate protein transport in and out of the nucleus of cells to obtain an enhanced anti-cancer effect.

[0006] BACKGROUND

[0007] The Karyopherin superfamily of proteins are soluble receptors that mediate nucleocytoplasmic shuttling of proteins and RNAs, and recently, nuclear transport receptors have been recognized as novel anticancer targets. The nuclear import and export receptors, Karyopherin Beta 1 (Kpnpi) and Chromosome Maintenance 1 (Crm1), are involved in the nucleo-cytoplasmic shutting of proteins. Both Kpnpi and Crm1 exhibit elevated expression in several cancer types. Accordingly, Kpnpi and Crm1 have each been separately suggested to be potential targets for treating proliferative diseases such as cancer.

[0008] Kpnpi , also known as Importin p, is a major nuclear transport receptor in the cell, which imports proteins containing nuclear localisation signals (NLS) into the nucleus [1], It also plays a role in regulating other key functions in the cell, including mitosis [1], Kpnpi has been reported to be overexpressed in various cancer types, including cervical cancer [2], prostate cancer [3], glioma [4] and ovarian cancer [5], amongst others. A summary of known inhibitors of Kpnpi / KPNA- and Kpnpi -dependent nuclear import is shown in Table 1.

[0009] Table 1. Inhibitors of KPNpi / KPNA- and KPNpi-dependent nuclear import.

[0010] Compound

[0011] Inhibitor Target Preclinical / Clinical anticancer activity type

[0012] Karyostatin Disrupts Blocked nuclear entry of NFAT cancer Small

[0013] 1A

[0031] RanGTP cell lines molecule binding to Importazole Disrupts Blocked nuclear entry of NFKB p65 and Small

[0014]

[0032] RanGTP c-MYC in cancer cell lines molecule binding to Antiproliferative and pro-apoptotic effects

[0015] KPNB1 in multiple myeloma, chronic myeloid leukemia, prostate, and breast cancer cell lines

[0016] Slowed tumor growth in mouse xenograft models of prostate cancer [3, 33, 34]

[0017] Ivermectin Binds I MPA and KPN B1 -dependent anti-proliferative and Natural

[0018]

[0035] inhibits KPNB1- pro-apoptotic effects in epithelial ovarian compound

[0019] KPNA-mediated carcinoma cell lines

[0036] (antibiotic, import Blocked nuclear import of HIF-1a, antiparasitic thereby downregulating hypoxia-induced agent) tumorigenic transcriptional responses

[0037]

[0020] 2- Potently binds Blocks nuclear entry of Erb2, EGFR and Small

[0021] Aminothiazole KPNB1 (Kd: STAT3 in several cancer cell lines. molecule derivative ~20nM); inhibits Anti-proliferative (G2 / M arrest) and pro-

[0022] Compound 1 classical and apoptotic effects in cancer cell lines

[0039] non-classical import pathways

[0038]

[0023] 2- Blocked tumor growth in murine Small

[0024] Aminothiazole xenograft model of pancreatic cancer molecule derivative

[0040]

[0025] Compound 6

[0026] INI-43 [6] Designed to Blocked nuclear entry of AP-1 , NFAT and Small target NFKB in cancer cell lines. molecule overlapping Anti-proliferative (G2 / M arrest) and pro-

[0027] RanGTP and apoptotic effects in breast, cervical and IMPA2 binding esophageal cancer cell lines; inhibits site of KPNB1 motility and invasive potential of cervical cancer cells

[0041] , Slowed tumor growth in cervical and oesophageal murine xenograft models INI-60

[0042] Designed Anti-proliferative (G1 / S arrest) and pro- Small target the apoptotic effects in cervical and molecule overlapping oesophageal cancer cell lines. RanGTP and Slowed tumor growth in esophageal

[0028] KPNA2 binding murine xenograft model site of KPNB1

[0029] A number of substituted pyrrolo[2,3-b]quinoxalines have been identified as inhibitors of nuclear import mediated by Kpnpi [6], Inhibitor of Nuclear Import 43 (INI-43), with chemical name 3-( 7 / 7- benzimidazol-2-yl)-1-(3-dimethylaminopropyl)pyrrolo[5,4-b]quinoxalin-2-amine, most potently inhibited nuclear entry and transcriptional activity of NFAT, AP-1 and NFKB, all representative cargoes of KPNB1 / KPNA-mediated import. INI-43 treatment of cancer cells results in G2 / M cell cycle arrest and cancer cell death via apoptosis. It also significantly inhibits the growth of tumours in vivo [6], Specific inhibition of Kpnpi has the potential to globally disrupt oncogenic nuclear import as both classical and nonclassical pathways may be targeted. It can lead to inhibition of lineage-defining transcription factors in small cell lung cancer

[0028] ,

[0030] Crm1 , also known as Exportin 1 (Xpo1), is the major nuclear export protein in the cell and cargoes proteins containing nuclear export signals (NES) out of the nucleus [7], Overexpression of Crm1 has been reported in many cancer types, including cervical cancer [2], oesophageal cancer [8], pancreatic cancer [9], ovarian cancer

[0010] and glioma

[0011] , amongst others. Crm1 is overexpressed in a broad range of cancer subtypes, where high mRNA or protein levels are observed in more advanced disease states and predict poor patient prognosis. Several small molecule inhibitors of Crm1 have been developed, with Selinexor (XPOVIO, KPT-330) displaying potent in vitro and in vivo anti-cancer effects against a broad range of cancer types [12a], Selinexor forms part of the group of Selective Inhibitors of Nuclear Export (SINE) compounds, which are a series of highly potent and orally bioavailable small molecules that modify Cys528 in a slowly reversible manner, resulting in transient Crm1 inhibition in normal cells and a significantly improved therapeutic window [12b], A summary of currently known XPO1 inhibitors is shown in Table 2.

[0031] Table 2. Inhibitors of Crm1 / XPO1 -dependent nuclear export.

[0032] Inhibitor Preclinical / Clinical anticancer activity Compound type

[0033] Leptomycin B Phase I clinical trial in advanced solid tumors Natural compound (Irreversibly binds (terminated early due to dose-limiting toxicities)

[0043] (antibiotic, XP01 Cys528) antifungal agent) KPT-330 >50 ongoing phase l / ll / lll clinical trials (monotherapy Small molecule

[0034] (Selinexor) or combination therapy) in solid and hematological

[0035] (Reversibly binds malignancies

[0030] ,

[0036] XP01 Cys528) FDA-approved for the following applications:

[0044]

[0037] • Combination treatment with dexamethasone in relapsed / refractory multiple myeloma (RRMM) patients who have received at least one prior therapy

[0038] • Combination treatment with dexamethasone and bortezomib in RRMM patients who have received at least one prior therapy

[0039] • As a single agent in relapsed or refractory diffuse B cell lymphoma patients

[0040] KPT-8602 Ongoing phase l / ll clinical trials (monotherapy or Second generation

[0041] (Eltanexor) combination therapy) for patients with relapsed or SINE (poor blood

[0042] (Reversibly binds refractory multiple myeloma, metastatic colorectal brain barrier

[0043] XP01 Cys528) cancer, metastatic castration resistant prostate penetrability; cancer and high-risk myelodysplastic syndrome

[0045] predicted improvement in tolerability profile)

[0046]

[0044] CBS9106 Ongoing phase I trial (single agent) for patients with Small molecule

[0045] (Felezonexor) advanced solid tumors

[0047]

[0046] (Reversibly binds XP01 Cys528; targets XP01 for proteasomal degradation

[0048] )

[0047] Several hydrazide containing SINE compounds are described in PCT international publication number WO2013 / 019548A1 , which is incorporated by reference herein. The hydrazide containing SINE compounds display excellent in vivo exposure as measured by area under the curve (AUC) tumour growth measurements in mouse, rat, dog and monkey, while exhibiting low levels of brain penetration. Selinexor, referred to as I-3 in WO2013 / 019548A1 , has undergone clinical trials for various hematologic malignancies and solid tumours and has been approved by the Food and Drug Administration (FDA) for use in the treatment of multiple myeloma. Early clinical studies involving Selinexor as a single agent demonstrated promising response rates in patients with hematopoietic malignancies

[0029] ,

[0048] While displaying significant anti-cancer activity on its own, it has been found that combining Crm1 inhibitors, like Selinexor, with other classes of therapeutics, is an effective strategy in the treatment of cancer

[0013] , Selinexor acts in synergistically sensitising multiple myeloma cells to the alkylating agent melphalan

[0014] , Furthermore, the combination of Selinexor and proteasome inhibitors, Bortezomib or Carfilzomib, is synergistic in various cancer types, including neuroblastoma cells

[0015] , sarcoma cells [16,17] and myeloma cells [18,19], Selinexor has also been found to act synergistically with cisplatin in ovarian cancer cells

[0020] and to enhance thyroid cancer cell sensitivity to doxorubicin

[0021] , Selinexor has been approved for combined treatment with dexamethasone in relapsed or refractory multiple myeloma patients who have received at least one prior therapy. Selinexor is currently being investigated in over 50 clinical trials in both solid tumours and haematological malignancies as a single agent or in combination with FDA- approved chemotherapeutic drugs

[0030] ,

[0049] Cancer chemotherapy is still the mainstay treatment modality in the management of disseminated malignancies. Patient responses and tolerability to treatment are generally poor and those that enter remission often return with refractory disease. Various cancers are becoming increasingly resistant or otherwise insensitive to traditional chemotherapeutic drugs. Consequently, there is a need for new anti-cancer therapies which aim to overcome chemoresistance mechanisms and / or address dose-limiting toxicities. There is therefore a need for new types of therapy that may alleviate at least some of the problems associated with existing approaches to cancer treatment.

[0050] The preceding discussion of the background is intended only to facilitate an understanding of the present disclosure. It should be appreciated that the discussion is not an acknowledgment or admission that any of the material referred to was part of the common general knowledge in the art as at the priority date of the application.

[0051] SUMMARY

[0052] In accordance with an aspect of the disclosure there is provided a combination for use in treating cancer comprising (a) a therapeutically effective amount of an inhibitor of nuclear import receptor Karyopherin Beta 1 (Kpnpi) or a pharmaceutically acceptable salt thereof and (b) a therapeutically effective amount of an inhibitor of nuclear export receptor Chromosome Maintenance 1 (Crm1) or a pharmaceutically acceptable salt thereof.

[0053] The inhibitor of nuclear import receptor Kpnpi may be / V-(1-phenylethyl)-2-(1-pyrrolidinyl)-4- quinazolinamine (Importazole), 9-[(1-methylpiperidin-3-yl)methoxy]-4-[(6-methylpyridin-2- yl)methyl]-7-(5-methylthiophen-2-yl)-3,5-dihydro-2H-1 ,4-benzoxazepine (INI-60) or a substituted pyrrolo[2,3-b]quinoxaline compound of Formula I below. The inhibitor of Crm1 may be a SINE compound that targets the XP01 protein such as (E)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1 ,2,4- triazol-1-yl]-2-pyrimidin-5-ylprop-2-enamide (Eltanexor, KPT-8602) or a hydrazide containing SINE compound of formula II below.

[0054] The inhibitor of nuclear import receptor Kpnpi may be a compound of Formula I: Formula I or a pharmaceutically acceptable salt thereof, in which

[0055] Ri is a branched or linear C2-C5 alkyl group optionally functionalised with a substituent selected from the group consisting of an amine, an imidazole, an alcohol or a morpholine; and

[0056] R2 is a hydrogen or a methyl group; or or a pharmaceutically acceptable salt thereof and the inhibitor of nuclear export receptor Crm1 may be a compound of Formula II: Formula II or a pharmaceutically acceptable salt thereof, in which

[0057] Xi is a hydrogen or a methyl group;

[0058] X2 is a pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrazin-2-yl, and quinoxaline-2-yl, pyrimidin- 4-yl, 1 ,1-dioxotetrahydrothiophen-3-yl, or cyclopropyl and wherein X2 is optionally substituted with one or more methyl or halogen substituents; or

[0059] Xi and X2together with their intervening atoms are 4-hydroxypiperidin-1-yl, pyrrolidin-1-yl, azepan-1-yl, 4-benzylpiperazin-1-yl, 4-ethylpiperazin-1-yl, 3-hydroxyazetidin-1-yl, or morpholin-4- yl;

[0060] X3 is a hydrogen or a halogen; and

[0061] ' / vvv' represents a single carbon-carbon bond bound to a carbon-carbon double bond which is either in an (E)- or (^-configuration.

[0062] R1 in the compound of Formula I may be selected from:

[0063] , an ethyl group, a propyl group, a butyl group, an / so-butyl group, an / so-pentyl group, a propanol group and

[0064] The compound of Formula I may be selected from the group consisting of:

[0065]

[0066] In particular, the compound of Formula I may be

[0067] The inhibitor of Kpnpi may be The compound of Formula II may be selected from

[0068] Preferably, the compound of Formula II is

[0069] The combination may provide an enhanced therapeutic anti-cancer effect. The combination may provide a synergistic therapeutic anti-cancer effect.

[0070] The cancer to be treated with the combination may be selected from the group consisting of cervical cancer, oesophageal cancer, multiple myeloma, ovarian cancer, uterine cancer, breast cancer, gastric cancer, lung cancer, leukemia, pancreatic cancer, colon cancer and melanoma. The cancer to be treated with the combination may be selected from the group consisting of cervical cancer, oesophageal cancer, ovarian cancer, uterine cancer, breast cancer and multiple myeloma.

[0071] In accordance with a second aspect of the invention, there is provided a method of treating cancer comprising administering to a subject in need thereof

[0072] (a) a therapeutically effective amount of an inhibitor of nuclear import receptor Kpnpi or a pharmaceutically acceptable salt thereof; and

[0073] (b) a therapeutically effective amount of an inhibitor of nuclear export receptor Crm1 or a pharmaceutically acceptable salt thereof.

[0074] The inhibitor of Kpnpi may be a compound of Formula I as defined above or pharmaceutically acceptable salt thereof or INI-60 or a pharmaceutically acceptable salt thereof and the inhibitor of Crm1 may be a compound of Formula II as defined above or pharmaceutically acceptable salt thereof.

[0075] In accordance with a third aspect of the invention, there is provided a use of a therapeutically effective amount of an inhibitor of nuclear import receptor Kpnpi or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of an inhibitor of nuclear export receptor Crm1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cancer.

[0076] The inhibitor of Kpnpi may be a compound of Formula I as defined above or pharmaceutically acceptable salt thereof or INI-60 or a pharmaceutically acceptable salt thereof and the inhibitor of Crm1 may be a compound of Formula II as defined above or pharmaceutically acceptable salt thereof. The medicament may be manufactured such that the compound of Formula I or INI-60 may be administered simultaneously with the compound of Formula II. The medicament may comprise both the compound of Formula I and the compound of Formula II. Alternatively, the medicament may be manufactured such that the compound of Formula I or INI-60 and the compound of Formula II, may be administered sequentially. Accordingly, the compound of Formula I or INI-60 may be separately manufactured for this use with the compound of Formula II.

[0077] In accordance with a fourth aspect of the invention, there is provided a kit for treating cancer comprising

[0078] (a) a therapeutically effective amount of an inhibitor of nuclear import receptor Kpnpi or a pharmaceutically acceptable salt thereof; and

[0079] (b) a therapeutically effective amount of an inhibitor of nuclear export receptor Crm1 or a pharmaceutically acceptable salt thereof.

[0080] The inhibitor of Kpnpi may be a compound of Formula I as defined above or a pharmaceutically acceptable salt thereof or INI-60 or a pharmaceutically acceptable salt thereof and the inhibitor of Crm1 may be a compound of Formula II as defined above or pharmaceutically acceptable salt thereof.

[0081] In accordance with a fifth aspect of the invention, there is provided a pharmaceutical composition comprising (a) a therapeutically effective amount of an inhibitor of nuclear import receptor Kpnpi or a pharmaceutically acceptable salt thereof; (b) a therapeutically effective amount of an inhibitor of nuclear export receptor Crm1 or a pharmaceutically acceptable salt thereof and (c) a pharmaceutically acceptable carrier.

[0082] The pharmaceutical composition may include a compound of Formula I as defined above or pharmaceutically acceptable salt thereof or INI-60 or a pharmaceutically acceptable salt thereof and a compound of Formula II as defined above or pharmaceutically acceptable salt thereof. Embodiments of the technology will now be described, by way of example only, with reference to the accompanying drawings.

[0083] BRIEF DESCRIPTION OF THE DRAWINGS

[0084] In the drawings:

[0085] Figure 1 is a bar graph of the relative mRNA expression of Kpnpi and Crm1 , respectively, both of which are significantly elevated in oesophageal tumour biopsies compared to matched normal epithelial tissue (n = 22);

[0086] Figure 2 is a plot of Kpnpi expression levels versus Crm1 expression levels in oesophageal tumour tissue;

[0087] Figure 3 is a plot of Kpnpi expression levels versus Crm1 expression levels in oesophageal tumour tissue by comparing fold change;

[0088] Figure 4 is a plot of Kpnpi expression levels versus Crm1 expression levels in cervical tumour tissue using microarray expression data;

[0089] Figure 5 is a plot of Kpnpi expression levels versus Crm1 expression levels in cervical tumour tissue using real-time RT-PCR expression data;

[0090] Figure 6 is a plot of Kpnpi expression levels versus Crm1 expression levels in cervical tumour tissue using gene expression datasets from the Oncomine database (Scotto et al., 2008);

[0091] Figure 7 is a plot of Kpnpi expression levels versus Crm1 expression levels in oesophageal tumour tissue using gene expression datasets from the Oncomine database (Hu et al., 2010);

[0092] Figure 8 is a plot of Kpnpi expression levels versus Crm1 expression levels in oesophageal tumour tissue using gene expression datasets from the Oncomine database (Aoyagi et al., 2011);

[0093] Figure 9 are Western blots showing inhibition of Crm1 expression using Crm1 siRNA and resultant effect on Kpnpi expression (A) and inhibition of Kpnpi expression using Kpnpi siRNA and resultant effect on Crm1 expression (B) with p-tubulin used as a control for even protein loading;

[0094] Figure 10 is a dose-response bar graph showing HeLa, WHC05 and ARPE19 cell viability in response to increasing concentrations of INI-43;

[0095] Figure 11 is a dose-response bar graph showing HeLa, WHC05 and ARPE19 cell viability in response to increasing concentrations of Selinexor;

[0096] Figure 12 is a Western blot showing Kpnpi and Crm1 expression in non-cancer and cancer cell lines; Figure 13 is a bar graph of the relative HeLa cell viability in response to treatment with INI-43 and Selinexor, individually and in combination (*p < 0.05);

[0097] Figure 14 is a bar graph of the relative WHC05 cell viability in response to treatment with INI-43 and Selinexor, individually and in combination (*p < 0.05);

[0098] Figure 15 is a bar graph of the relative ARPE19 cell viability in response to treatment with INI-43 and Selinexor, individually and in combination (*p < 0.05);

[0099] Figure 16 is a combination index (Cl) plot showing Cl values below 1 after treatment of cervical cancer cells with INI-43 and Selinexor;

[0100] Figure 17 is a Cl plot showing Cl values below 1 after treatment of oesophageal cancer cells with INI-43 and Selinexor;

[0101] Figure 18 is a plot of relative Caspase-3 / 7 activity from a Caspase-3 / 7 assay showing significantly increased Caspase-3 / 7 activity 40 hours after treatment of HeLa cells with INI-43 and Selinexor, compared to treatment with either inhibitor alone (*p < 0.05);

[0102] Figure 19 is a Western blot showing enhanced Parp-1 / 2 cleavage after treatment of HeLa cells with INI-43 and Selinexor, compared to treatment with either inhibitor alone;

[0103] Figure 20 includes phase contrast and fluorescence images showing structural alterations of cancer cell spheroids after treatment with INI-43 and Selinexor, compared to treatment with either inhibitor alone and without treatment;

[0104] Figure 21 is a bar graph of the relative luminescence observed via the three-dimensional spheroid viability assay showing synergistically enhanced inhibition of spheroid viability after combination treatment with INI-43 and Selinexor (*p < 0.05);

[0105] Figure 22 includes images of a clonogenic assay showing the effect of INI-43, Selinexor and combination treatment with both INI-43 and Selinexor on the colonyforming ability of cancer cells;

[0106] Figure 23 is a bar graph of the number of colonies counted in the in clonogenic assay (*p

[0107] < 0.05);

[0108] Figure 24 includes IC50 curves for INI-43 in (A) AN3CA and (B) SK-UT-1 uterine cancer cell lines and bar graphs of relative cell viability of (C) SKOV-3 ovarian cancer, (D) SK-UT-1 uterine cancer and (E) MDA-M-231 breast cancer cell lines in response to individual or combination treatment with INI-43 and Selinexor (*p

[0109] < 0.05);

[0110] Figure 25 is a bar graph of the relative cell viability of cervical cancer HeLa cells treated with INI-43, Selinexor or the combination of INI-43 and Selinexor (*p < 0.05);

[0111] Figure 26 is a bar graph of the relative cell viability of uterine cancer SK-UT-1 cells treated with INI-43, Selinexor or the combination of INI-43 and Selinexor (*p < 0.05); Figure 27 is a bar graph of the relative cell viability of ovarian cancer SKOV-3 cells treated with INI-43, Selinexor or the combination of INI-43 and Selinexor (*p < 0.05);

[0112] Figure 28 includes plots of relative cell viability of (A) HeLa and (B, C) WHC05 cells with a range of Bortezomib concentrations to determine a suitable concentration of Bortezomib to use in subsequent combination treatment experiments; and bar graphs of relative cell viability 24 hours after treating (D) HeLa cells and (E) WHC05 cells with INI-43, Selinexor, or Bortezomib, individually or in combination with the percentage cell viability inhibition indicated above the bar graphs;

[0113] Figure 29 includes bar graphs of relative cell viability 24 hours after treating HeLa cells with INI-43, Selinexor, or Cisplatin, individually or in combination, with percentage cell viability inhibition indicated above the bar graphs (treatment was with (A) 15 pM Cisplatin or (B) 30 pM Cisplatin, individually and in combination);

[0114] Figure 30 includes plots of the relative cell viability of (A) HeLa and (B) CaSki cells with a range of concentrations of INI-60, Selinexor and the combination of INI-60 and Selinexor and Cl plots showing Cl values less than 1 after treatment of (C) HeLa cells and (D) CaSki cells with INI-60 and Selinexor;

[0115] Figure 31 is a Western blot showing enhanced PARP cleavage after treatment of HeLa cells with INI-60 and Selinexor, compared to treatment with either inhibitor alone; and

[0116] Figure 32 is a bar graph of the higher relative Caspase-3 / 7 activity observed after combination treatment compared to treatment with INI-60 and Selinexor alone (*p < 0.05).

[0117] DETAILED DESCRIPTION WITH REFERENCE TO THE DRAWINGS

[0118] A combination of a nuclear import inhibitor and a nuclear export inhibitor for use in treating a disease associated with nuclear transport receptor activity is provided. The disease may be associated with Karyopherin Beta 1 (Kpnpi) and Chromosome Maintenance 1 (Crm1) activity. The disease may be a proliferative disease such as cancer. The effects of the combined administration of a nuclear import inhibitor and nuclear export inhibitor on cancer biology have not previously been determined. It has now been surprisingly found that the inhibition of nuclear import protein Kpnpi and nuclear export protein Crm1 with small molecule inhibitors of Kpnpi and Crm1 results in enhanced, and synergistic anti-cancer effects. Without wishing to be bound by any particular theory or mechanism of action, the synergistic effect may be attributed to enhanced cancer cell death mediated through NFKB function, which is reduced even further when both nuclear transport proteins are inhibited. Specific inhibitors of Kpnpi that may find use in the anti-cancer combination therapy include the molecules listed in Table 1 and derivatives thereof. The inhibitors of Crm1 that may find use in this anti-cancer therapy include the molecules listed in Table 2 and derivatives thereof.

[0119] With reference to Tables 1 and 2, the inhibitor of nuclear import protein Kpnpi may be selected from the group consisting of (Karyostatin 1A), / \ / -(1- phenylethyl)-2-(1-pyrrolidinyl)-4-quinazolinamine (Importazole), ivermectin (22,23- dihydroavermectin B1a + 22,23-dihydroavermectin B1b) (Ivermectin), (E)- / V-(5-benzylthiazol-2- yl)-3-(furan-2-yl)acrylamide (aminothiazole derivative Compound 1), A / -|(5-(4- fluorophenyl)thiozol-2-yl)-3-(furan-2-yl)propenamide (Aminothiazole derivative Compound 6), 3- (7 / 7-benzimidazol-2-yl)-1-(3-dimethylaminopropyl)pyrrolo[5,4-b]quinoxalin-2-amine (INI-43) and derivatives thereof and 9-[(1-methylpiperidin-3-yl)methoxy]-4-[(6-methylpyridin-2-yl)methyl]-7-(5- methylthiophen-2-yl)-3,5-dihydro-2H-1 ,4-benzoxazepine (INI-60) and the inhibitor of nuclear export receptor Crm1 selected from the group consisting of (2E,5S,6R,7S,9R, 1 OE, 12E, 15R, 16Z, 18E)- 17-ethyl-6-hydroxy-3,5,7,9, 11,15-hexamethyl-19- [(2S,3S)-3-methyl-6-oxo-2,3-dihydropyran-2-yl]-8-oxononadeca-2, 10, 12, 16, 18-pentaenoic acid (Leptomycin B), (Z)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]- / \ / -pyrazin-2-ylprop-2- enehydrazide (Selinexor) and derivatives thereof, (E)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4- triazol-1-yl]-2-pyrimidin-5-ylprop-2-enamide (Eltanexor) and 1-[[6-chloro-5-

[0120] (trifluoromethyl)pyridin-2-yl]amino]-3-(3,3-dimethylbutoxymethyl)-4-methylpyrrole-2, 5-dione (Felezonexor).

[0121] Small molecule inhibitors of Kpnpi that may be used in the combination treatment of cancer include Importazole or pharmaceutically acceptable salts thereof, the substituted pyrrolo[2,3- b]quinoxalines of the Formula I: Formula I or pharmaceutically acceptable salts thereof, wherein Ri is a branched or linear C2-C5 alkyl group optionally functionalised with a substituent selected from the group consisting of an amine, an imidazole, an alcohol or a morpholine; and R2is a hydrogen or a methyl group and INI-60 or a pharmaceutically acceptable salt thereof.

[0122] Small molecule inhibitors of Crm1 that may be used in the combination treatment of cancer include the hydrazide-containing derivatives of the Formula II Formula II or pharmaceutically acceptable salts thereof, wherein

[0123] Xi is a hydrogen or a methyl group;

[0124] X2is a pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrazin-2-yl, and quinoxaline-2-yl, pyrimidin- 4-yl, 1 ,1-dioxotetrahydrothiophen-3-yl, or cyclopropyl and wherein X2 is optionally substituted with one or more methyl or halogen substituents; or

[0125] Xi and X2together with their intervening atoms are 4-hydroxypiperidin-1-yl, pyrrolidin-1-yl, azepan-1-yl, 4-benzylpiperazin-1-yl, 4-ethylpiperazin-1-yl, 3-hydroxyazetidin-1-yl, or morpholineyl;

[0126] X3is a hydrogen or a halogen; and represents a single carbon-carbon bond bound to a carbon-carbon double bond which is either in an (E)- or (^-configuration and in some instances (E)-3-[3-[3,5- bis(trifluoromethyl)phenyl]-1 ,2,4-triazol-1-yl]-2-pyrimidin-5-ylprop-2-enamide (Eltanexor).

[0127] Such combined use of the small molecule inhibitors including the compounds of Formula I, INI- 60 and the compounds of Formula II or their pharmaceutically acceptable salts may produce greater anti-cancer effects compared to the effect of each when used individually, and in particular may produce greater anti-cancer effects compared to the additive effects of each used individually, i.e. synergistic anti-cancer effects. The terms "treating", "treatment" and the like are used herein to mean affecting a subject, tissue, or cell to obtain a desired pharmacological and / or physiological effect. The effect may be therapeutic in terms of a partial or complete cure of the cancer. "Treating" as used herein covers any treatment of cancer in a subject; inhibiting the cancer, i.e., arresting its development; or relieving or ameliorating the effects of the cancer, i.e., cause regression of the tumour or of the effects of the cancer.

[0128] The term "subject" as used herein refers to any mammal having cancer which requires treatment, typically a human.

[0129] The term "anti-cancer" as used herein in reference to "anti-cancer agent", "anti-cancer therapeutic effect", "anti-cancerous effect" and the like is meant in its broadest scope as in known in the art, and includes the activities of arrest of cell growth, induction of apoptosis, induction of differentiation, cell death and the like.

[0130] As used herein the terms “therapeutically effective amount” or “effective amount” refers to an amount of a nuclear transport inhibitor that is effective in treating cancer. The specific "effective amount" will vary according to the particular condition being treated, the physical condition and clinical history of the subject, the duration of the treatment and the nature of the combination of agents applied and its specific formulation.

[0131] The terms "enhanced effect" or “greater effect” is used herein to refer to a combined effect caused by the combined action and / or interaction between the nuclear import inhibitor and the nuclear export inhibitor, wherein the observed effect (e.g., cytotoxicity) in the presence of the drugs together is significantly higher than the effect of each individual drug (e.g., cytotoxicities) administered separately. In some embodiments, the observed combined effect of the drugs is significantly higher than each of the individual effects. In certain embodiments the term significant means that the observed p<0.05.

[0132] The terms "synergistic" and “synergism” are used herein to refer to a combined effect caused by the combined action and / or interaction between the nuclear import inhibitor and the nuclear export inhibitor wherein the observed effect (e.g., cytotoxicity) in the presence of the drugs together is higher than the sum of the individual effects (e.g., cytotoxicities) of each drug administered separately. In some embodiments, the observed combined effect of the drugs is significantly higher than the sum of the individual effects. In certain embodiments the term significant means that the observed p<0.05. Accordingly, a combination for use in treating cancer is provided comprising component (a), namely a therapeutically effective amount of an inhibitor of nuclear import receptor Kpnpi or a pharmaceutically acceptable salt or derivative thereof and component (b) namely a therapeutically effective amount of an inhibitor of nuclear export receptor Crm1 or a pharmaceutically acceptable salt or derivative thereof. The inhibitor of nuclear import receptor Kpnpi may be a compound of Formula I as defined above, INI-60, Importazole or pharmaceutically acceptable salts or derivatives of each. The inhibitor of nuclear export receptor Crm1 may be a compound of Formula II as defined above, Eltanexor, Felezonexor or Leptomycin B or pharmaceutically acceptable salts or derivatives of each.

[0133] The inhibitor of nuclear import receptor or protein Kpnpi may be a compound of Formula I or a pharmaceutically acceptable salt thereof as defined above. Ri in Formula I may be selected from:

[0134] , an ethyl group, a propyl group, a butyl group, an / so-butyl group, an / so-pentyl group, a propanol group and

[0135] More particularly, the compound of Formula I or a pharmaceutically acceptable salt thereof may be a quinoxaline derivative represented by any one of the following structural formulae:

[0136]

[0137] 5 Preferably, the compound of Formula I is pharmaceutically acceptable salt thereof.

[0138] Alternatively, the inhibitor of nuclear import protein Kpnpi is pharmaceutically acceptable salt thereof.

[0139] The inhibitor of nuclear export receptor or protein Crm1 may be a compound of Formula II or a pharmaceutically acceptable salt thereof as defined above. In particular, the single carbon to carbon bond represented by ^ / W jnthe compound of Formula II is in the (^-configuration as Formula I l(a).

[0140] Xi in Formula II or Formula 11 (a) may be hydrogen or methyl. X2 in Formula II or Formula 11 (a) may be pyridin-2-yl, pyridin-4-yl, pyrazin-2-yl, or pyrimidin-4-yl, wherein X2 is optionally substituted with a single methyl or chloro substituent; or Xi and X2 together form 4-hydroxypi peridi n-1-yl. X3 in Formula II or Formula ll(a) may be a hydrogen.

[0141] The compound of Formula II may be represented by any one of the following structural formulae or a pharmaceutically acceptable salt thereof:

[0142] Preferably the compound of Formula II is represented by one of the following structural formulae or a pharmaceutically acceptable salt thereof: More preferably, the compound of Formula II is (Selinexor), or a pharmaceutically acceptable salt thereof. Selinexor (KPT-330, Xpovio) has the IIIPAC name

[0143] (Z)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]-N'-pyrazin-2-ylprop-2-enehydrazide. Alternatively, the inhibitor of nuclear export receptor or protein Crm1 may be (E)-3-[3-[3,5- bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]-2-pyrimidin-5-ylprop-2-enamide (Eltanexor, KPT- 8602) or a pharmaceutically acceptable salt thereof. In one embodiment, a combination for use in treating cancer comprises INI-43 and Selinexor, providing a synergistic anti-cancer effect. In a second embodiment, a combination for use in treating cancer comprises INI-60 and Selinexor, providing a synergistic anti-cancer effect.

[0144] The combination may include one or more further anti-cancer agents. In particular, the combination may include a further platinum based chemotherapeutic agent such as cisplatin or Bortezomib. A combination comprising INI-43, Selinexor and cisplatin may provide a synergistic anti-cancer effect so that can be used for treating cancer, particularly cervical cancer. A combination comprising INI-43, Selinexor and Bortezomib may provide a synergistic anti-cancer effect so that it can be used for treating cancer, particularly cervical or oesophageal cancer.

[0145] Use of a therapeutically effective amount of an inhibitor of nuclear import receptor Kpnpi or a pharmaceutically acceptable salt thereof; and a therapeutically effective amount of an inhibitor of nuclear export receptor Crm1 or a pharmaceutically acceptable salt thereof for the treatment of cancer is further provided. Similarly, a method of treating cancer with a combination of therapeutically effective amount of an inhibitor of nuclear import receptor Kpnpi or a pharmaceutically acceptable salt thereof; and a therapeutically effective amount of an inhibitor of nuclear export receptor Crm1 or a pharmaceutically acceptable salt thereof is provided.

[0146] Use of the compound of Formula I as defined above or INI-60 in combination with the compound of Formula II as defined above for the treatment of cancer and methods for treating cancer with a combination of a therapeutically effective amount a compound of Formula I or a pharmaceutically acceptable salt thereof or INI-60 or a pharmaceutically acceptable salt thereof and a therapeutically effective amount a compound of Formula II or a pharmaceutically acceptable salt thereof are also provided. With such use and in a method of treating cancer, the combination therapy (CT) has an enhanced therapeutic effect compared to the effect of the compound of Formula I and the compound of Formula II administered alone, more particularly a synergistically enhanced anti-cancer therapeutic effect.

[0147] Determining the dosage and duration of treatment is within the skill set of a person skilled in the art. For example, dosages of the inhibitor compounds (i.e., compounds of Formula I and II, INI- 60 etc.) are suitably determined depending on the individual cases taking symptoms, age and sex of the subject and the like into consideration. The amount of the inhibitor compound to be incorporated into a pharmaceutical composition varies with dosage route, solubility of the compound, administration route, administration scheme and the like. An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient and the method, route and dose of administration. The clinician using parameters known in the art makes determination of the appropriate dose. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved.

[0148] The inhibitor of Kpnpi , such as a compound of Formula I as defined above may be administered in an amount sufficient to allow the reduction of an established normal dose of the inhibitor of Crm1 , such as a compound of Formula II and vice versa. The reduction of the normal dose of either one of the compounds of Formula I, INI-60 or Formula II, for example, to effect the same degree of treatment may be reduced by at least any of 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or more.

[0149] Each of the inhibitor compounds, in particular the compounds of Formula I or II as defined above or INI-60, may be administered in an amount of 0.1 mg / kg body weight to 100 mg / kg body weight, preferably 0.1 mg / kg to 50 mg / kg as may be found to be appropriate. Lower or higher concentrations may be used, depending on the type, number and amount the compound of Formula I or INI-60 and the compound of Formula II co-administered to a subject, the subject to be treated (age, gender, weight etc.), the type of cancer to be treated and the stage of the disease. As exemplified hereinbelow, the combination of a compound of Formula I or INI-60 and a compound of Formula II significantly reduces the survival of cancer cell lines. The inhibiting activity of the exemplary combinations was significantly higher as compared to the additive inhibition activity of each of the components of the respective combinations, thereby providing a synergistic anti-cancer effect.

[0150] Each of component (a) and (b) of the combination as defined herein may be administered simultaneously (concurrently) or sequentially (separately) to treat cancer. When the components are administered simultaneously, the two components may be contained in the same composition (e.g., a composition comprising both the compound of Formula I and the compound of Formula II together with a pharmaceutically acceptable carrier, excipient, diluent or the like) or in separate compositions administered shortly (within minutes) after one another. With sequential administration component (a) and component (b) are administered with a time separation. The time separation may be 15 minutes or more and either one of component (a) or component (b) may be administered first. The components of the combination will then be in separate pharmaceutical compositions, each comprising the respective component together with a pharmaceutically acceptable carrier, excipient, diluent or the like.

[0151] Each of the above-described combinations may provide an enhanced therapeutic anti-cancer effect. In particular, the combinations may provide a synergistic therapeutic anti-cancer effect. The combinations may find use in treating a wide range of different types of cancers. Generally, the combination treatment may be used to treat cancers in which KPN i is overexpressed, Crm1 is overexpressed, or both KPN i and Crm1 are overexpressed in terms of mRNA expression levels relative to that of normal cells. In particular, the cancer to be treated with the combination may be selected from the group consisting of cervical cancer, oesophageal cancer, multiple myeloma, ovarian cancer, uterine cancer, breast cancer, gastric cancer, lung cancer, leukemia, pancreatic cancer, colon cancer, melanoma, liver cancer (e.g., liver hepatocellular carcinoma), prostate cancer (e.g., prostate adenocarcinoma), stomach cancer (e.g., stomach adenocarcinoma), fibrosarcoma and bone cancer (e.g., osteosarcoma). Preferably the cancer to be treated is selected from cervical cancer, oesophageal cancer, multiple myeloma, ovarian cancer, uterine cancer, breast cancer, gastric cancer, lung cancer, leukemia, pancreatic cancer, colon cancer and melanoma. More preferably, the combination treatment may be used for cervical cancer, oesophageal cancer, ovarian cancer, uterine cancer, breast cancer and multiple myeloma. The cancer may also be selected from a type of cancer that is presently being treated or researched to be treated with Selinexor or other current or researched combination therapies employing Selinexor.

[0152] Cancer subtypes that may be treated with the combination include glioblastoma, rectosigmoid adenocarcinoma, ductal breast carcinoma, pancreatic ductal adenocarcinoma, T-cell childhood acute lymphoblastic leukemia, invasive lobular breast carcinoma, hepatocellular carcinoma, colon adenocarcinoma, germinal center B-cell like diffuse large B-cell lymphoma, hepatocellular carcinoma, skin basal cell carcinoma, skin squamous cell carcinoma, gastric cancer, infiltrating bladder urothelial carcinoma, colorectal carcinoma, nasopharyngeal carcinoma, squamous cell lung carcinoma, large cell lung carcinoma, cervical squamous cell carcinoma, anaplastic oligodendroglioma, anaplastic oligoastrocytoma, clear cell renal cell carcinoma, lung adenocarcinoma, cervical squamous cell carcinoma, Burkitt’s lymphoma, gastric adenocarcinoma, colon carcinoma, leiomyosarcoma, mycofibrosarcoma, T-cell acute lymphoblastic leukemia, Pro-B acute lymphoblastic leukemia, B-cell childhood acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, T-cell acute lymphoblastic, prostate carcinoma, leiomyosarcoma, fibrosarcoma, malignant fibrous histocytoma, pleomorphic liposarcoma, synovial sarcoma and melanoma. These cancer subtypes have a 1-to- 5-fold change in KPNpi levels relative to non-cancer cell lines based on data in the microarray database, Oncomine.

[0153] A therapeutically effective amount of an inhibitor of nuclear import receptor Kpn i or a pharmaceutically acceptable salt thereof may also be used for the preparation of a medicament for treating cancer to be administered in combination with a therapeutically effective amount of an inhibitor of nuclear export receptor Chromosome Maintenance 1 (Crm1) or a pharmaceutically acceptable salt thereof. Alternatively, a therapeutically effective amount of an inhibitor of nuclear export receptor Crm1 or a pharmaceutically acceptable salt thereof may be used for the preparation of a medicament for treating cancer to be administered in combination with a therapeutically effective amount of an inhibitor of nuclear import receptor Kpnpi or a pharmaceutically acceptable salt thereof. Further alternatively, both a therapeutically effective amount of an inhibitor of nuclear import receptor Kpnpi or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of an inhibitor of nuclear export receptor Chromosome Maintenance 1 (Crm1) or a pharmaceutically acceptable salt thereof may be used for the preparation of a combination medicament for treating cancer.

[0154] The nuclear import inhibitor in the medicament may be a compound of Formula I as defined above, INI-60 or pharmaceutically acceptable salts thereof. The nuclear export inhibitor in the medicament may be a compound of Formula 11 as defined above or a pharmaceutically acceptable salt thereof. As previously mentioned with respect to the method of treatment, the medicament comprising component (a) or more particularly, the compound of Formula I or INI-60, may be administered simultaneously with component (b) or more particularly, the compound of Formula II. The medicament may be in the form of two separate compositions, each containing one of the components of the combination or a single composition containing both active components of the combination. Alternatively, the medicament comprising the components (a) and (b) may be administered sequentially and be in the form of separate pharmaceutical compositions. Such medicaments can have any suitable form and may include other ingredients including one or more pharmaceutically acceptable excipients, diluents, adjuvants and / or carriers.

[0155] A kit for treating cancer comprising (a) a therapeutically effective amount of an inhibitor of nuclear import receptor Kpnpi or a pharmaceutically acceptable salt thereof; and (b) a therapeutically effective amount of an inhibitor of nuclear export receptor Crm1 or a pharmaceutically acceptable salt thereof is also provided. Each of the components (a) or (b) may be formulated with at least one pharmaceutically acceptable adjuvant such as a diluent or carrier. Each of the components (a) or (b) may be adapted for administration simultaneously or sequentially. Component (a) in the kit may be a compound of Formula I as defined above, INI-60 or pharmaceutically acceptable salts thereof. Component (b) in the kit may be a compound of Formula II as defined above or a pharmaceutically acceptable salt thereof.

[0156] EXAMPLES

[0157] The examples demonstrate the correlation between Kpnpi and Crm1 expression in cancer cells and evaluate the impact of concomitant Kpnpi and Crm1 inhibition on cancer cell biology with exemplary inhibitors, INI-43, INI-60 and Selinexor. Three-drug combinations were also tested of the exemplary inhibitors INI-43 and Selinexor with either the platinum-based chemotherapeutic agent, Cisplatin, or with Bortezomib ([(7R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2- [(pyrazinylcarbonyl)amino]propyl]amino]butyl]boronic acid).

[0158] Example 1

[0159] Materials and Methods

[0160] Cell culture

[0161] Human cervical cancer cell lines (HeLa, ME180 and CaSki) were obtained from the American Type Culture Collection (ATCC) and grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Life Technologies) containing 10% Fetal Bovine Serum (FBS), penicillin (100 p / ml), and streptomycin (100 pg / ml) (HyClone Laboratories). The human oesophageal squamous cell carcinoma cell lines, WHCO5 and WHCO6 were originally established from a South African patient with oesophageal squamous cell carcinoma and acquired from Professor Rob Veale at the University of Witwatersrand

[0023] while the human oesophageal squamous cell carcinoma (KYSE30) cell line was acquired from DSMZ. All cells were maintained in a humidified incubator at 37 °C and in 5% carbon dioxide. Cell lines were authenticated by DNA profiling within the last three years, using the Cell ID system (Promega) and mycoplasma tested by the Hoescht DNA staining method. All experiments were performed with mycoplasma-free cells.

[0162] Test compounds

[0163] Stock solutions of Inhibitor of nuclear import-43 (INI-43) and Selinexor (KPT-330) (obtained from Sigma Aldrich) were prepared in DMSO and stored at room temperature (INI-43) or 4°C (KPT- 330), away from light.

[0164] IC50 determination

[0165] Cells were seeded in 96-well plates at a density of 5000 cells / well and the next day treated with various concentrations of nuclear import and export inhibitors for 24 to 48 hours. MTT (Sigma) assays was used to measure viable cells. Absorbance was measured using a BioTek microplate spectrophotometer at 595 nm. IC50 curves were constructed using GraphPad Prism software. The drug combination index was determined using the method described by Chou-Talalay

[0024] and calculated using CompuSyn software (ComboSyn, Inc.).

[0166] Western blot analysis Cells were treated with INI-43 or KPT-330 independently, or in combination and after 24 hours the floating and adherent cells were collected, centrifuged, and lysed using RIPA buffer, containing 1 X complete protease inhibitor cocktail (Roche) and 1 mM sodium orthovanadate. Protein concentrations were quantified using the BCA protein assay kit (Pierce, Thermo Scientific), according to the manufacturer’s instructions. Western blots were performed using primary antibodies: rabbit anti-Kpnpi (H-300) (sc-11367, Santa Cruz Biotechnology), rabbit ant- Crm1 (H-300) (sc-5595, Santa Cruz Biotechnology), rabbit anti-p-tubulin (H-235) (sc-9104, Santa Cruz Biotechnology), rabbit anti-Parp-1 / 2 (H-250, sc-7150, Santa Cruz Biotechnology) or rabbit anti-GAPDH.

[0167] Caspase-3 / 7 activity

[0168] Cells were seeded in 96-well plates and treated with INI-43 plus KPT-330 singly and in combination for 40 hours. Caspase-3 / 7 activity was monitored using the Caspase-Gio® 3 / 7 assay, according to the manufacturer's instructions. Luminescence was measured using the Veritas™ microplate luminometer (Promega) and normalized to cell viability measured by the MTT performed in parallel.

[0169] Statistical analysis

[0170] Experiments were performed in triplicate and expressed as the mean ± standard error of the mean (SEM). For data analysis, the Student's t-test was performed using Microsoft Excel. A paired and two-tailed distribution was used. The p-value of < 0.05 (indicated by *) was considered statistically significant.

[0171] Spheroid assay

[0172] HeLa cells were plated in Millicell ultra-low attachment plates (Merck) at 5000 cells / well and, after the establishment of spheroids (24-48 hours), were treated with drugs for 48 hours. Spheroid viability was determined using the CellTiter-Glo 3D reagent (Promega), according to the manufacturer’s instructions, and luminescence recorded using the Veritas™ microplate luminometer. For spheroid imaging, cells were stained with Hoechst 33258 (Sigma) and viewed under the EVOS M5000 fluorescent microscope (ThermoFisher Scientific). Images in Figure 20 show spheroid integrity after treatment with INI-43 (5 pM), Selinexor (10 pM), or INI-43 / Selinexor combination.

[0173] Colony-forming assay

[0174] HeLa cells were plated in 12-well plates at 500 cells / well and the next day treated with drugs. 24 hours after treatment, drug was removed and replaced with fresh media. Eight days after treatment, colonies were fixed in methanol and stained with crystal violet. Colonies were counted using the Image J cell counter plugin. Representative images in Figure 22 show Crystal Violet- stained colonies formed 8 days after treatment with INI-43 (4 pM) or Selinexor (1 pM), individually or in combination, for 24 hours.

[0175] Results

[0176] Expression of the nuclear importer, Kpn / 31, and nuclear exporter, Crm1, positively correlate with each other in cancer tissue biopsies

[0177] The nuclear transport proteins, Kpnpi and Crm1 , have each separately been determined to be overexpressed in several cancer types. Here, the expression of both Kpnpi and Crm1 in oesophageal tumour tissue biopsies were analysed and compared to that found in matched normal oesophageal epithelium by real-time RT-PCR (qPCR). mRNA expression levels of both Kpnpi and Crm1 were significantly increased in oesophageal tumour tissue compared to normal as shown in Figure 1. Since both genes function in nuclear transport pathways and are upregulated in cancer, it remained to be determined if their expression levels positively correlate with each other. Correlation analyses were therefore performed, where Kpnpi expression levels were compared to Crm1 expression levels and Pearson correlation coefficients and corresponding p-values calculated. Interestingly, using Iog2-transformed qPCR data, expression of Kpnpi was found to significantly associate with expression of Crm1 in oesophageal cancer patient tissue specimens, where samples with high expression of one nuclear transport receptor similarly had high expression of the other nuclear transport receptor as is evident from Figure 2. A positive Pearson correlation coefficient of 0.47 was determined. As oesophageal tumour tissue samples were provided with matched normal oesophageal epithelium from the same patient, the expression data was next analysed whereby the fold change in Kpnpi expression in tumour tissue compared to matched normal was compared to the fold change in Crm1 expression in tumour tissue compared to matched normal. The data is represented in Figure 3. Here, Pearson correlation analysis revealed a strong correlation coefficient of 0.59, suggesting that tumour samples similarly upregulate expression of both Kpnpi and Crm1 nuclear transport receptors (Figure 3).

[0178] To expand these findings to another cancer type, Kpnpi and Crm1 expression was analysed in cervical tumour samples. It has previously been shown that Kpnpi and Crm1 are overexpressed in cervical cancer tissue specimens by microarray and real-time RT-PCR analyses (van der Watt et al., 2009). Pearson correlation analyses were thus performed using these mRNA expression datasets, and strong Pearson correlation coefficients of 0.83 and 0.62 were obtained when Kpnpi and Crm1 expression levels were compared using microarray and real-time RT-PCR expression data, respectively as shown in Figures 4 and 5. Findings were further corroborated by analysing gene expression datasets from the Oncomine database

[0025] , Kpnpi and Crm1 expression levels were compared in tumour samples from various studies, and Pearson correlation analyses confirmed significant correlations in Kpnpi and Crm1 gene expression levels in cervical cancer and oesophageal cancer samples as illustrated in Figures 6 to 8. Together, these findings reveal that Kpnpi and Crm1 expression levels correlate with each other in distinct cancer types.

[0179] Reciprocal regulation of Kpnpi and Crm1 expression

[0180] As the expression of both Kpnpi and Crm1 were found to correlate with each other, it was next determined whether inhibition of the one would influence expression of the other. siRNA was employed to knockdown expression of Crm1 or Kpnpi in cervical cancer cells and knockdown was confirmed by Western blot analysis. Interestingly, when Kpnpi expression was analysed in Crm1 knock-down cells it was found to be decreased, and when Crm1 expression was analysed in Kpnpi knock-down cells it was similarly found to be decreased as shown in Figure 9 (A and B). This suggests that there is reciprocal regulation of Kpnpi and Crm1 expression in cancer cells, which may explain why their expression correlates in tumour tissue.

[0181] Inhibition of Kpnpi and Crm1 results in cancer cell death, while non-cancer cells are more resistant to single and combined treatment

[0182] Since Kpnpi and Crml expression was found to correlate in tumour tissue, it was next determined whether combined targeting of Kpnpi and Crm1 could have enhanced anti-cancer effects. Firstly, the effects of inhibition of Kpnpi and Crm1 individually was determined, where Kpnpi was inhibited with the small molecule inhibitor, INI-43, and Crm1 inhibited with the SINE compound, Selinexor. The cytotoxicity of INI-43 and Selinexor in representative cancer (HeLa and WHC05) and non-cancer (ARPE19) cell lines was determined using the MTT assay. All cell lines showed decreases in cellular viabilities in a dose-dependent manner after 48-hour drug treatment, with a range of IC50 concentrations from 0.13 to 12.56 pM as shown in Figure 10 (A and B). Results showed that the non-cancer ARPE19 cells were significantly more resistant to treatment of both INI-43 and Selinexor, compared to cancer cell lines. This finding coincides with the endogenous expression levels of Kpnpi and Crm1 in these cells, where non-cancer cells with low protein expression are less sensitive to Kpnpi and Crm1 inhibition, compared to cancer cells with high protein expression as is evident from Figures 11 and 12.

[0183] As cells were shown to respond to single treatment with Kpnpi and Crm1 inhibitors, it was next determined whether combined treatment would result in enhanced anti-cancer effects. HeLa, WHC05 and APRE19 cells were treated with Selinexor, either in the presence or absence of INI- 43, for 24 hours (this reduced treatment time was to limit the cell death that occurs in response to the individual treatments). Cells were significantly more sensitive to Selinexor when treated in the presence of INI-43 as shown in Figures 13 to 15. Consistent with their sensitivity to single agents, ARPE19 cells were less sensitive to combined inhibition than cancer cells (Figures 13 to 15).

[0184] Co-treatment of INI-43 and Selinexor synergistically kills cancer cells

[0185] As co-treatment of cancer cells with INI-43 and Selinexor resulted in enhanced inhibition of cancer viability, it was next determined whether the two drugs act synergistically, by calculating the combination index (Cl), using the Chou-Talalay method

[0024] , after treatment with a fixed dose ratio. Representative cervical cancer and oesophageal cancer cells were treated with INI-43 and Selinexor for 24 hours at varying concentrations to give I NI-43-to- Selinexor ratios of 1 :2 to 1 :5 (depending on cell sensitivity to each drug), and cell viability determined. Based on the cell viability results, the Cl values were calculated using CompuSyn software (ComboSyn, Inc) and plotted against fraction affected (Fa). Combination index (Cl) values below 1 were achieved in all cell lines at all concentrations, revealing synergistically enhanced cell death (Figures 16 and 17).

[0186] Co-treatment with Kpn / 31 and Crm1 inhibitor increases apoptosis

[0187] Given the fact that both drugs are known to induce cancer cell apoptosis [2,26], we next measured markers of apoptosis after single and dual treatment of cells. Caspase-3 / 7 activity was the most elevated upon combined treatment of cells with both INI-43 and Selinexor, where Caspase-3 / 7 activity was significantly greater in co-treated cells compared to cells treated with either drug alone as shown in Figure 18. Cleaved Parp-1 , a marker of late apoptosis, was also more pronounced in co-treated cells, compared to cells treated with individual drugs as shown in Figure 19.

[0188] The in vitro results further suggest that even a low dose of INI-43 in combination with Selinexor has improved cell killing effects. Figure 18 shows that cell killing effects are improved when INI- 43 concentration is lowered to a non-toxic dose and combined with a partially toxic dose of Selinexor.

[0189] Taken together, co-treatment of cancer cells with INI-43 and Selinexor results in synergistically enhanced anti-cancer effects and more potently induced apoptosis than either single treatment alone.

[0190] A positive correlation between Kpnpi and Crm1 nuclear transport receptor expression in cervical and oesophageal cancer tissue was identified for the first time. The high expression of Kpnpi and Crm1 in tumour tissue suggests that Kpnpi may collaborate with Crm1 in the promotion of cancer, by enhancing overall nuclear transport rates. The example demonstrates that Kpnpi and Crm1 inhibitors such as INI-43 and Selinexor act synergistically in inducing cancer cell death, whereby their combined action is greater than the sum of their individual effects. There could be several mechanisms accounting for this effect. One such mechanism could be the resultant dysregulation of the NFKB pathway. In sarcoma cells, it was determined that Selinexor acts by inducing the nuclear localisation of IKB, thereby resulting in the inhibition of NFKB and transcriptional suppression of the antiapoptotic protein, survivin

[0016] , Similarly, Kpnpi inhibition with siRNA results in the inhibition of NFKB subunit p50 translocation to the nucleus in prostate cancer cells [3], and Kpnpi inhibition using INI-43 results in the inhibition of NFKB p65 and p50 nuclear translocation in cisplatin-treated cervical cancer cells

[0022] , Accordingly both Crm1 and Kpnpi inhibition reduce NFKB activity, but via different mechanisms. NFkB activity is surprisingly further inhibited with the combination of a Crm1 inhibitor and a Kpnpi inhibitor. The anti-cancer synergism seen with the combined administration of a Crm1 inhibitor and a Kpnpi inhibitor is surprising as both inhibitors target the same general pathway of nuclear transport, yet their combined effect is not redundant or merely additive.

[0191] The effect of combined treatment of INI-43 and Selinexor on 3D cancer spheroid viability

[0192] Cancer cell spheroids provide a more complex three-dimensional model, therefore cancer spheroids were treated with INI-43 and Selinexor individually and in combination. Figure 20 shows the structural alterations observed after individual and combination treatment, with the spheroids darker (loss of cell viability and cell debris within the structure) and widespread disintegration after the combination treatment. Fluorescence was observed throughout the treatment with INI-43 which demonstrates that it penetrates the aggregation of cells. Figure 21 includes the results of a 3D cell viability assay and shows significantly enhanced inhibition of spheroid viability after combination treatment.

[0193] The effect of combined treatment of INI-43 and Selinexor on cancer colony-forming ability Clonogenic assays were carried out to determine if cancer cells maintain their proliferative capacity after combined treatment with INI-43 and Selinexor. Cells were treated for 24 hours, and thereafter the drug(s) were removed and the ability of cells to form colonies monitored over 8 days. As shown in Figures 22 and 23, low dose INI-43 and Selinexor on their own had little effect on colony-forming ability, but the combination treatment completely abolished colony-formation, thereby demonstrating their synergistic anti-colony forming effect.

[0194] Example 2

[0195] Example 1 demonstrates combined treatment of cancer cells with the nuclear import inhibitor, I N I- 43, and nuclear export inhibitor, Selinexor, results in enhanced cancer cell death (a synergistic response) that is greater than the sum of the individual drug-induced responses using cervical cancer and oesophageal cancer cell lines. Further tests were conducted to determine whether this effect is observed in other cancer types, including multiple myeloma, as Selinexor has been FDA-approved for the treatment of multiple myeloma.

[0196] Referring to Figure 24 (A-E), a combination of INI-43 and Selinexor was tested in other cancer types. Uterine cancer, ovarian cancer, breast cancer and multiple myeloma cell lines were grown for combination treatment experiments, to determine the combined effect of INI-43 and Selinexor on the viability of cancer cells of various tissue origin. Before testing the effect of the combination treatment, IC50 values for INI-43 were first determined in the uterine cancer cell lines, AN3CA and SK-UT-1 to establish their sensitivity to INI-43. Results showed that the IC50 value for INI-43 in AN3CA and SK-UT-1 cells was 11 pM (Figure 24 (A)) and 6 pM (Figure 24 (B)), respectively, which are both in the range of that observed in other cancer cell lines.

[0197] The different cancer cell lines were next subjected to the combination treatment, and viability determined using the MTT assay. Results showed that there was significantly enhanced SKOV3 ovarian cancer and SK-UT-1 uterine cancer cell death after treatment with INI-43 and Selinexor, compared to that observed after individual treatments (Figure 24 (C, D)). Results were also obtained showing a small but significant increase in cell death of MDA-MB231 breast cancer cells treated with a combination of INI-43 and Selinexor (Figure 24 (E)).

[0198] The effect of combined treatment of INI-43 and Selinexor on gynaelogical cancers are further demonstrated in Figures 25 to 27. The different cancer cell lines were subjected to no treatment, individual treatment and the combination treatment, and viability determined using an MTT assay. Results demonstrate that there is significantly enhanced HeLA cervical cancer (Figure 25), SK- UT-1 uterine cancer (Figure 26) and SKOV-3 ovarian cancer (Figure 27) cell death after treatment with INI-43 and Selinexor, compared to cell death observed after individual treatments.

[0199] Example 3

[0200] The combination of Selinexor and Bortezomib has been FDA-approved. Tests were conducted to determine whether the combination of INI-43, Selinexor and Bortezomib results in even greater inhibition of cancer viability.

[0201] Referring to Figure 28 (A-C), a range of Bortezomib concentrations were tested to identify a suitable concentration of Bortezomib to use that would result in minimal cell death on its own. HeLa cells were moderately affected by Bortezomib in the concentration range of 0.5 - 100 pM (Figure 28 (A)), therefore a concentration of 0.25 pM was chosen for subsequent experiments. WHC05 cells were much more sensitive to Bortezomib, with 0.5 pM killing 80% of the cells (Figure 28 (B)), hence a lower concentration range was tested to find a concentration which had only a minor cell killing effect. 10 nM was chosen as a suitable concentration (Figure 28 (C)).

[0202] HeLa and WHC05 cells were next treated with INI-43, Selinexor and Bortezomib, individually or in combination, and the cell killing effect determined by an MTT assay. Results showed that while the drugs had minor cell killing effects on their own, the three-drug combination resulted in the greatest inhibition of cell viability (Figure. 28 (D, E)), with the combined effect being greater than the sum of the individual effects, i.e. the combination treatment with INI-43, Selinexor and Bortezomib resulted in a synergistic cell killing effect.

[0203] Example 4

[0204] It has been previously shown that INI-43 enhances the cell death response induced by the chemotherapeutic drug, Cisplatin

[0022] , Tests were conducted to determine whether the combination of INI-43, Selinexor and Cisplatin results in even greater inhibition of cancer viability.

[0205] Referring to Figure 29, HeLa cells were treated with INI-43, Selinexor, or Cisplatin, individually, or in combination and the effect on cell viability determined using the MTT assay. Cells were treated with either 15 pM (A) or 30 pM (B) Cisplatin individually, and together with INI-43 and Selinexor in combination. The three-drug combination resulted in significantly enhanced cervical cancer cell death, compared to treatment with individual drugs. The combined cell killing effect of INI-43, Selinexor, or Cisplatin was synergistic, i.e. greater than the sum of the individual effects.

[0206] Example 5

[0207] Methods and Materials

[0208] Cell viability experiments

[0209] Cells were seeded in 96-well plates at a density of 5000 cells / well and the next day treated with various concentrations of INI-60 and Selinexor, at a constant ratio of 1 :10, for 24 hours. MTT (Sigma) assays were used to measure viable cells. Absorbance was measured using a BioTek microplate spectrophotometer at 595 nm. The drug combination index was determined using the method described by Chou-Talalay

[0024] and calculated using CompuSyn™ software (ComboSyn, Inc.). PARP cleavage

[0210] Cells were treated with INI-60 (7.5 uM) and Selinexor (0.75 uM) independently, or in combination, and after 24 hours the floating and adherent cells were collected, centrifuged, and lysed using RIPA buffer, containing 1 X complete protease inhibitor cocktail (Roche) and 1 mM sodium orthovanadate. Protein concentrations were quantified using the BCA protein assay kit (Pierce, Thermo Scientific), according to the manufacturer’s instructions. Western blots were performed using primary antibodies: rabbit anti-Parp-1 / 2 (H-250, sc-7150, Santa Cruz Biotechnology) or rabbit anti-GAPDH (#G9545, Sigma).

[0211] Caspase-3 / 7 activity

[0212] Cells were seeded in 96-well plates and treated with INI-60 (7.5 uM) plus Selinexor (0.75 uM) singly or in combination for 20 hours. Caspase-3 / 7 activity was monitored using the Caspase- Glo™ 3 / 7 assay, according to the manufacturer's instructions. Luminescence was measured using the Veritas™ microplate luminometer (Promega) and normalized to cell viability measured by the MTT performed in parallel.

[0213] Results

[0214] Effect of combined treatment of INI-60 and Selinexor on cervical cancer cells

[0215] Figures 30 (A) and (B) show that the combination of INI-60 and Selinexor reduced the viability of HeLa and CaSki cervical cancer cells. The combination index (Cl) values are in both cases less than 1 , thereby showing a synergistic effect (Figures 30 (C) and (D)). Figure 31 shows that more PARP cleavage was observed after combination treatment compared with the treatment with INI- 60 and Selinexor on their own. Figure 32 shows that significantly higher Caspase 3 / 7 activity was observed after combination treatment, compared to INI-60 and Selinexor treatment alone. Therefore, there was greater cell death via apoptosis in cells treated with the INI-60-Selinexor combination.

[0216] The nuclear transport inhibitors described herein display broad spectrum anticancer activity. Importantly, siRNA depletion of both Crm1 and Kpnpi receptors has limited effects on normal cell viability. Therefore, inhibitors of Kpnpi and Crm1 (including the compounds of Formula I and II) provide a more targeted anti-cancer effect. A further advantage of the therapeutic targeting of proteins like Crm1 and Kpnpi is that unlike in the case of many other targeted therapies, a mutated target is not required, hence the spectrum of cancers that can be treated is much broader. The combination therapy described herein results in an anti-cancerous effect that is enhanced compared to the effect of each compound / agent alone. This also means that the dosage of each agent in the combination therapy can be reduced as compared to monotherapy with each agent, while still achieving an overall anti-cancerous effect. This is particularly advantageous when one or both agents have adverse side effects. Selinexor, for example, has adverse side effects such as thrombocytopenia that could lead to haemorrhage. The combination therapy of Selinexor with a selective inhibitor of nuclear import protein Kpnpi such as INI-43 allows for the reduction of Selinexor dosage to minimise or avoid its adverse side effects, whilst achieving the same overall anti-cancerous effect. Due to the synergistic anti-cancer effect seen with the combination treatment, the total amount of drugs administered to a patient can be reduced, which may result in minimizing undesired side effects stemming from either drug. The combination therapy may overcome chemoresistance mechanisms and address dose-limiting toxicities.

[0217] The treatment may be particularly effective when targeting a specific cancer or tumour type since the functional relevance of targeting nuclear receptors in cancer may be tumour-type specific. Specific types of cancer exhibiting positively correlated increased Kpnpi and Crm1 nuclear transport receptor expression, for example, can be treated by administering therapeutically effective amounts of inhibitors of Kpnpi and Crm1 , including the compounds of Formula I and II described herein.

[0218] Both Crm1 and Kpnpi inhibitors are separately known to engage in further synergistic interactions with other chemotherapeutic agents including platinum-based chemotherapeutic agents such as cisplatin. Selinexor, for example, has shown synergism with a range of anticancer agents including with proteosome inhibitors such as bortezomib, carfilzomib and ixazombin, a BCR-ABL- targeted tyrokinase inhibitor called imatinib, and steroids such as dexamethasone, and a nth racy clines such as doxorubicin and idarubicin amongst others. A combination of a substituted pyrrolo[2,3-b]quinoxalines of the Formula I such as INI-43 and imatinib may be used for the treatment of cancer and in particular for the treatment of chronic myeloid leukemia. A synergistic anti-cancer effect is expected when the quinoxalines of the Formula I and imatinib are administered to a subject in combination.

[0219] The combination of Kpnpi and Crm1 inhibitors such as a compound of Formula I and a compound of Formula II, may thus be administered or combined with a further, third chemotherapeutic agent for the treatment of cancer. The further chemotherapeutic agent may be a platinum-based chemotherapeutic agent such as cisplatin. Treatment with the compounds of Formula I and II together with cisplatin may be especially effective in subjects with cervical cancer, oesophageal cancer, ovarian cancer, breast cancer and uterine cancer, for example or other gynaecological cancers Treatment with Crm1 and Kpnpi inhibitors such as the compounds of Formula I and II together with dexamethasone or bortezomib may target a cancer such as lymphoma (including mantle cell lymphoma), leukemia or multiple myeloma. Treatment with Crm1 and Kpnpi inhibitors such as the compounds of Formula I and II together with imatinib may be especially effective in a cancer such as chronic myeloid leukemia.

[0220] The foregoing description has been presented for the purpose of illustration; it is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Persons skilled in the relevant art can appreciate that many modifications and variations are possible in light of the above disclosure.

[0221] The language used in the specification has been principally selected for readability and instructional purposes, and it may not have been selected to delineate or circumscribe the inventive subject matter. It is therefore intended that the scope of the invention be limited not by this detailed description, but rather by any claims that issue on an application based hereon. Accordingly, the disclosure of the embodiments of the invention is intended to be illustrative, but not limiting, of the scope of the invention, which is set forth in the following claims.

[0222] Finally, throughout the specification and accompanying claims, unless the context requires otherwise, the word ‘comprise’ or variations such as ‘comprises’ or ‘comprising’ will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

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Claims

CLAIMS:

1. A combination for use in treating cancer comprising(a) a therapeutically effective amount of an inhibitor of nuclear import receptor Karyopherin Beta 1 (Kpnpi), wherein the inhibitor of Kpnpi is a compound of Formula I:Formula I or a pharmaceutically acceptable salt thereof, in whichRi is a branched or linear C2-C5 alkyl group optionally functionalised with a substituent selected from the group consisting of an amine, an imidazole, an alcohol or a morpholine;R2 is a hydrogen or a methyl group; or the inhibitor of Kpnpi ispharmaceutically acceptable salt thereof; and(b) a therapeutically effective amount of an inhibitor of nuclear export receptor Chromosome Maintenance 1 (Crm1), wherein the inhibitor of Crm1 is a compound of Formula II:Formula II or a pharmaceutically acceptable salt thereof, in whichXi is a hydrogen or a methyl group;X2 is a pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrazin-2-yl, and quinoxaline-2-yl, pyrimidin-4-yl, 1,1-dioxotetrahydrothiophen-3-yl, or cyclopropyl and wherein X2 is optionally substituted with one or more methyl or halogen substituents; orXi and X2together with their intervening atoms are 4-hydroxypiperidin-1-yl, pyrrolidin-1-yl, azepan-1-yl, 4-benzylpiperazin-1-yl, 4-ethylpiperazin-1-yl, 3- hydroxyazetidin-1-yl, or morpholin-4-yl;X3is a hydrogen or a halogen; and x / wv- represents a single carbon-carbon bond bound to a carbon-carbon double bond which is either in an (E)- or (^-configuration.

2. The combination of claim 1 , wherein Ri is selected from:, an ethyl group, a propyl group, a butyl group, an / so-butyl group, an / so-pentyl group, a propanol group and3. The combination of claim 1 or claim 2, wherein the compound of Formula I is selected from the group consisting of:

4. The combination of any one of claims 1 to 3, wherein the compound of Formula I is5. The combination of claim 1, wherein the inhibitor of Kpnpi is6. The combination of any one of claims 1 to 5, wherein the compound of Formula II is7. The combination of claim 6, wherein the compound of Formula II is8. The combination of any one of claims 1 to 7, wherein the combination provides a synergistic therapeutic anti-cancer effect.

9. The combination of any one of claims 1 to 8, wherein the cancer is selected from the group consisting of cervical cancer, oesophageal cancer, multiple myeloma, ovarian cancer, uterine cancer, breast cancer, gastric cancer, lung cancer, leukemia, pancreatic cancer, colon cancer, and melanoma.

10. The combination of claim 9, wherein the cancer is selected from the group consisting of cervical cancer, oesophageal cancer, ovarian cancer, uterine cancer, breast cancer, and multiple myeloma.

11. A method of treating cancer comprising administering to a subject in need thereof(a) a therapeutically effective amount of an inhibitor of nuclear import receptor Karyopherin Beta 1 (Kpnpi), wherein the inhibitor of Kpnpi is a compound of Formula I:Formula I or a pharmaceutically acceptable salt thereof, in whichRi is a branched or linear C2-C5 alkyl group optionally functionalised with a substituent selected from the group consisting of an amine, an imidazole, an alcohol or a morpholine;R2is a hydrogen or a methyl group; or the inhibitor of Kpnpi isor a pharmaceutically acceptable salt thereof; and(b) a therapeutically effective amount of an inhibitor of nuclear export receptor Chromosome Maintenance 1 (Crm1), wherein the inhibitor of Crm1 is a compound of Formula II:Formula II or a pharmaceutically acceptable salt thereof, in whichXi is a hydrogen or a methyl group;X2is a pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrazin-2-yl, and quinoxaline-2-yl, pyrimidin-4-yl, 1 ,1-dioxotetrahydrothiophen-3-yl, or cyclopropyl and wherein X2 is optionally substituted with one or more methyl or halogen substituents; orXi and X2together with their intervening atoms are 4-hydroxypiperidin-1-yl, pyrrolidin-1-yl, azepan-1-yl, 4-benzylpiperazin-1-yl, 4-ethylpiperazin-1-yl, 3- hydroxyazetidin-1-yl, or morpholin-4-yl;X3 is a hydrogen or a halogen; and' / vv' represents a single carbon-carbon bond bound to a carbon-carbon double bond which is either in an (E)- or (^-configuration.

12. Use of (a) a therapeutically effective amount of an inhibitor of nuclear import receptor Karyopherin Beta 1 (Kpnpi), wherein the inhibitor of Kpnpi is a compound of Formula I:or a pharmaceutically acceptable salt thereof, in whichRi is a branched or linear C2-C5 alkyl group optionally functionalised with a substituent selected from the group consisting of an amine, an imidazole, an alcohol or a morpholine;R2is a hydrogen or a methyl group; or the inhibitor of Kpnpi isor a pharmaceutically acceptable salt thereof; and(b) a therapeutically effective amount of an inhibitor of nuclear export receptor Chromosome Maintenance 1 (Crm1), wherein the inhibitor of Crm1 is a compound of Formula II:Formula II or a pharmaceutically acceptable salt thereof, in whichXi is a hydrogen or a methyl group;X2is a pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrazin-2-yl, and quinoxaline-2-yl, pyrimidin-4-yl, 1,1-dioxotetrahydrothiophen-3-yl, or cyclopropyl and wherein X2 is optionally substituted with one or more methyl or halogen substituents; orXi and X2together with their intervening atoms are 4-hydroxypiperidin-1-yl, pyrrolidin-1-yl, azepan-1-yl, 4-benzylpiperazin-1-yl, 4-ethylpiperazin-1-yl, 3- hydroxyazetidin-1-yl, or morpholin-4-yl;X3is a hydrogen or a halogen; and' / vvv' represents a single carbon-carbon bond bound to a carbon-carbon double bond which is either in an (E)- or (^-configuration, in the manufacture of a medicament for treating cancer.

13. The use of claim 12, wherein the inhibitor of Kpnpi is to be administered simultaneously with the inhibitor of Crm 1.

14. The use of claim 12, wherein the inhibitor of Kpnpi and the inhibitor of Crm1 are to be administered sequentially.

15. A kit for treating cancer comprising(a) a therapeutically effective amount of an inhibitor of nuclear import receptor Karyopherin Beta 1 (Kpnpi), wherein the inhibitor of Kpnpi is a compound of Formula I:Formula I or a pharmaceutically acceptable salt thereof, in whichRi is a branched or linear C2-C5 alkyl group optionally functionalised with a substituent selected from the group consisting of an amine, an imidazole, an alcohol or a morpholine;R2 is a hydrogen or a methyl group; or the inhibitor of Kpnpi ispharmaceutically acceptable salt thereof; and(c) a therapeutically effective amount of an inhibitor of nuclear export receptor Chromosome Maintenance 1 (Crm1), wherein the inhibitor of Crm1 is a compound of Formula II:Formula II or a pharmaceutically acceptable salt thereof, in whichXi is a hydrogen or a methyl group;X2is a pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrazin-2-yl, and quinoxaline-2-yl, pyrimidin-4-yl, 1,1-dioxotetrahydrothiophen-3-yl, or cyclopropyl and wherein X2 is optionally substituted with one or more methyl or halogen substituents; orXi and X2together with their intervening atoms are 4-hydroxypiperidin-1-yl, pyrrolidin-1-yl, azepan-1-yl, 4-benzylpiperazin-1-yl, 4-ethylpiperazin-1-yl, 3- hydroxyazetidin-1-yl, or morpholin-4-yl;X3 is a hydrogen or a halogen; and' / vvv' represents a single carbon-carbon bond bound to a carbon-carbon double bond which is either in an (E)- or (^-configuration.

Images