Combination therapy of cancer

Abstract

The present application relates to the use of a VRAC modulator of formula I or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of cancer in combination with an anti-hormone agent selected from the group consisting of: a. An anti-estrogen selected from fulvestrant, tamoxifen, toremifene and clomifene; and b. An anti-progestational hormone selected from the group consisting of mifepristone, ulipristal acetate, ensipristone, liropristone, and onapristone. # imgabs0 #

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201780043536.3 and invention name “Combined Treatment of Cancer”. Technical Field

[0002] The present invention relates to the treatment of cancer, and in particular to methods of sensitizing cancer cells to anti-cancer treatment by administering an effective amount of a VRAC modulator. background

[0004] Cancer represents an overwhelming burden on our society, with approximately 14 million new cancer cases diagnosed in 2014. Despite the introduction of many new treatment modalities / options, de novo or acquired resistance to applied treatments still represents the leading cause of cancer mortality.

[0005] Volume-regulated anion channels (VRACs) are present in most mammalian cells. An important function of VRACs is cell volume regulation. When cells swell in a hypotonic solution, these channels are activated and chloride ions flow out of the cell in parallel with potassium ions (via potassium channels) and water, thereby restoring the original cell volume.

[0006] Compounds capable of blocking VRAC activity are characterized in Helix et al., J. Membrane Biol. 196 (2003). For example, NS3728 (Endovion; SCO-101) is a potent, orally active VRAC blocker.

[0007] WO 98 / 47879 and WO 00 / 24707 disclose VRAC modulators, including NS3728, and their use in treating sickle cell anemia.

[0008] WO 2004 / 012733 discloses VRAC modulators, including NS3728, and their use in treating diseases responsive to anti-angiogenic therapy, particularly anti-metastatic therapy.

[0009] Previous studies have suggested a role for VRAC in the ability of cancer cells to undergo apoptosis, and NS3728 has been shown to inhibit cisplatin-mediated apoptosis in vitro (Poulsen et al., Am J Physiol Cell Physiol, 2010; et al., Am J Physiol Cell Physiol, 2016; Planells_Cases, R. et al., EMBO J., 2015).

[0010] Chemotherapy for metastatic colorectal cancer (mCRC) is usually based on the antimetabolite drug 5-fluorouracil (5FU) combined with the DNA-binding agent oxaliplatin (e.g., FOLFOX regimen) or the topoisomerase I inhibitor irinotecan (e.g., FOLFIRI regimen) and is usually administered in combination with biological therapy (e.g., EGFR-targeted drugs or anti-angiogenic drugs). Despite the efficacy of these combined regimens, which have significantly increased the response rate and survival rate of patients with mCRC (Cunningham et al., 2010; Gallagher and Kemeny, 2010), only 30-50% of treated patients show an objective response to one of these two combined therapies, and cancer progression remains a common outcome despite chemotherapy treatment (Gallagher and Kemeny, 2010; Goldberg et al., 2004). Worse still, the objective response rate of patients treated with prior FOLFIRI to second-line chemotherapy for mCRC FOLFOX is reduced to 10-20%, and vice versa, and there is almost no objective response to third-line treatment. It is therefore clear that innate (pre-existing) or acquired drug resistance constitutes a major problem in the management of mCRC. Similar conclusions can be drawn when discussing primary CRC, where a large proportion (30-40%) of stage III patients who receive adjuvant chemotherapy (most commonly FOLFOX or XELOX) will experience disease recurrence following chemotherapy.

[0011] Breast cancer (BC) is a heterogeneous disease, which is also reflected in the heterogeneous response patterns to various types of chemotherapy. In the adjuvant treatment of BC, the most commonly used drugs are cytotoxic drugs, including anthracyclines (topoisomerase 2 inhibitors) and taxanes. These drugs are most often administered with cyclophosphamide. For patients with estrogen receptor-positive BC, adjuvant therapy most commonly includes anti-estrogens, such as aromatase inhibitors, estrogen receptor antagonists (tamoxifen and fulvestrant). However, about 18-20% of patients receiving adjuvant or neoadjuvant systemic anticancer therapy will experience disease recurrence. In patients with metastatic BC (mBC), the response rate range for systemic therapy is rapidly reduced to 20-25% from 60% of first-line treatment when 3-line to 4-line treatment is reached. These figures clearly show that a high degree of innate tolerance and acquired tolerance lead to disease progression and thousands of women die each year.

[0012] Therefore, the identification, characterization, and preclinical and clinical validation of new drugs and drug combinations for the treatment of cancer, especially drug-resistant cancers, constitutes a high unmet medical need. Summary of the invention

[0013] The present invention provides a new cancer treatment regimen, and is particularly suitable for treating chemotherapy-resistant cancers. The treatment regimen of the present invention includes treatment with a compound that can modulate the activity of the volume-regulated anion channel (VRAC), i.e., a VRAC modulator, such as Endovion (NS3728). Co-treatment with VRAC modulators has surprisingly been shown to enhance the effect of anti-cancer treatment. In addition, co-treatment with VRAC modulators has been shown to resensitize chemotherapy-resistant cancer cells to treatment with certain chemotherapeutic agents.

[0014] In a first aspect, the present invention relates to a method of sensitizing cancer cells to an anti-cancer therapy, the method comprising administering to a subject in need thereof an effective amount of a VRAC modulator. The VRAC modulator may be administered before, during and / or after administration of the anti-cancer therapy.

[0015] In a second aspect, the present invention relates to a method of enhancing the therapeutic effect of an anti-cancer therapy, the method comprising administering to a subject in need thereof an effective amount of a VRAC modulator. The VRAC modulator may be administered before, during and / or after administration of the anti-cancer therapy.

[0016] In a third aspect, the invention is directed to a method of treating cancer comprising administering to a subject in need thereof an effective amount of a VRAC modulator and an effective amount of an anti-cancer therapy, such as a chemotherapeutic agent.

[0017] In a fourth aspect, the invention relates to a composition comprising an effective amount of a VRAC modulator and a chemotherapeutic agent.

[0018] In a fifth aspect, the present invention relates to a composition comprising an effective amount of a VRAC modulator and an effective amount of a chemotherapeutic agent for use as a medicament.

[0019] In a sixth aspect, the present invention is directed to a composition comprising an effective amount of a VRAC modulator and an effective amount of a chemotherapeutic agent for use in treating cancer.

[0020] In a seventh aspect, the present invention is directed to VRAC modulators for use in sensitizing cancer cells to anti-cancer treatments.

[0021] In an eighth aspect, the present invention relates to VRAC modulators for use in enhancing the therapeutic effect of anti-cancer therapy.

[0022] In a ninth aspect, the invention relates to a kit comprising a VRAC modulator and a chemotherapeutic agent, and optionally instructions for use, wherein the VRAC modulator and the chemotherapeutic agent are formulated for simultaneous, sequential or separate administration.

[0023] In a tenth aspect, the present invention relates to the use of a VRAC modulator in the preparation of a medicament for sensitizing cancer cells to an anti-cancer treatment or for enhancing the therapeutic effect of an anti-cancer treatment.

[0024] In an eleventh aspect, the present invention relates to the use of a VRAC modulator and an anti-cancer therapy, such as a chemotherapeutic agent, for treating a patient, wherein the patient has previously been determined to be resistant to treatment with the anti-cancer agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Effects of daily doses of NS3728 (40 mg / kg PO), temozolomide (Tedo) (25 mg / kg IP), and their combination on the growth of C9 glioblastoma xenografts in vivo.

[0026] Figure 2 Effects of daily doses of NS3728 (40 mg / kg), temozolomide (Tedo) (5 mg / kg IP), and their combination on the growth of C9 glioblastoma cells in the subcutaneous air pouch (SAS) model in vivo.

[0027] Figure 3 Effects of NS3728 (80 mg / kg, PO), 5-fluorouracil (30 mg / kg, IP), and their combination on the growth of HT29 colon tumor xenografts in vivo compared with vehicle-treated mice.

[0028] Figure 4 Effects of NS3728 (80 mg / kg, PO), paclitaxel (5 mg / kg, IP), and their combination on the growth of HT29 colon tumor xenografts in vivo compared with vehicle-treated mice.

[0029] Figure 5 Effects of NS3728, docetaxel and their combination on cell viability of docetaxel-resistant MCF7 breast cancer cells in vitro. Error bars are standard deviations.

[0030] Figure 6 Effects of NS3728, docetaxel, and their combination on cell viability of docetaxel-resistant MDA-MB-231 breast cancer cells in vitro. Error bars are standard deviations.

[0031] Figure 7 Effects of NS3728, oxaliplatin and their combination on cell viability of parental HCT116 CRC cancer cells in vitro. Error bars are standard deviations.

[0032] Figure 8 Effects of NS3728, SN-38, and their combination on cell viability of parental HCT116 CRC cancer cells in vitro. Error bars are standard deviations.

[0033] Fig. 9Effects of NS3728, SN-38, and their combination on cell viability of parental HT29 CRC cancer cells in vitro. Error bars are standard deviations.

[0034] Fig.10 Effects of NS3728, SN-38, and their combination on cell viability of parental LoVo CRC cancer cells in vitro. Error bars are standard deviations.

[0035] Fig.11 Effects of NS3728, SN-38, and their combination on cell viability of SN-38-resistant HCT116 CRC cancer cells in vitro. Error bars are standard deviations.

[0036] Fig.12 Effects of NS3728, SN-38, and their combination on cell viability of SN-38-resistant HT29 CRC cancer cells in vitro. Error bars are standard deviations.

[0037] Fig.13 Effects of NS3728, SN-38, and their combination on cell viability of SN-38-resistant LoVo CRC cancer cells in vitro. Error bars are standard deviations.

[0038] Fig.14 Effects of NS3728, SN-38, and their combination on cell viability of oxaliplatin-resistant HT29 CRC cancer cells in vitro. Error bars are standard deviations.

[0039] Fig.15 Effects of NS3728, SN-38, and their combination on cell viability of oxaliplatin-resistant LoVo CRC cancer cells in vitro. Error bars are standard deviations.

[0040] Fig.16 Effects of NS3728, tamoxifen and their combination on cell viability of tamoxifen-resistant MCF7 / LCC2 breast cancer cells in vitro. Error bars are standard deviations.

[0041] Fig.17 Effects of NS3728, ICI182,780 (Fulvestrant / Faslodex) and their combination on cell viability of ICI182,780-resistant MCF7 / LCC9 breast cancer cells in vitro. Error bars are standard deviations. DETAILED DESCRIPTION OF THE INVENTION

[0043] The present inventors surprisingly discovered that co-treatment of cancer with VRAC modulators and anti-cancer therapies (e.g., chemotherapeutic agents) has increased efficacy compared to monotherapy and further resensitizes chemoresistant cancer cells to treatment, thus proposing a new cancer treatment regimen in which VRAC modulators are administered to enhance the therapeutic effect of anti-cancer therapy and / or sensitize cancer cells to anti-cancer therapy.

[0044] VRAC

[0045] Volume-regulated anion channels (VRACs) are present in most mammalian cells. An important function of VRACs is cell volume regulation: when cells swell in a hypotonic solution, these channels are activated and chloride ions flow out of the cell in parallel with potassium ions (via potassium channels) and water, thus restoring the original cell volume.

[0046] The gene family encoding VRAC was identified as the LRRC8 family by two independent groups in 2014 (Voss et al., 2014, Science 344(6184):634-8 and Qui et al., 2014, Cell 157(2):447-58). VRAC has historically been referred to in the literature as volume-sensitive organic osmolyte anion channel (VSOAC), volume-sensitive outward rectifier (VSOR), voltage-dependent anion channel (VDAC), human volume-sensitive outward rectifier (HSVOR), volume-regulated chloride channel (VRClC), and I Cl,膨胀 These phenotypic manifestations are currently postulated to be the result of different compositions of LRRC8 subunits within the functional VRACs expressed in different cell types.

[0047] VRAC is a nearly ubiquitously distributed anion channel that is activated by cell swelling and / or reductions in intracellular ionic strength. It is the channel that mediates increased anion conduction during regulated volume decrease (RVD). In electrophysiological recordings, VRAC exhibits outward rectification and, in most cells, shows a pronounced voltage-dependent inactivation at positive membrane potentials. VRAC is highly anion selective, with a unique selectivity order for small inorganic ions: SCN - - =NO3 - <Br - <Cl - <HCO3 - <F - (Eisenman type I halide selectivity sequence) and - depending on the specific subunit composition - possess a wide pore which also allows the penetration of a large number of negatively charged or neutral organic molecules such as cisplatin.

[0048] ​Activation of VRAC is mandatory, depends on the presence of intracellular ATP, and is supported by a complex intracellular signaling cascade involving protein kinases.

[0049] VRAC blockers

[0050] In one embodiment, the VRAC modulator is a VRAC blocker. The term "VRAC blocker" is used interchangeably herein with the term "VRAC inhibitor." VRAC blockers are compounds that inhibit the transmembrane transport of chloride ions or any other anion or neutral molecule in response to cell swelling or a decrease in intracellular ionic strength.

[0051] Compounds that are VRAC inhibitors bind to the channel protein and exert their discernible inhibitory effect via one or more of the methods for measuring VRAC function as described herein. Inhibitory compounds may act via one or more theoretical modes of action (MOA), the main of which are:

[0052] 1) One way to obtain inhibition is to directly block (block) the current through the open channel by binding somewhere in the transmembrane pore region of the channel. Such molecules are sometimes referred to in the art as "blockers".

[0053] 2) Another - more heterogeneous - way to obtain inhibition is by preventing or delaying the natural activation process of the channel and / or by stimulating / accelerating its natural inactivation or deactivation process. Such molecules are generally expected to result in a reduced probability of finding the individual channel proteins in their open configuration (reduced open state probability, P o ). Such molecules can be collectively referred to as "negative gating modulators", regardless of which microscopic process they affect. Negative gating modulators are sometimes also called "negative allosteric modulators" or "NAMs".

[0054] Elucidating the detailed MOA of a specific compound is a very challenging task and may require a combination of highly sensitive functional measurements (e.g. single channel recordings as described herein) and high resolution structural information (e.g. crystallography, sequence analysis, mutagenesis analysis, etc.). In the current case, it has not been clearly determined which MOA is responsible for the VRAC inhibitory effect of the VRAC modulators described herein. Therefore, they are collectively referred to as VRAC blockers.

[0055] The potential of a given substance to act as a VRAC blocker can be determined using standard laboratory test methods such as

[0056] a) Whole-cell or single-channel patch clamp technique, or its automated HTC version

[0057] b) microelectrode electrophysiology (penetrating, sharp electrodes),

[0058] c) Flux measurements (e.g. radioisotopes and non-radioisotopes),

[0059] d) fluorescent dyes (e.g. membrane potential indicators or chloride concentration indicators),

[0060] e) Cell volume measurement (eg light scattering, Coulter counter measurement or optical methods such as image analysis).

[0061] To measure VRAC activity, it is necessary to activate the channel protein by swelling the cells (e.g. by exposure to hypotonic extracellular Ringer's solution) or by making the intracellular solution hypertonic or low in ionic strength (only a few techniques allow this, i.e. whole-cell patch clamping is described under 1.a).

[0062] The most direct way to record VRAC activity is via electrophysiological methods, which allow direct measurement of the swelling-activated transmembrane anion current at a fixed membrane potential (voltage clamp). The possibilities are:

[0063] Manual technology

[0064] a) Manual whole-cell patch clamp (measures anion currents across the entire cell membrane). This is the most realistic configuration for drug screening. In the literature, cell-attached configurations (measure currents through relatively few ion channels expressed in a small patch of cell membrane) have been used in a few cases, and their use in excised configurations, where a membrane fragment has been isolated on the tip of a patch pipette (inside-out and outside-out configurations). These methods are primarily relevant for biophysical studies of specific compounds (low throughput) or for elucidation of the mode of action.

[0065] b) Perforated patch recordings (essentially the same as the whole-cell configuration described under 1a, except that no pores are created in the membrane, but a low-resistance connection to the cell interior is made by using a permeabilizing agent, usually a bacterial pore-forming peptide such as amphotericin B). Throughput is substantially lower than the whole-cell approach, but has the advantage of keeping the cellular environment closer to the physiological composition during recording. (In the context of VRAC, which depends on intracellular ATP, the molecule does not need to be applied to the pipette solution as it is in the whole-cell configuration).

[0066] c) Manual "classical" electrophysiology using membrane-penetrating "sharp" electrodes. Not easily applicable to drug screening, as the method is difficult to use with typical small laboratory cell lines (HEK, CHO, etc.).

[0067] Automatic Technology

[0068] d) Various forms of HTS electrophysiology, now exemplified by QPatch or QUBE from Sophion Bioscience (Korsgaard, MP et al., Comb Chem High Troughput Screen, 2009; Chambers, C et al., Assay Drug Dev Technol., 2016) or Nanion systems. All methods essentially use flat glass or silicon substrates mounted on a chip for whole-cell measurements (see a) or perforated membrane patch measurements (see b), which allow for highly or automated measurements and compound / fluid handling in a parallel setup.

[0069] More indirect effects of VRAC activation can be measured by:

[0070] a) Expression (stable, transient or inducible) of a halide-sensitive fluorescent protein or peptide in the cell in question (i.e. HEK293). The most commonly used is the yellow fluorescent protein (YFP), whose light emission is extremely sensitive to the type and concentration of halide in the culture medium. In this case, it is exploited that the effect of iodide ions is much more pronounced than that of chloride ions. By applying iodide ions (which are more permeable through VRAC than Cl ions) to the extracellular solution, it is achieved that these ions enter the cell interior through the membrane when the VRAC is open (in the opposite direction of the chloride ions leaving the cell). VRAC activation is therefore reported by increased light emission from YFP. Inhibitors of VRAC are expected to inhibit this emission (less influx of iodide ions), so this method can be used to quantify the potency and degree of inhibition.

[0071] b) The extent of swelling of cells expressing VRAC can be followed directly by measuring cell size, for example by Coulter counter, light scattering techniques, microscopy or impedance measurements. VRAC inhibition does not affect the swelling process, but prevents or delays the volume recovery process called RVD (Regulated Volume Depletion) driven by VRAC activation. It is important to recognize that by measuring YFP and cell size, one becomes increasingly reliant on indirect "downstream" effects of VRAC activation, which significantly increases the risk of recording false positive and false negative signals. Therefore, such results are often validated by using electrophysiological measurements.

[0072] c) Many other approaches could be combined with VRAC activation, but these are more indirect. Effects on migration, proliferation, cytotoxicity, apoptosis assays of specific cell lines may all be important in terms of predicting in vivo cancer effects, but none rely solely on VRAC.

[0073] VRAC expression can be measured, for example, by qPCR, in situ hybridization or antibodies.

[0074] In one embodiment, the VRAC blocker is a diphenylurea derivative, such as those described in WO 98 / 47879 and / or WO 00 / 24707.

[0075] In one embodiment, the VRAC blocker exhibits an IC50 value of inhibition in vitro according to standard test procedures of less than 10 μM, such as less than 1000 nM, for example less than 100 nM, such as less than 50 nM, for example less than 10 nM.

[0076] In one embodiment, the VRAC blocker has an IC50 of 5 μM or less, such as 3 μM or less, for example 1 μM or less, such as 0.5 μM or less, for example 0.1 μM or less, for example 10 nM or less, for example 5 nM or less.

[0077] In one embodiment, the VRAC blocker has an IC50 of 2 μM or less, such as 1 μM or less, such as 0.5 μM or less, for example 0.1 μM or less, such as 10 nM or less, for example 5 nM or less. In a preferred embodiment, the VRAC blocker has an IC50 of 0.5 μM or less.

[0078] An example of a VRAC blocker is a compound of formula I or a pharmaceutically acceptable salt thereof:

[0079]

[0080] Where R 2 represents a tetrazolyl group; and

[0081] R 3 , R 4 , R 5 , R 6 , R 12 , R 13 , R 14 , R 15 and R 16 independently represent hydrogen, halogen, trifluoromethyl, nitro, alkyl, alkylcarbonyl, -NR a R b 、-NR a -CO-R b , phenyl or heteroaryl;

[0082] The phenyl group is optionally substituted with halogen, trifluoromethyl, nitro, -CO-NHR c 、-CO-OR c or -CO-NR'R";

[0083] Where R c is hydrogen, alkyl or phenyl;

[0084] R' and R" are independently hydrogen or alkyl; or

[0085] R' and R" together with the nitrogen to which they are attached form a 5- to 7-membered heterocyclic ring which may optionally contain an oxygen atom and / or an additional nitrogen atom, and / or a carbon-carbon double bond and / or a carbon-nitrogen double bond as ring members;

[0086] and the heterocycle may be optionally substituted with an alkyl group;

[0087] R a and R b are independently hydrogen or alkyl; or

[0088] R 15 and R 16 or R 14 and R 15 Together with the benzene ring to which they are attached, they form a naphthalene ring or an indane ring; and R 3 , R 4 , R 5 , R 6 , R 12 and R 13 and R 14 , R 15 and R 16 The remaining one is as defined above.

[0089] In one embodiment, the VRAC blocker is a compound of Formula I or a pharmaceutically acceptable salt thereof:

[0090]

[0091] Where R 2 represents a tetrazolyl group;

[0092] R 3 , R 4 , R 5 , R 6 , R 12 , R 13 , R 14 , R 15 and R 16 independently of one another represent hydrogen, halogen, trifluoromethyl, nitro or phenyl;

[0093] The phenyl group is optionally substituted with halogen, trifluoromethyl, nitro or -CO-NHR c replace;

[0094] Where R c is hydrogen, alkyl or phenyl.

[0095] In one embodiment of the compound of formula I, R 2 Represents tetrazolyl; R3 , R 4 , R 5 , R 6 , R 12 , R 13 , R 14 , R 15 and R 16 independently of one another represent hydrogen, halogen, trifluoromethyl or nitro.

[0096] In one embodiment of the compound of formula I, R 3 , R 5 and R 6 represents hydrogen; and R 4 Represents a halogen, such as bromine.

[0097] In one embodiment of the compound of formula I, R 3 , R 4 , R 5 and R 6 Represents hydrogen.

[0098] In another embodiment of the compound of Formula I, R 3 , R 5 and R 6 represents hydrogen; and R 4 Indicates -NR a R b , such as amino.

[0099] In yet another embodiment of the compounds of Formula I, R 3 , R 5 and R 6 represents hydrogen; and R 4 Indicates -NR a -CO-R b , such as acetylamino.

[0100] In yet another embodiment of the compound of Formula I, R 3 , R 5 and R 6 represents hydrogen; and R 4 Indicates trifluoromethyl, nitro or -CO-NHR c Substituted phenyl; wherein R c In one embodiment, R 4 In another embodiment, R 4 In another embodiment, R 4 Indicates being-CO-NHR c Substituted phenyl, such as anilinocarbonylphenyl, especially 4-anilinocarbonylphenyl.

[0101] In another embodiment of the compound of Formula I, R 3 , R 5 and R 6 represents hydrogen; and R 4 Indicates being-CO-OR c or -CO-NR'R" substituted phenyl. In a specific embodiment, R 4 Indicates being-CO-OR c Substituted phenyl, wherein R c is hydrogen. In a particular embodiment, R4 represents phenyl substituted by -CO-NR'R", such as 4-dimethylcarbamoylphenyl or 4-(4-methyl-1-piperazine-carbonyl)-phenyl.

[0102] In yet another embodiment, R 15 In one embodiment, R 15 represents trifluoromethyl, and R 12 , R 13 , R 14 and R 16 In yet another embodiment, R 13 In one embodiment, R 13 and R 15 represents trifluoromethyl, and R 12 , R 14 and R 16 Represents hydrogen.

[0103] In yet another embodiment, R 16 In one embodiment, R 16 represents trifluoromethyl, and R 12 , R 13 , R 14 and R 15 Represents hydrogen.

[0104] In yet another embodiment, R 15 represents halogen, such as chlorine or bromine. In a specific embodiment, R 15 represents a halogen, such as chlorine or bromine, and R 12 , R 13 , R 14 and R 16 In yet another embodiment, R 13 and R 15 represents a halogen, such as chlorine, and R 12 , R 14 and R 16 In yet another embodiment, R 14and R 15 represents a halogen, such as chlorine, and R 12 , R 13 and R 16 Represents hydrogen.

[0105] In yet another embodiment, R 16 represents halogen, such as fluorine. In a specific embodiment, R 16 represents a halogen, such as fluorine, and R 12 , R 13 , R 14 and R 15 Represents hydrogen.

[0106] In yet another embodiment, R 16 In one embodiment, R 16 represents an alkyl group, such as a methyl group or an ethyl group, and R 12 , R 13 , R 14 and R 15 Represents hydrogen.

[0107] In yet another embodiment, R 14 In one embodiment, R 14 represents nitro, and R 12 , R 13 , R 15 and R 16 Represents hydrogen.

[0108] In yet another embodiment, R 14 In one embodiment, R 14 represents an alkylcarbonyl group, such as acetyl, and R 12 , R 13 , R 15 and R 16 Represents hydrogen.

[0109] In yet another embodiment, R 15 In yet another embodiment, R 14 In one embodiment, R 14 or R 15 One of them represents a phenyl group, and R 12 , R 13 , R 14 , R 15 and R 16 The rest of represents hydrogen.

[0110] In yet another embodiment, R 15 In one embodiment, R15 represents a pyridyl group, such as pyridin-3-yl, and R 12 , R 13 , R 14 and R 16 Represents hydrogen.

[0111] In yet another embodiment, R 15 and R 16 Together with the benzene ring to which they are attached they form a naphthalene ring.

[0112] In yet another embodiment, R 14 and R 15 Together with the benzene ring to which they are attached they form an indane ring.

[0113] In yet another embodiment, the compound of formula I is selected from:

[0114] N-4-Nitrophenyl-Nˊ-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;

[0115] N-3,5-Bis(trifluoromethyl)phenyl-Nˊ-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;

[0116] N-3-Trifluoromethylphenyl-N'-[4-(3-nitrophenyl)-2-(1-H-tetrazol-5-yl)phenyl]urea;

[0117] N-3-Trifluoromethylphenyl-N'-[4-(4-anilinocarbonylphenyl)-2-(1-H-tetrazol-5-yl)phenyl]urea;

[0118] N-3-Trifluoromethylphenyl-N'-[4-(4-trifluoromethylphenyl)-2-(1-H-tetrazol-5-yl)phenyl]urea;

[0119] N-(3-Trifluoromethyl-phenyl)-Nˊ-[2-(1-H-tetrazolyl-5-yl)-phenyl]urea;

[0120] N-(3-Trifluoromethyl-phenyl)-Nˊ-[4-bromo-2-(1-H-tetrazol-5-yl)-phenyl]urea;

[0121] N-(3-Trifluoromethyl-phenyl)-Nˊ-[4-phenyl-2-(1-H-tetrazol-5-yl)-phenyl]urea;

[0122] N-(3-Chloro-phenyl)-Nˊ-[2-(1-H-tetrazol-5-yl)-phenyl]urea;

[0123] N-(3-Trifluoromethyl-phenyl)-Nˊ-[4-amino-2-(1-H-tetrazolyl-5-yl)-phenyl]urea;

[0124] N-(3-Trifluoromethyl-phenyl)-Nˊ-[4-acetylamino-2-(1-H-tetrazol-5-yl)-phenyl]urea;

[0125] N-(3-Trifluoromethyl-phenyl)-Nˊ-[4-carbamoyl-2-(1-H-tetrazol-5-yl)-phenyl]urea;

[0126] N-(3-trifluoromethyl-phenyl)-Nˊ-[4-(Nˊˊ,Nˊˊ-dimethylcarbamoyl)-2-(1-H-tetrazol-5-yl)-phenyl]urea;

[0127] 3ˊ-(1-H-tetrazolyl-5-yl)-4ˊ-[3-(3-trifluoromethyl-phenyl)-ureido]-biphenyl-4-carboxylic acid;

[0128] N-(Indan-5-yl)-Nˊ-[2-(1-H-tetrazolyl-5-yl)-phenyl]urea;

[0129] N-(Biphenyl-4-yl)-Nˊ-[2-(1-H-tetrazolyl-5-yl)-phenyl]urea;

[0130] N-(Biphenyl-3-yl)-Nˊ-[2-(1-H-tetrazolyl-5-yl)-phenyl]urea;

[0131] N-(3-Acetyl-phenyl)-Nˊ-[2-(1-H-tetrazol-5-yl)-phenyl]urea;

[0132] N-(Biphenyl-3-yl)-Nˊ-[2-(1-H-tetrazolyl-5-yl)-phenyl]urea;

[0133] N-[3-(Pyridin-3-yl)-phenyl]-Nˊ-[2-(1-H-tetrazol-5-yl)-phenyl]urea;

[0134] N-(3-Bromo-phenyl)-Nˊ-[4ˊ-(4-methyl-piperazine-1-carbonyl)-3-(1-H-tetrazol-5-yl)-biphenyl-4-yl]urea;

[0135] N-(3,5-dichloro-phenyl)-Nˊ-[4-bromo-2-(1-H-tetrazol-5-yl)-phenyl]urea;

[0136] N-(3,4-dichloro-phenyl)-Nˊ-[4-bromo-2-(1-H-tetrazol-5-yl)-phenyl]urea;

[0137] N-(Naphthalen-1-yl)-Nˊ-[4-bromo-2-(1-H-tetrazol-5-yl)-phenyl]urea;

[0138] N-(2-Trifluoromethyl-phenyl)-Nˊ-[4-bromo-2-(1-H-tetrazol-5-yl)-phenyl]urea;

[0139] N-(2-Fluoro-phenyl)-Nˊ-[4-bromo-2-(1-H-tetrazol-5-yl)-phenyl]urea;

[0140] N-(2-ethyl-phenyl)-Nˊ-[4-bromo-2-(1-H-tetrazol-5-yl)-phenyl]urea;

[0141] or a pharmaceutically acceptable salt thereof.

[0142] In a particular embodiment of the invention, the compound is:

[0143] N-4-Nitrophenyl-Nˊ-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;

[0144] N-3,5-Bis(trifluoromethyl)phenyl-Nˊ-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;

[0145] N-3-Trifluoromethylphenyl-N'-[4-(3-nitrophenyl)-2-(1-H-tetrazol-5-yl)phenyl]urea;

[0146] N-3-Trifluoromethylphenyl-N'-[4-(4-anilinocarbonylphenyl)-2-(1-H-tetrazol-5-yl)phenyl]urea;

[0147] N-3-Trifluoromethylphenyl-N'-[4-(4-trifluoromethylphenyl)-2-(1-H-tetrazol-5-yl)phenyl]urea;

[0148] or a pharmaceutically acceptable salt thereof.

[0149] In one embodiment, the VRAC blocker is NS3728:

[0150]

[0151] or a pharmaceutically acceptable salt thereof. NS3728 is also known as Endovion and SCO-101. NS3728 is a potent, orally active VRAC blocker, IC 50 is 0.40 μM (Helix et al., J Membrane Biol. 196, 2003).

[0152] In one embodiment, the VRAC blocker is NS3623:

[0153]

[0154] or a pharmaceutically acceptable salt thereof. NS3623 is a VRAC blocker, IC 50 is 1.8 μM (Helix et al., J Membrane Biol. 196, 2003).

[0155] In one embodiment, the VRAC blocker is NS3749:

[0156]

[0157] or a pharmaceutically acceptable salt thereof. NS3749 is a VRAC blocker, IC 50 is 1.4 μM (Helix et al., J Membrane Biol. 196, 2003).

[0158] In one embodiment, the VRAC blocker is tamoxifen (TMX):

[0159]

[0160] or a pharmaceutically acceptable salt thereof. Tamoxifen is a drug used to prevent breast cancer in women and to treat breast cancer in women and men. It is also being studied for other types of cancer. Tamoxifen is a voltage-dependent VRAC blocker, IC 50 =2.2 μM (Helix et al., J Membrane Biol. 196, 2003).

[0161] In one embodiment, the VRAC blocker is not tamoxifen. In particular, when the anti-cancer treatment is an anti-estrogen, more particularly tamoxifen, the VRAC blocker is not tamoxifen.

[0162] Other examples of VRAC blockers include DIDS (IC 50 =26 μM) and NPPB (IC 50 =21 μM) (Helix et al., J Membrane Biol. 196, 2003).

[0163] In one embodiment, VRAC modulators are identified as described in EP 2 919 009, the teachings of which are incorporated by reference in their entirety.

[0164] Definition of Substituents

[0165] In the context of the present invention, halogen denotes fluorine, chlorine, bromine or iodine.

[0166] In the context of the present invention, alkyl refers to a monovalent saturated straight or branched hydrocarbon chain. The hydrocarbon chain preferably contains 1 to 6 carbon atoms (C 1-6-alkyl), including pentyl, isopentyl, neopentyl, tert-pentyl, hexyl and isohexyl. In one embodiment, alkyl represents C 1-4 -alkyl, including butyl, isobutyl, sec-butyl and tert-butyl. In another embodiment of the present invention, alkyl represents C 1-3 - alkyl, which may in particular be methyl, ethyl, propyl or isopropyl.

[0167] In the context of the present invention, heteroaryl refers to an aromatic mono-, bi- or poly-heterocyclic group which possesses one or more heteroatoms in its ring structure. Preferred heteroatoms include nitrogen (N), oxygen (O) and sulfur (S). Preferred monocyclic heteroaryl groups of the present invention include aromatic 5- and 6-membered heterocyclic monocyclic groups including furanyl, especially 2- or 3-furanyl; thienyl, especially 2- or 3-thienyl; pyrrolyl (oxazolyl), especially 1, 2 or 3-pyrrolyl; Azolyl, especially oxazol-2, 4 or 5-yl; thiazolyl, especially thiazol-2, 4 or 5-yl; imidazolyl, especially 1, 2 or 4-imidazolyl; pyrazolyl, especially 1, 3 or 4-pyrazolyl; isocyanate Azolyl, especially iso Oxazol-3, 4 or 5-yl; isothiazolyl, in particular isothiazol-3, 4 or 5-yl; Oxazolyl, especially 1,2,3-, 1,2,4-, 1,2,5- or 1,3,4- oxadiazole-3, 4 or 5-yl; triazolyl, in particular 1,2,3-, 1,2,4-, 2,1,3- or 4,1,2-triazolyl; thiadiazolyl, in particular thiadiazol-3, 4 or 5-yl; pyridinyl, in particular 2, 3 or 4-pyridinyl; pyridazinyl, in particular 3 or 4-pyridazinyl; pyrimidinyl, in particular 2, 4 or 5-pyrimidinyl; pyrazinyl, in particular 2 or 3-pyrazinyl; and triazinyl, in particular 1,2,3-, 1,2,4- or 1,3,5-triazinyl.

[0168] 5-7 membered heterocyclic rings containing one nitrogen atom include, for example, but are not limited to, pyrrolidine, piperidine, homopiperidine, pyrroline, tetrahydropyridine, pyrazolidine, imidazolidine, piperazine, homopiperazine and morpholine.

[0169] Pharmaceutically acceptable salts

[0170] VRAC blockers as described herein may be provided in any form suitable for the intended administration. Suitable forms include pharmaceutically (ie physiologically) acceptable salts and prodrug forms.

[0171] Examples of pharmaceutically acceptable addition salts include, but are not limited to, nontoxic inorganic and organic acid addition salts such as hydrochlorides derived from hydrochloric acid, hydrobromides derived from hydrobromic acid, nitrates derived from nitric acid, perchlorates derived from perchloric acid, phosphates derived from phosphoric acid, sulfates derived from sulfuric acid, formate derived from formic acid, acetates derived from acetic acid, aconites derived from aconitic acid, ascorbates derived from ascorbic acid, benzenesulfonates derived from benzenesulfonic acid, benzoates derived from benzoic acid, cinnamates derived from cinnamic acid, citrates derived from citric acid, pamoates derived from pamoic acid, heptanoates derived from heptanoic acid. The salts may be formed by procedures well known and described in the art.

[0172] Other acids, such as oxalic acid, which might not be considered pharmaceutically acceptable, may be used to prepare salts useful as intermediates in obtaining compounds for use in accordance with the present invention and their pharmaceutically acceptable acid addition salts.

[0173] Examples of pharmaceutically acceptable cationic salts of the compounds of the invention include, but are not limited to, sodium, potassium, calcium, magnesium, zinc, aluminum, lithium, choline, lysine and ammonium salts of the compounds of the invention containing anionic groups, etc. Such cationic salts can be formed by procedures well known and described in the art.

[0174] In the context of the present invention, the term " Salts" are also considered pharmaceutically acceptable salts (aza- Salts). Preferred aza Salts include alkyl- Salts, especially methyl- and ethyl- Salt; Cycloalkyl- Salts, especially cyclopropyl- Salts; and cycloalkylalkyl- Salts, especially cyclopropyl-methyl- Salt.

[0175] Pharmaceutical composition

[0176] In one embodiment, the invention relates to a pharmaceutical composition comprising an effective amount of a VRAC modulator and a chemotherapeutic agent.

[0177] Although the VRAC blockers and / or chemotherapeutic agents disclosed herein can be used in the form of raw compounds, it is preferred to introduce the active ingredient (optionally in the form of a physiologically acceptable salt) into a pharmaceutical composition together with one or more adjuvants, excipients, carriers, buffers, diluents and / or other conventional pharmaceutical auxiliaries.

[0178] In one embodiment, the present invention provides a pharmaceutical composition comprising a VRAC blocker according to the present invention, or a pharmaceutically acceptable salt or derivative thereof, and one or more pharmaceutically acceptable carriers, and optionally other therapeutic and / or prophylactic ingredients known and used in the art. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient thereof. In another embodiment, the present invention provides a pharmaceutical composition comprising more than one compound or prodrug for use according to the present invention, such as two different compounds or prodrugs for use according to the present invention.

[0179] The pharmaceutical compositions of the present invention may be those suitable for oral, rectal, bronchial, nasal, pulmonary, topical (including buccal and sublingual), transdermal, vaginal or parenteral (including cutaneous, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intracerebral, intraocular injection or infusion) administration, or those in a form suitable for administration by inhalation or insufflation, including powder and liquid aerosol administration, or those administered by a sustained release system. Suitable examples of sustained release systems include semipermeable matrices of solid hydrophobic polymers containing the compounds of the present invention, which matrices may be in the form of shaped articles, for example, films or microcapsules.

[0180] Therefore, the VRAC blocker of the present invention can be placed into the form of pharmaceutical compositions and unit dosage forms thereof together with conventional adjuvants, carriers or diluents. Such forms include solids, and in particular tablets, filled capsules, powders and pellets; and liquids, in particular aqueous or non-aqueous solutions, suspensions, emulsions, elixirs and capsules filled with them, all for oral use, suppositories for rectal administration and sterile injectable solutions for parenteral use. Such pharmaceutical compositions and unit dosage forms thereof may contain conventional ingredients in conventional proportions, with or without other active compounds or principles, and such unit dosage forms may contain any suitable effective amount of active ingredients, which is equivalent to the expected daily dose range to be used.

[0181] VRAC blockers as disclosed herein can be administered in a variety of oral and parenteral dosage forms.

[0182] In order to prepare a pharmaceutical composition from the chemical compounds of the present invention, a pharmaceutically acceptable carrier can be a solid or a liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories and dispersible granules. A solid carrier can be one or more substances that can also act as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, preservative, tablet disintegrating agent or encapsulating material.

[0183] In powders, the carrier is a finely divided solid, which is in admixture with the finely divided active component.

[0184] In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.

[0185] Powders and tablets preferably contain 5% or 10% to about 70% active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, and the like. The term "preparation" is intended to include formulating the active compound with encapsulating material as a carrier to provide a capsule in which the active ingredient (with or without a carrier) is surrounded by a carrier, thereby being combined with the carrier. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.

[0186] To prepare suppositories, a low melting wax such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active ingredient is dispersed uniformly therein by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.

[0187] Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

[0188] Liquid preparations include solutions, suspensions, and emulsions, such as water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.

[0189] Thus, the VRAC blocker according to the present invention can be formulated for parenteral administration (e.g., by injection, such as bolus or continuous infusion), and can be in the form of a unit dose for ampoules, prefilled syringes, small volume infusions, or in multiple dose containers with added preservatives. The composition can take the form of a suspension, solution, or emulsion, such as in an oily or aqueous vehicle, and can contain a formulation agent, such as a suspending agent, a stabilizer, and / or a dispersant. Alternatively, the VRAC blocker can be in powder form, obtained by aseptic isolation of a sterile solid or by lyophilization from a solution, for reconstitution with a suitable vehicle (e.g., sterile, pyrogen-free water) prior to use.

[0190] Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavoring agents, stabilizers, and thickening agents as desired.

[0191] Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well known suspending agents.

[0192] Also included are solid form preparations that are intended to be converted into liquid form preparations for oral administration shortly before use. Such liquid forms include solutions, suspensions, and emulsions. In addition to the active ingredient, these preparations may also contain colorants, flavorings, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, etc.

[0193] For topical application to the epidermis, the VRAC blocker can be formulated as an ointment, cream or lotion, or as a transdermal patch. Ointments and creams can be formulated, for example, with an aqueous or oily base, and with the addition of a suitable thickener and / or gelling agent. Lotions can be formulated with an aqueous or oily base, and typically also contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickeners or colorants.

[0194] Compositions suitable for topical administration in the mouth include lozenges comprising the active agent in a flavored basis, usually sucrose and acacia or tragacanth; lozenges comprising the active ingredient in an inert basis such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0195] Solutions or suspensions may be applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray.The compositions may be presented in single or multi-dose form.

[0196] Administration to the respiratory tract may also be accomplished by means of an aerosol formulation in which the active ingredient is provided in a pressurized package having a suitable propellant, such as a chlorofluorocarbon (CFC), for example dichlorodifluoromethane, trichlorofluoromethane or dichlorotetrafluoroethane, carbon dioxide or other suitable gas. The aerosol may conveniently also contain a surfactant, such as lecithin. The dosage of the drug may be controlled by providing a metering valve.

[0197] Alternatively, the active ingredient may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethylcellulose and polyvinylpyrrolidone (PVP). Conveniently, the powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form, for example in capsules or cartridges of, for example, gelatin or blister packs, from which the powder may be administered with the aid of an inhaler.

[0198] In compositions intended for administration to the respiratory tract, including intranasal compositions, the compound will typically have a small particle size, for example about 5 microns or less. Such a particle size can be obtained by means known in the art, for example by micronization.

[0199] When desired, compositions adapted to provide sustained release of the VRAC active ingredient may be employed.

[0200] The pharmaceutical formulation is preferably in unit dosage form. In this form, the formulation is subdivided into unit doses containing appropriate quantities of the active ingredient. The unit dosage form can be a packaged formulation containing discrete quantities of the formulation, such as packaged tablets, capsules, and powders in vials or ampoules. Furthermore, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

[0201] Tablets or capsules for oral administration and liquids for intravenous administration and continuous infusion are preferred compositions.

[0202] In other embodiments, the VRAC inhibitor is formulated for topical administration.

[0203] Further details on formulation and administration techniques may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton, PA).

[0204] A therapeutically effective dose is the amount of active ingredient that improves a symptom or condition. Therapeutic efficacy and toxicity, such as ED 50 and LD 50The dose ratio between therapeutic and toxic effects is the therapeutic index and can be expressed as the ratio LD 50 / ED 50 Pharmaceutical compositions that exhibit large therapeutic indices are preferred.

[0205] The dose administered must, of course, be carefully adjusted according to the age, weight and condition of the individual being treated as well as the route of administration, dosage form and regimen and the desired results, and, of course, the exact dose should be determined by the practitioner.

[0206] The actual dosage of the VRAC modulator and / or anticancer agent will depend on the nature and severity of the condition being treated and will be determined by the physician, and may be varied by titrating the dosage on a case-by-case basis to produce the desired therapeutic effect.

[0207] For example, the VRAC blocker can be administered in one or several doses per day. An exemplary range is 10-200 mg / day po (oral), administered in one or two doses, such as 25-50 mg po, twice a day.

[0208] The subject to be treated according to the present invention may be any human or animal subject, preferably a human suffering from cancer.

[0209] cancer

[0210] The present invention is directed to sensitizing cancer cells to anti-cancer treatments or enhancing the therapeutic effects of anti-cancer treatments.

[0211] In one embodiment, the cancer is resistant to treatment with a chemotherapeutic agent. If the cancer is resistant, co-treatment with a VRAC blocker can resensitize the cancer to the chemotherapeutic agent in question. The resistance of the cancer can be innate resistance or acquired resistance. In general, if a patient treated with a clinically accepted dose of an anticancer agent does not respond to the anticancer agent as expected, i.e., in the case of worsening, growth or spread of the cancer (progressive disease), the cancer is considered to be resistant to a particular anticancer therapy. One skilled in the art can determine whether a cancer is drug sensitive or resistant.

[0212] In one embodiment, the cancer to be treated according to the present invention can be selected from lung cancer (non-small cell lung cancer and small cell lung cancer), glioblastoma, head and neck cancer, malignant melanoma, basal cell skin cancer, squamous cell skin cancer, breast cancer, liver cancer, pancreatic cancer, prostate cancer, colorectal cancer, anal cancer, cervical cancer, bladder cancer, uterine corpus cancer, ovarian cancer, gallbladder cancer, sarcoma, leukemia (myeloid and lymphoid), lymphoma, myeloma.

[0213] In one embodiment, the cancer is metastatic.

[0214] In one embodiment, the cancer is colorectal cancer.

[0215] In one embodiment, the cancer is metastatic colorectal cancer.

[0216] In one embodiment, the cancer is breast cancer.

[0217] In one embodiment, the cancer is metastatic breast cancer.

[0218] In one embodiment, the cancer is glioblastoma.

[0219] In one embodiment, the cancer is a solid tumor, such as a solid tumor selected from the group consisting of a sarcoma, a carcinoma, and a lymphoma.

[0220] In one embodiment, the cancer is not a solid tumor.For example, the cancer can be a hematological malignancy, including but not limited to leukemia.

[0221] In one embodiment, the cancer is prostate cancer, such as metastatic prostate cancer.

[0222] In one embodiment, the cancer is a steroid hormone receptor-positive cancer, e.g., an estrogen receptor-positive cancer, a progesterone receptor-positive cancer, or an androgen receptor-positive cancer.

[0223] Cancer treatment

[0224] The anti-cancer treatment according to the present invention may be treatment with one or more chemotherapeutic agents and / or radiotherapy. In a preferred embodiment, the anti-cancer treatment comprises treatment with a chemotherapeutic agent.

[0225] Chemotherapeutic drugs can be divided into several groups based on factors such as how they work, their chemical structure, and their relationship to other drugs. Some drugs work in more than one way and may belong to more than one group. In one embodiment, the anti-cancer treatment of the present invention includes treatment with more than one chemotherapeutic agent.

[0226] In one embodiment, the anti-cancer treatment is a chemotherapeutic agent selected from cytotoxic agents, cytostatic agents, anti-hormonal agents, anti-angiogenic agents, and anti-cancer biological agents with well-defined targets.

[0227] In one embodiment, the chemotherapeutic agent is a cytotoxic or cytostatic agent.

[0228] In a preferred embodiment, the anti-cancer treatment is a chemotherapeutic agent and the VRAC modulator is co-administered with the chemotherapeutic agent.

[0229] The VRAC modulator can be administered prior to, concurrently with, and / or after the anti-cancer therapy is administered. In one embodiment, the VRAC modulator is administered prior to the anti-cancer therapy. In one embodiment, the VRAC modulator is administered concurrently with the anti-cancer therapy.

[0230] Co-administration, as used herein, refers to administering a VRAC modulator and an anti-cancer therapy to a subject, wherein the VRAC modulator may be administered prior to, concurrently with, and / or after the anti-cancer therapy.

[0231] Administration of a VRAC modulator enhances the effect of an anti-cancer therapy. Thus, the therapeutic effect of using a VRAC blocker and an anti-cancer therapy is additive or synergistic. In one embodiment, the therapeutic effect is synergistic.

[0232] In one embodiment, administration of a VRAC modulator allows for administration of an anti-cancer therapy at doses lower than normal doses, ie, doses generally considered subtherapeutic doses.

[0233] In one embodiment, administration of a VRAC modulator enhances the clinical effect of an anti-cancer therapy. The clinical effect can be determined by a clinician.

[0234] In one embodiment, the anti-cancer treatment is a chemotherapeutic agent selected from topoisomerase inhibitors, antihormonal agents, alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, corticosteroids, targeted anticancer therapies, differentiation agents, and immunotherapy.

[0235] Alkylating agents

[0236] In one embodiment, the chemotherapeutic agent is an alkylating agent. Alkylating agents directly damage DNA (the genetic material in each cell) to prevent cells from multiplying. These drugs work at all stages of the cell cycle and are used to treat many different cancers, including glioblastoma, leukemia, lymphoma, Hodgkin's disease, multiple myeloma, and sarcoma, as well as lung, breast, and ovarian cancers.

[0237] Alkylating agents are divided into different categories, including:

[0238] Nitrogen mustards: such as mechlorethamine, nitrogen mustard, chlorambucil, cyclophosphamide Ifosfamide and melphalan

[0239] Nitrosoureas: such as streptozotocin, carmustine (BCNU), and lomustine

[0240] Alkyl sulfonates: busulfan

[0241] Triazines: dacarbazine (DTIC) and temozolomide

[0242] Ethylenimines: thiotepa and altretamine, hexamethylmelamine

[0243] In one embodiment, the alkylating agent is selected from nitrogen mustards, nitrosoureas, alkyl sulfonates, triazines, ethyleneimines.

[0244] In one embodiment, the alkylating agent is a triazine, such as temozolomide.

[0245] Platinum drugs (such as cisplatin, carboplatin, and oxaliplatin) are sometimes grouped with alkylating agents because they kill cells in a similar way. However, for the purposes of this article, platinum drugs are not considered alkylating agents.

[0246] In one embodiment, the anti-cancer therapy does not include or consists of treatment with metal-based anti-cancer drugs, such as platinum-, ruthenium-, gold-, or titanium-based anti-cancer drugs.

[0247] In one embodiment, the anti-cancer treatment does not include or consists of treatment with a platinum-based anti-cancer drug such as cisplatin, carboplatin, oxaliplatin, or nedaplatin.

[0248] In one embodiment, an alkylating agent is used in combination with a VRAC modulator to treat glioblastoma, particularly glioblastoma that is resistant to treatment with an alkylating agent.

[0249] In a specific embodiment, the alkylating agent is a triazine, such as temozolomide (Tedo), the VRAC modulator is NS3728, and the cancer to be treated is glioblastoma, particularly temozolomide-resistant glioblastoma.

[0250] Antimetabolites

[0251] In one embodiment, the chemotherapeutic agent is an antimetabolite. Antimetabolites interfere with the growth of DNA and RNA by replacing the normal building blocks of RNA and DNA. These agents damage cells during the S phase when the chromosomes of the cell are replicated. They are commonly used to treat leukemia, breast cancer, ovarian cancer, and intestinal cancer, for example, colorectal cancer and other types of cancer.

[0252] In one embodiment, the antimetabolite is selected from 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine Cytarabine Floxuridine, fludarabine, gemcitabine Hydroxyurea, methotrexate, pemetrexed

[0253] In one embodiment, the antimetabolite is 5-fluorouracil (5-FU).

[0254] In one embodiment, antimetabolites are used in combination with VRAC modulators to treat colorectal cancer, particularly colorectal cancer that is resistant to antimetabolite treatment. In one embodiment, the colorectal cancer is metastatic colorectal cancer.

[0255] In a specific embodiment, 5-FU is co-administered with a VRAC modulator such as NS3728 for the treatment of colorectal cancer, particularly 5-FU-resistant colorectal cancer. In one embodiment, the colorectal cancer is metastatic colorectal cancer.

[0256] Antitumor antibiotics

[0257] These drugs work by changing the DNA inside cancer cells to prevent them from growing and multiplying.

[0258] In one embodiment, the chemotherapeutic agent is an anti-tumor antibiotic agent. In other embodiments, the chemotherapeutic agent is not an anti-tumor antibiotic agent.

[0259] In one embodiment, the antitumor antibiotic agent is an anthracycline. Anthracyclines are anticancer antibiotics that interfere with enzymes involved in DNA replication. These drugs work at all stages of the cell cycle. They are widely used in a variety of cancers. Anthracyclines can also inhibit topoisomerase II.

[0260] Examples of anthracyclines include:

[0261] ·Daunorubicin

[0262] Adriamycin

[0263] ·Birubicin

[0264] Idabi

[0265] Anticancer antibiotics that are not anthracyclines include:

[0266] Actinomycin-D

[0267] Bleomycin

[0268] Mitomycin-C

[0269] Mitoxantrone (also used as a topoisomerase II inhibitor)

[0270] Topoisomerase inhibitors

[0271] In one embodiment, the chemotherapeutic agent is a topoisomerase inhibitor, which can be a topoisomerase I inhibitor or a topoisomerase II inhibitor. These drugs interfere with enzymes called topoisomerases, which help separate DNA strands so they can replicate during the S phase. Topoisomerase inhibitors are used to treat colorectal cancer, certain leukemias, as well as lung, ovarian, gastrointestinal, and other cancers.

[0272] Topoisomerase inhibitors are grouped according to the type of enzyme they affect.

[0273] Topoisomerase I inhibitors include:

[0274] Topotecan

[0275] Irinotecan (CPT-11). The active metabolite of irinotecan is SN-38.

[0276] Topoisomerase II inhibitors include:

[0277] Etoposide (VP-16)

[0278] Teniposide

[0279] Mitoxantrone (also used as an antitumor antibiotic)

[0280] Anthracyclines

[0281] In one embodiment, the topoisomerase inhibitor is a topoisomerase I inhibitor, such as irinotecan or its active metabolite SN-38.

[0282] In one embodiment, the topoisomerase inhibitor is a topoisomerase II inhibitor, such as an anthracycline.

[0283] In one embodiment, a topoisomerase inhibitor, such as a topoisomerase I inhibitor, is used in combination with a VRAC modulator to treat colorectal cancer, particularly colorectal cancer that is resistant to treatment with the topoisomerase inhibitor. In one embodiment, the colorectal cancer is metastatic colorectal cancer.

[0284] In a specific embodiment, irinotecan / SN-38 is co-administered with a VRAC modulator such as NS3728 for the treatment of colorectal cancer, particularly irinotecan / SN-38 resistant colorectal cancer. In one embodiment, the colorectal cancer is metastatic colorectal cancer.

[0285] Mitotic inhibitors

[0286] In one embodiment, the chemotherapeutic agent is a mitotic inhibitor. Mitotic inhibitors are often plant alkaloids and other compounds derived from natural products. They work by stopping mitosis in the M phase of the cell cycle, but can damage cells at all stages by preventing enzymes from forming proteins required for cell reproduction.

[0287] Examples of mitotic inhibitors include:

[0288] Taxanes: Paclitaxel and docetaxel

[0289] Epothilone: ​​Ixabepilone

[0290] Vinca alkaloids: vinblastine Vincristine Vinorelbine

[0291] ·Estrogen mustard

[0292] Mitotic inhibitors are used to treat many different types of cancer, including breast cancer, lung cancer, myeloma, lymphoma, and leukemia.

[0293] In one embodiment, the mitotic inhibitor is a taxane, such as paclitaxel or docetaxel.

[0294] In one embodiment, a mitotic inhibitor is used in combination with a VRAC modulator to treat breast cancer, particularly breast cancer that is resistant to treatment with a mitotic inhibitor. In one embodiment, the breast cancer is metastatic breast cancer.

[0295] In a specific embodiment, a taxane such as paclitaxel or docetaxel is co-administered with a VRAC modulator such as NS3728 for the treatment of breast cancer, particularly paclitaxel- or docetaxel-resistant breast cancer. In one embodiment, the breast cancer is metastatic breast cancer.

[0296] Corticosteroids

[0297] In one embodiment, the chemotherapeutic agent is a corticosteroid.Corticosteroids, often simply called steroids, are naturally occurring hormones and hormone-like drugs that are used to treat many types of cancer as well as other diseases.

[0298] When these drugs are used as part of cancer treatment, they are considered chemotherapy drugs.

[0299] Examples of corticosteroids include:

[0300] Prednisone

[0301] Methylprednisolone

[0302] Dexamethasone

[0303] Steroids are also commonly used to help prevent nausea and vomiting caused by chemotherapy. They are used before chemotherapy and can also help prevent severe allergic reactions.

[0304] In some embodiments, the chemotherapeutic agent is not a corticosteroid.

[0305] Other chemotherapy drugs

[0306] Some chemotherapy drugs work in slightly different ways and don't fit well into any of the other categories. Examples include drugs such as L-asparaginase (which is an enzyme) and the proteasome inhibitor bortezomib. of medicines.

[0307] Targeted anticancer therapy

[0308] In one embodiment, the chemotherapeutic agent is a targeted anti-cancer agent, such as an antibody-based therapy, which acts on a well-defined target or biological pathway.

[0309] Examples of targeted therapies include:

[0310] Imatinib

[0311] Gefitinib

[0312] Sunitinib

[0313] Bortezomib

[0314] In one embodiment, the targeted anti-cancer therapy is an anti-angiogenic agent, such as an anti-VEGF agent. For example, the anti-angiogenic agent can be a humanized anti-VEGF monoclonal antibody, such as Avastin (bevacizumab). If the anti-cancer therapy of the present invention is an anti-angiogenic agent, the anti-angiogenic agent is not a VRAC blocker, such as NS3728.

[0315] In some embodiments, the chemotherapeutic agent is not a targeted anti-cancer agent.

[0316] Differentiation Agents

[0317] These drugs act on cancer cells to make them mature into normal cells. Examples include retinoids, retinoic acid (ATRA or ) and bexarotene and arsenic trioxide

[0318] In one embodiment, the chemotherapeutic agent is a differentiation agent. In some embodiments, the chemotherapeutic agent is not a differentiation agent.

[0319] Antihormonal agents

[0320] In one embodiment, chemotherapeutic agents are agents used for anti-hormonal therapy. Such drugs are sex hormones or hormone-like drugs that change the effect or production of female or male hormones. They are used to slow the growth of breast cancer, prostate cancer, and endometrial (uterine) cancer, which normally grow in response to natural sex hormones in the body. These cancer treatment hormones do not work in the same way as standard chemotherapy drugs. They work by making it impossible for cancer cells to use the hormones they need for growth or by preventing the body from producing hormones.

[0321] Examples of antihormonal therapies include:

[0322] Anti-estrogen: Fulvestrant Tamoxifen and toremifene Clomiphene and raloxifene.

[0323] Antiprogestins: mifepristone, ulipristal acetate, alepristone, lilopristone, and onapristone

[0324] Antiandrogen: Bicalutamide Flutamide and nilutamide

[0325] Aromatase inhibitors: Anastrozole Exemestane and letrozole

[0326] Progestogen: megestrol acetate

[0327] Estrogen

[0328] Gonadotropin-releasing hormone (GnRH), also known as luteinizing hormone-releasing hormone (LHRH) agonists or analogs: Leuprorelin Goserelin

[0329] In one embodiment, the anti-cancer treatment comprises anti-estrogen therapy.Antiestrogens, also known as estrogen receptor antagonists or estrogen receptor blockers, are a class of drugs that prevent estrogens, such as estradiol, from mediating their biological effects in the body.

[0330] In one embodiment, the anti-estrogen is fulvestrant.

[0331] In one embodiment, the anti-estrogen is tamoxifen.

[0332] In one embodiment, the anti-cancer treatment comprises anti-progestin therapy.Anti-progestogens or anti-progestins, also known as progesterone receptor antagonists or progesterone blockers, are a class of drugs that prevent progestins such as progesterone from mediating their biological effects in the body.

[0333] Examples of antiprogestins include mifepristone, ulipristal acetate, alepristone, lilopristone, and onapristone.

[0334] In one embodiment, the anti-cancer treatment comprises anti-androgen therapy.Anti-androgens, also known as androgen receptor antagonists or testosterone blockers, are a class of drugs that prevent androgens such as testosterone and dihydrotestosterone (DHT) from mediating their biological effects in the body.

[0335] In some embodiments, the chemotherapeutic agent is not an anti-hormonal agent.

[0336] Anti-hormonal therapy is particularly useful in treating steroid hormone receptor-positive cancers, such as anti-estrogens used to treat ER-positive breast or uterine cancer.

[0337] In one embodiment, an antiestrogen is co-administered with a VRAC modulator, such as NS3728, for the treatment of ER-positive cancers, such as ER-positive breast cancer. In one embodiment, the breast cancer is metastatic breast cancer.

[0338] In one embodiment, an antiprogestin is co-administered with a VRAC modulator, such as NS3728, for the treatment of a PR-positive cancer, such as PR-positive breast cancer. In one embodiment, the breast cancer is metastatic breast cancer.

[0339] In one embodiment, an antiandrogen is co-administered with a VRAC modulator, such as NS3728, for the treatment of an AR-positive cancer, such as an AR-positive prostate cancer. In one embodiment, the prostate cancer is metastatic prostate cancer.

[0340] Immunotherapy

[0341] In one embodiment, the chemotherapeutic agent is an immunotherapy agent. Immunotherapy drugs are given to people with cancer to help their immune system recognize and attack cancer cells.

[0342] There are different types of immunotherapy. Active immunotherapy stimulates the body's own immune system to fight the disease. Passive immunotherapy doesn't rely on the body to attack the disease; they are immune system components (like antibodies) produced outside the body and used to fight cancer.

[0343] Examples of active immunotherapy include:

[0344] Monoclonal antibody therapy, such as rituximab and alemtuzumab

[0345] Nonspecific immunotherapies and adjuvants (other substances or cells that enhance the immune response), such as BCG, interleukin-2 (IL-2), and interferon-alpha

[0346] Immunomodulatory drugs, such as thalidomide and lenalidomide

[0347] In one embodiment, the chemotherapeutic agent is a PD-1 or PD-L1 inhibitor, such as an antibody capable of inhibiting PD-1 or PD-L1.

[0348] Cancer vaccines are a type of active specific immunotherapy.

[0349] In some embodiments, the chemotherapeutic agent is not an immunotherapy agent.

[0350] Radiation therapy

[0351] In one embodiment, anticancer therapy is radiotherapy. Radiotherapy is a therapy using ionizing radiation, usually used as a part of cancer treatment to control or kill malignant cells. If some types of cancer cells are confined to an area of ​​the body, radiotherapy may be curative in these types of cancer. It can also be used as a part of adjuvant therapy to prevent postoperative tumor recurrence of the removal of primary malignant tumors (e.g., early stages of breast cancer). Radiotherapy has a synergistic effect with chemotherapy and is used before, during and after chemotherapy in susceptible cancers. The dosage and treatment regimen of radiotherapy vary according to the type and stage of the cancer being treated, and can be determined by a clinician.

[0352] Medical Uses

[0353] In one embodiment, the invention relates to a method of sensitizing cancer cells to an anti-cancer therapy comprising administering to a subject in need thereof an effective amount of a VRAC modulator.

[0354] In one embodiment, the invention is directed to a method of enhancing the therapeutic effect of an anti-cancer therapy comprising administering to a subject in need thereof an effective amount of a VRAC modulator.

[0355] In one embodiment, the invention is directed to a method of treating cancer comprising administering to a subject in need thereof an effective amount of a VRAC modulator and an effective amount of an anti-cancer therapy, such as a chemotherapeutic agent.

[0356] In one embodiment, the invention relates to a composition comprising an effective amount of a VRAC modulator and an effective amount of a chemotherapeutic agent for use as a medicament.

[0357] In one embodiment, the invention is directed to a composition comprising an effective amount of a VRAC modulator and an effective amount of a chemotherapeutic agent for use in treating cancer.

[0358] In one embodiment, the invention relates to VRAC modulators for use in sensitizing cancer cells to anti-cancer treatments.

[0359] In one embodiment, the present invention relates to VRAC modulators for use in enhancing the therapeutic effect of anti-cancer therapy.

[0360] In one embodiment, the invention relates to the use of a VRAC modulator in the preparation of a medicament for sensitizing cancer cells to an anti-cancer treatment or for enhancing the therapeutic effect of an anti-cancer treatment.

[0361] In one embodiment, the invention relates to a kit comprising a VRAC modulator and a chemotherapeutic agent and optionally instructions for use, wherein the VRAC modulator and the chemotherapeutic agent are formulated for simultaneous, sequential or separate administration.

[0362] In one particularly interesting embodiment, the invention relates to a VRAC modulator and an anticancer therapy, such as a chemotherapeutic agent, for use in treating cancer in a subject, wherein the cancer has been determined to be resistant to treatment with the anticancer therapy. Treatment with a VRAC modulator resensitizes the resistant cancer cells to the anticancer therapy. Resistance can be innate resistance or acquired resistance.

[0363] Co-treatment with a VRAC modulator as described herein can be considered an adjuvant therapy to an anti-cancer therapy because the VRAC modulator maximizes the effectiveness of the anti-cancer therapy.

[0364] In one embodiment, treatment with a VRAC modulator described herein can be considered neoadjuvant therapy when administered prior to anti-cancer therapy. Example

[0365] Example 1. Temozolomide and NS3728 in glioblastoma

[0366] The in vivo antitumor effect of NS3728 on C6 glioma cells was tested in two separate experiments as a monotherapy (40 mg / kg / day) or in combination with temozolomide (Tedo, 25 or 5 mg / kg / day) using a xenograft approach or a subcutaneous air sac model (SAS; see Lichtenberg, J., PJ Hjarnaa, PE Kristjansen, D. Hansen & L. Binderup: The rat subcutaneous air sac model: A quantitative assay of antiangiogenesis in induced vessels Pharmacology & Toxicology, 1999, 84, 34-40).

[0367] In the first study, 5 million C6 rat glioma cells were inoculated into the flanks of Fisher rats. After three days, the tumors grew to 2-5 mm in diameter and treatment was initiated. C6 cells were observed to have aggressive growth behavior and quickly formed palpable solid tumors. Oral administration of NS3728 (40 mg / kg / day) in combination with temozolomide (25 mg / kg / day) resulted in a significant reduction in growth rate, while neither NS3728 (40 mg / kg / day) nor temozolomide (25 mg / kg / day) showed any significant antitumor effect when given as monotherapy (see Figure 1 ). Obvious signs of toxicity were observed in all animals treated with temozolomide, including submissive behavior, hunched appearance, and weight loss. The study was terminated on day 4 of treatment for ethical reasons.

[0368] In the second study, tumor growth was slowed by inoculating only half the number of cells (2.5 million C6 cells per animal) and using the subcutaneous air bag (SAS) technique. Briefly, rats were anesthetized with CO2 / O2 and 10-15 ml of air was introduced in the back by subcutaneous injection to create an air bag located approximately 4-5 cm behind the head. Over time, the wall of the air bag became thicker and thicker, and after 10-14 days, sufficient cell lining with a transparent membrane appearance had been established. C6 glioma cells were inoculated directly on this membrane in rats. Treatment was started on the day of inoculation, and the study was terminated on the 10th day of treatment. C6 cells showed less aggressive growth, and oral administration of NS3728 (40 mg / kg / day), whether as a monotherapy or in combination with temozolomide (5 mg / kg / day), significantly inhibited tumor growth ( Figure 2 ), whereas treatment with temozolomide alone did not result in significant tumor growth inhibition, as measured by tumor weight.

[0369] The experiments shown here demonstrate that oral administration of NS3728 in combination with temozolomide (the "first-line" therapy for glioblastoma in Denmark) slows the growth of C6 glioma cells by 50-55%, regardless of the site of inoculation. The effects are clearly additive or synergistic compared to monotherapy with either compound. Thus, the results show that treatment with NS3728 sensitizes cancer cells to treatment and enhances the effects of temozolomide in vivo.

[0370] Example 2. Paclitaxel or 5-fluorouracil and NS3728 in colon cancer

[0371] In two independent experimental series, NS3728 was tested as a monotherapy (80 mg / kg) in combination with paclitaxel (5 mg / kg) or 5-fluorouracil (30 mg / kg) for its in vivo antitumor effect on the human HT29 colon cancer cell line. NS3728 was administered orally (PO), while paclitaxel and 5-fluorouracil were administered by intraperitoneal injection (IP). Cancer cells were inoculated subcutaneously at t=0 (one animal in the vehicle group did not develop a tumor and was excluded from the 5-fluorouracil experiment), treatment started on day 4 and ended on day 22. The experiment included the following dosing groups (n=9-10 / group):

[0372] 1) solvent,

[0373] 2) NS3728,

[0374] 3) Paclitaxel or 5-fluorouracil,

[0375] 4) NS3728+paclitaxel or NS3728+5-fluorouracil.

[0376] The drugs were administered in cycles (days 4-8; days 11-15, days 18-22), with NS3728 and cytotoxic drugs administered daily, interrupted by dosing holidays. Figure 3 (5-fluorouracil) and Figure 4 In both experiments, tumor volume in the vehicle group increased in a superlinear manner until the final tumor volume in the 5-fluorouracil experiment was 166 mm 3 and 122 mm in the paclitaxel experiment 3 A slightly (non-significant) inhibitory growth curve was observed when both NS3728 and the cytotoxic drug were given as monotherapy. In contrast, the growth curve was further reduced when the combination therapy with these two cytotoxic drugs (NS3728 and cytotoxic drugs) was adopted, and both combinations reached significance (t test) relative to the control group (for NS3728 + 5-fluorouracil, P = 0.03; for NS3728 + paclitaxel, P = 0.003).

[0377] In conclusion, we have demonstrated that human xenografted HT29 tumors exhibit increased sensitivity to combination treatment with NS3728 and either 5-fluorouracil or paclitaxel compared to monotherapy with one of the chemotherapeutic drugs. Thus, NS3728 is able to enhance the effects of 5-fluorouracil and paclitaxel in vivo.

[0378] Example 3. Docetaxel and NS3728 in docetaxel-resistant MCF-7 and MDA-MB231 human breast cancer cell lines.

[0379] Breast cancer is a heterogeneous disease and at least 6 subtypes of breast cancer have been previously described by Soerlie et al. (PMID: 11553815). As a major distinction, breast cancer can be divided into hormone (estrogen and progesterone) receptor-positive and hormone receptor-negative breast cancer. We included these two major subtypes in our experiments. We established human breast cancer cell lines that are resistant to the chemotherapy docetaxel as described by Hansen et al. 2015 (PMID: 25596703). These cell lines are MCF-7 and MDA-MB-231.

[0380] MCF-7: estrogen and progesterone receptor positive

[0381] MDA-MB-231: Estrogen and progesterone receptor negative and HER2 receptor negative (so-called triple-negative phenotype)

[0382] We have examined the effects of NS3728 as a single agent or in combination with docetaxel in docetaxel-resistant breast cancer cell lines.

[0383] We used a standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay. Cells were seeded in 96-well plates (10,000 cells / well) and allowed to adhere for 24 hours. Subsequently, NS3728, docetaxel, or a combination of these two drugs was added for 72 hours, and then cell viability was determined. All values ​​are expressed as a percentage of untreated cells (named "no treatment"). "DMSO" refers to solvent control because both NS3728 and docetaxel were diluted in DMSO. Therefore, a DMSO (dimethyl sulfoxide) control was included to study any potential effects of organic solvents on cell viability. Each data point was performed in triplicate.

[0384] DMSO does not affect the cell viability of docetaxel-tolerant MCF-7 breast cancer cells. 65nM docetaxel was selected because this is the concentration at which docetaxel-tolerant MCF-7 breast cancer cells develop tolerance. 65nM docetaxel and 40μM NS3728 provided as monotherapy do not reduce cell viability. The combination of 40μM NS3728 and 65nM docetaxel produces a combined effect on the cell viability of docetaxel-tolerant MCF-7 breast cancer cells.

[0385] The effects of NS3728, docetaxel and their combination on the cell viability of docetaxel-resistant MCF-7 breast cancer cells are shown in Figure 5 middle.

[0386] DMSO does not affect the cell viability of docetaxel-tolerant MDA-MB-231 breast cancer cells. 150nM docetaxel was selected because this is the concentration at which cells develop tolerance. 150nM docetaxel reduces viability to about 50% of the untreated control. 50μM NS3728 does not reduce cell viability. Under 50μM NS3728 and 150nM docetaxel, a combined effect on the cell viability of docetaxel-tolerant MDA-MB-231 breast cancer cells was observed.

[0387] The effects of NS3728, docetaxel and their combination on the cell viability of docetaxel-resistant MDA-MB-231 breast cancer cells are shown in Figure 6 middle.

[0388] in conclusion

[0389] These preclinical studies showed that the VRAC inhibitor NS3728 could enhance the effect of docetaxel in docetaxel-resistant breast cancer cell lines. Thus, the data suggest that NS3728 is able to resensitize docetaxel-resistant cancer cells to docetaxel treatment.

[0390] Example 4. Oxaliplatin or SN38 and NS3728 in parental HCT116, HT29 and LoVo human colorectal cancer (CRC) cell lines.

[0391] CRC cells exhibit genetic instability due to chromosomal instability, microsatellite instability (MSI) and CpG island methylation phenotype (CIMP), which leads to the activation of proto-oncogenes such as KRAS and the inactivation of tumor suppressor genes such as TP53 (p53). The presence or absence of certain genetic alterations can determine the response of CRC patients to treatment, so it is important to study CRC cell lines with different combinations of the above genetic alterations. Therefore, we selected three cell lines to represent the main genetic alterations observed in CRC patients:

[0392] -HCT116: MSI, KRAS mutation, TP53 wild-type, CIMP+, CIN-, from primary tumor (Dukes C)

[0393] -HT29: MSS, KRAS wild-type, TP53 mutation, CIMP+, CIN+ from primary tumor (Dukes C)

[0394] -LoVo: MSI, KRAS mutation, TP53 wild-type, CIMP-, CIN-, from metastasis (Dukes C)

[0395] We examined the effects of NS3728 as a single agent or in combination with OXP or SN-38 in parental (i.e., drug-sensitive) cell lines. We used a standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay. Cells were seeded in 96-well plates (10,000 cells / well) and allowed to adhere for 24 hours. Subsequently, NS3728, OXP, SN-38, or a combination of NS3728 and one of the cytotoxic drugs was added for 48 hours, and then cell viability was determined. All values ​​are expressed as a percentage of untreated cells (named "no treatment"). "DMSO" refers to solvent control because both NS3728 and chemotherapeutic drugs were diluted in DMSO. Therefore, a DMSO (dimethyl sulfoxide) control was included to study any potential effects of organic solvents on cell viability. Each data point was performed in triplicate.

[0396] In parental cells, we tested the effects of NS3728 as a single agent and in combination with OXP or SN-38.

[0397] Parental CRC cell lines - NS3728 and OXP

[0398] DMSO caused a minor decrease in cell viability of HCT116 parental cells. NS3728 reduced cell viability to approximately 50% of untreated cells, and OXP dose-dependently reduced cell viability of HCT116 parental cells. The combination of 40 or 50 μM NS3728 and 0.16 / 0.8 / 4 / 20 μM oxaliplatin did not inhibit cell viability more than either monotherapy in HCT116 parental cells.

[0399] The effect of the combination of NS3728 and OXA on the cell viability of parental HCT116 CRC cancer cells is shown in Figure 7 middle.

[0400] We also did not observe any combined inhibitory effect of NS3728 and OXP on cell viability in HT29 parental and LoVo parental cells (data not shown).

[0401] Therefore, combining NS3728 with oxaliplatin does not appear to have any effect in OXP-sensitive colorectal cancer.

[0402] HCT116 parental CRC cell line - NS3728 and SN-38

[0403] DMSO caused a slight decrease in cell viability. NS3728 reduced cell viability to about 50% of untreated cells at two concentrations (40 and 50 μM). SN-38 (0.16 μM) also reduced viability to about 50% of untreated cells. The combination of 40 or 50 μM NS3728 and 0.16 μM SN-38 had a combined inhibitory effect on cell viability, with 40 μM NS3728+SN-38 reducing cell viability to about 30% of untreated cells and 50 μM NS3728+SN-38 reducing cell viability to about 23% of untreated cells.

[0404] The effect of the combination of NS3728 and SN-38 on the cell viability of parental HCT116 CRC cancer cells is shown in Figure 8 middle.

[0405] HT29 parental CRC cell lines - NS3728 and SN-38

[0406] DMSO reduced cell viability in this cell line to about 78% of untreated cells. NS3728 reduced cell viability to about 90% of untreated cells at 40 μM and to about 77% of untreated cells at 50 μM. SN-38 (0.032 μM) reduced viability to about 77% of untreated cells. The combination of 40 or 50 μM NS3728 and 0.032 μM SN-38 caused a combined inhibitory effect on cell viability, with 40 μM NS3728+SN-38 reducing cell viability to about 50% of untreated cells and 50 μM NS3728+SN-38 reducing cell viability to about 38% of untreated cells.

[0407] The effect of the combination of NS3728 and SN-38 on the cell viability of parental HT29 CRC cancer cells is shown in Fig. 9 middle.

[0408] LoVo parental CRC cell lines - NS3728 and SN-38

[0409] DMSO reduced cell viability in this cell line to about 75% of untreated cells. NS3728 reduced cell viability to about 45% of untreated cells at 40 μM and to about 38% of untreated cells at 50 μM. SN-38 (0.0064 μM) reduced viability to about 53% of untreated cells. The combination of 40 or 50 μM NS3728 and 0.0064 μM SN-38 caused a combined inhibitory effect on cell viability, with 40 μM NS3728+SN-38 reducing cell viability to about 35% of untreated cells and 50 μM NS3728+SN-38 reducing cell viability to about 22% of untreated cells.

[0410] The effect of the combination of NS3728 and SN-38 on the cell viability of parental LoVo CRC cancer cells is shown in Fig.10 middle.

[0411] in conclusion

[0412] These preclinical studies showed that the VRAC inhibitor NS3728 can 1) inhibit CRC cell growth when administered as a monotherapy, and 2) enhance the effects of certain types of chemotherapy on human CRC cells. No combined effect of NS3728 and oxaliplatin was found. Therefore, the results suggest that treatment with a VRAC inhibitor can enhance the effects of certain types of chemotherapy.

[0413] Example 5. Oxaliplatin or SN-38 and NS3728 in oxaliplatin (OXP) or SN-38-resistant HCT116, HT29, and LoVo human colorectal cancer (CRC) cell lines.

[0414] As described by Jensen et al. 2015 (PMID: 25759163), we have established human CRC cancer cell lines that are resistant to the chemotherapeutic agents oxaliplatin (OXP) or SN-38 (the active metabolite of irinotecan). These cell lines are HCT116, HT29, and LoVo.

[0415] We examined the effects of NS3728 as a single agent or in combination with OXP or SN-38 in OXP- and SN-38-resistant cell lines. We used a standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay. Cells were seeded in 96-well plates (10,000 cells / well) and allowed to adhere for 24 hours. Subsequently, NS3728, OXP, SN-38, or drug combinations were added for 48 hours, and then cell viability was measured. All values ​​are expressed as a percentage of untreated cells (named "no treatment"). "DMSO" refers to solvent control because both NS3728 and chemotherapeutic drugs were diluted in DMSO. Therefore, a DMSO (dimethyl sulfoxide) control was included to study any potential effects of organic solvents on cell viability. Each data point was performed in triplicate.

[0416] In OXP-resistant cells, we tested the effects of NS3728 as a single agent and in combination with SN-38 or OXP.

[0417] In SN-38-resistant cells, we tested the effects of NS3728 as a single agent and in combination with SN-38 or OXP.

[0418] SN-38-resistant CRC cell lines - NS3728 and oxaliplatin

[0419] Compared with monotherapy with NS3728 or oxaliplatin, no effect of combining NS3728 and oxaliplatin was found in SN-38-resistant HCT116, HT29, and LoVo CRC cancer cell lines (data not shown). Therefore, NS3728 does not seem to enhance the therapeutic effect of oxaliplatin in oxaliplatin-sensitive CRC cells that are resistant to SN-38.

[0420] HCT116 SN-38-resistant CRC cell line - NS3728 and SN-38

[0421] In this cell line, DMSO had no effect on cell viability. NS3728 (30 μM) reduced cell viability to about 85% of untreated cells, and SN-38 (4 μM) had little effect on cell viability. However, the combination of 4 μM SN-38 and 30 μM NS3728 caused a combined decrease in cell viability, and thus significantly reduced cell viability to about 60%.

[0422] The effect of the combination of NS3728 and SN-38 on the cell viability of SN-38-resistant HCT116 CRC cancer cells was shown in Fig.11 middle.

[0423] HT29 SN-38-resistant CRC cell lines - NS3728 and SN-38

[0424] DMSO had no effect on cell viability. NS3728 (30 μM) increased cell viability to about 120% of untreated cells. NS3728 (40 μM) had no effect on cell viability, and NS3728 (50 μM) reduced viability to about 85% of untreated cells. SN-38 (0.8 μM) reduced cell viability to about 78% of untreated cells. The combination of 0.8 μM SN-38 and 30 / 40 / 50 μM NS3728 caused a significant combined reduction in cell viability, which was reduced to less than 30% of untreated cells for all three NS3728 concentrations.

[0425] The effect of the combination of NS3728 and SN-38 on the cell viability of SN-38-resistant HT29 CRC cancer cells was shown in Fig.12 middle.

[0426] LoVo SN-38-resistant CRC cell lines - NS3728 and SN-38

[0427] DMSO reduced cell viability in this cell line to about 80% of untreated cells. NS3728 (30 μM) reduced viability to about 80% of untreated cells, NS3728 (40 μM) reduced viability to about 57% of untreated cells, and NS3728 (50 μM) reduced viability to about 50% of untreated cells. SN-38 (0.16 μM) reduced viability to about 78% of untreated cells. The combination of 0.16 μM SN-38 and 30 / 40 / 50 μM NS3728 caused a significant combined reduction in cell viability, to less than 20% of untreated cells for all three NS3728 concentrations.

[0428] The effect of the combination of NS3728 and SN-38 on the cell viability of SN-38-resistant LoVo CRC cancer cells was shown in Fig.13 middle.

[0429] HT29 oxaliplatin-resistant CRC cancer cells - NS3728 and SN-38

[0430] DMSO caused essentially no change in cell viability. NS3728 (20 μM) reduced viability to about 85% of untreated cells, and SN-38 (0.1 μM) reduced viability to about 43% of untreated cells. The combination of 20 μM NS3728 and 0.1 μM SN-38 caused a combined effect on cell viability.

[0431] The effect of the combination of NS3728 and SN-38 on the cell viability of oxaliplatin-resistant HT29 CRC cancer cells was shown in Fig.14 middle.

[0432] LoVo oxaliplatin-resistant CRC cancer cells - NS3728 and SN-38

[0433] DMSO reduced viability to approximately 85% of untreated cells. Combinations of 20 / 30 / 40 / 50 μM NS3728 and SN-38 (0.005 μM) all had significant combined effects on cell viability.

[0434] The effect of the combination of NS3728 and SN-38 on the cell viability of oxaliplatin-resistant LoVo CRC cancer cells was shown in Fig.15 middle.

[0435] HT116 oxaliplatin-resistant CRC cancer cells - NS3728 and N-38

[0436] In oxaliplatin-resistant HCT116 CRC cells, a minor inhibitory combined effect was observed when NS38728 was combined with SN-38 (data not shown).

[0437] Oxaliplatin-resistant CRC cancer cells-NS3728 and oxaliplatin

[0438] Compared with monotherapy with NS3728 or oxaliplatin, no effect of the combination of NS3728 and oxaliplatin was found in oxaliplatin-resistant HCT116, HT29 and LoVo CRC cancer cell lines (data not shown). Therefore, NS3728 does not seem to resensitize oxaliplatin-resistant CRC cells to oxaliplatin treatment.

[0439] in conclusion

[0440] These preclinical studies demonstrate that the VRAC inhibitor NS3728 can enhance the effects of certain types of chemotherapy in resistant CRC cell lines. Of particular interest is that NS3728 also enhances the effects of SN-38 in cancer cells with acquired SN-38 resistance or acquired oxaliplatin resistance. The data suggest that NS3728 is able to resensitize SN-38 resistant cancer cells to SN-38 treatment.

[0441] Example 6. Combination of tamoxifen or fulvestrant with NS3728 in tamoxifen-resistant MCF-7 / LCC2 or fulvestrant MCF-7 / LCC9 human breast cancer cell lines.

[0442] We have established human breast cancer cell lines that are resistant to the anti-estrogen tamoxifen as described by Brünner et al. 1993 (PMID: 8324732). These cell lines are MCF-7 / LCC2. We have also established human breast cancer cell lines that are resistant to the anti-estrogen fulvestrant as described by Brünner et al. 1997 (PMID: 9270017).

[0443] MCF-7 is an estrogen and progesterone receptor positive cell line.

[0444] We investigated the effects of NS3728 as a single agent or in combination with tamoxifen or fulvestrant in drug-resistant breast cancer cell lines (MCF-7 / LCC2 or MCF-7 / LCC9).

[0445] We used a standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay. Cells were seeded in 96-well plates (10,000 cells / well) and allowed to adhere for 24 hours. Subsequently, NS3728, docetaxel, or a combination of both drugs was added for 72 hours, and then cell viability was measured. All values ​​are expressed as a percentage of untreated cells (designated "no treatment"). Each data point was performed in triplicate.

[0446] 0.1 μM tamoxifen was selected because this was the concentration that showed only a minor cell growth effect on MCF-7 / LCC2 cells in the original publication, while the growth of tamoxifen-sensitive cells was significantly more inhibited. 40 μM NS3728 caused a slight decrease in cell viability to 70% of the untreated control. 0.1 μM tamoxifen had no effect on the cell viability of tamoxifen-tolerant MCF-7 / LCC2 cells. The combination of 40 μM NS3728 and 0.1 μM tamoxifen caused a strong and significant (P < 0.05) decrease in MCF-7 / LCC2 cell viability, which was reduced to less than 40% (0.1% of untreated cells) of the untreated cells. Fig.16 ).

[0447] 1 μM of Fulvestrant was selected because this was the concentration used as the final concentration in the original publication to establish the MCF-7 / LCC9 cell line, and this concentration strongly inhibited the growth of Fulvestrant-sensitive MCF-7 cells (Brünner et al., 1997, PMID 9270017). 40 μM NS3728 caused a reduction in cell viability to 60% of the untreated control. 1 μM Fulvestrant had no effect on the cell viability of Fulvestrant-tolerant MCF-7 / LCC9 cells. The combination of 40 μM NS3728 and 1 μM Fulvestrant resulted in a strong and significant (P<0.05) reduction in MCF-7 / LCC9 cell viability, down to 24% (P<0.05) of untreated cells. Fig.17 ).

[0448] in conclusion

[0449] These preclinical studies demonstrated that the VRAC inhibitor NS3728 can enhance the effects of two types of antiestrogens (i.e., tamoxifen and fulvestrant) on MCF-7 breast cancer cells that are resistant to these types of antiestrogens. Therefore, the data suggest that NS3728 can resensitize anti-estrogen-resistant breast cancer cells to anti-estrogen therapy, particularly tamoxifen and fulvestrant.

[0450] Example 7. In vivo study of the resensitization effect of NS3728 in docetaxel-resistant xenograft tumors (breast cancer)

[0451] Mouse xenograft experiments were performed using a pair of docetaxel-sensitive and docetaxel-resistant triple-negative human breast cancer cell lines (MDA-MB-231). The mice used for the experiments were severely immunocompromised to prevent rejection of the human cancer cells.

[0452] The experimental setup consists of three stages:

[0453] 1. “Growth”

[0454] 2. “Dose-Response”

[0455] 3. "Drug treatment"

[0456] Phase 1: Growth of sensitive (parental) and docetaxel-resistant MDA-MB-231 cells

[0457] To determine the optimal cell number for xenograft tumor growth of docetaxel-sensitive and docetaxel-resistant MDA-MB-231 cells, two groups of mice were inoculated with different numbers of sensitive or docetaxel-resistant MDA-MB-231 cells. This experiment determined the optimal number of cancer cells, which is important for subsequent dose response experiments in phases 2 and 3.

[0458] Phase 2: Tolerance phenotype, docetaxel dose-response

[0459] To confirm the docetaxel-sensitive and -resistant phenotypes in xenografted MDA-MB-231 cells, mice were treated with docetaxel when tumors were already established. MDA-MB-231 cells that were sensitive or docetaxel-resistant were inoculated into mice at the same cell number as determined in phase 1. This experiment is important to demonstrate that cancer cells are also sensitive or resistant, respectively, in an in vivo setting.

[0460] Phase 3: Docetaxel resensitizes NS3728 treatment

[0461] To determine the resensitization effect of NS3728 in combination with docetaxel in docetaxel-resistant xenograft tumors, parental and docetaxel-resistant MDA-MB-231 cells were inoculated into mice and treated with vehicle (control group), docetaxel alone, NS3728 alone, or a combination of NS3728 and docetaxel.

[0462] During all steps, tumor formation was monitored by palpation and measured by calipers. Tumor volume was calculated and growth curves were plotted against time. Mice were euthanized when tumors approached maximum size (12 mm) or if mice showed any signs of discomfort or illness.

[0463] in conclusion

[0464] Based on our in vitro experiments ( Figure 6 ) and paclitaxel xenograft experiments ( Figure 4 ), we expected to observe that the combination of docetaxel and NS3728 would restore docetaxel sensitivity in xenografted docetaxel-resistant MDA-MB-231 cells, reflected by a reduction in tumor volume compared with NS3728 alone or docetaxel alone.

Claims

1. Use of a VRAC modulator of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer in combination with an anti-hormone agent, Wherein the anti-hormone agent is selected from: a. an anti-estrogen selected from the group consisting of fulvestrant, tamoxifen, toremifene and clomiphene; and b. an antiprogestin selected from the group consisting of mifepristone, ulipristal acetate, alepristone, lilopristone and onapristone.

2. Use of a VRAC modulator of formula I or a pharmaceutically acceptable salt thereof and an anti-hormone agent in the preparation of a medicament for treating cancer, in, The anti-hormone agent is selected from: a. an anti-estrogen selected from the group consisting of fulvestrant, tamoxifen, toremifene and clomiphene; and b. an antiprogestin selected from the group consisting of mifepristone, ulipristal acetate, alepristone, lilopristone and onapristone.

3. The method of claim 1 or 2, wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, lung cancer (non-small cell lung cancer and small cell lung cancer), glioblastoma, head and neck cancer, malignant melanoma, basal cell skin cancer, squamous cell skin cancer, liver cancer, pancreatic cancer, prostate cancer, anal cancer, cervical cancer, bladder cancer, uterine corpus cancer, ovarian cancer, gallbladder cancer, sarcoma, leukemia (myeloid and lymphoid), lymphoma and myeloma.

4. The use according to claim 1 or 2, wherein the cancer is breast cancer, prostate cancer, cervical cancer or uterine body cancer.

5. The use according to claim 1 or 2, wherein the cancer is glioblastoma, such as glioblastoma resistant to temozolomide treatment.

6. The use according to claim 1 or 2, wherein the VRAC modulator and the anti-hormonal agent are formulated for simultaneous, sequential or separate administration.

7. The use according to claim 1 or 2, wherein the VRAC modulator is in the form of a tablet or capsule for oral administration.

8. The use according to claim 1 or 2, wherein the VRAC modulator is in the form of a liquid for intravenous administration or continuous infusion.

9. The use according to claim 1 or 2, wherein the cancer is an estrogen receptor positive cancer, such as fulvestrant or tamoxifen resistant cancer, preferably wherein the estrogen receptor positive cancer is breast cancer, optionally wherein the breast cancer is metastatic breast cancer.

10. The use according to claim 9, wherein the resistance is innate resistance.

11. The use according to claim 9, wherein the resistance is acquired resistance.

12. A pharmaceutical composition comprising, separately or together, effective amounts of a VRAC modulator of Formula I or a pharmaceutically acceptable salt thereof and an anti-hormone agent, and one or more pharmaceutically acceptable adjuvants, excipients, carriers, buffers and / or diluents The anti-hormone agent is selected from: a. an anti-estrogen selected from the group consisting of fulvestrant, tamoxifen, toremifene and clomiphene; and b. an antiprogestin selected from the group consisting of mifepristone, ulipristal acetate, alepristone, lilopristone and onapristone.

13. Use of a VRAC modulator of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for sensitizing cancer cells to an anti-hormonal agent Wherein the anti-hormone agent is selected from: a. an anti-estrogen selected from the group consisting of fulvestrant, tamoxifen, toremifene and clomiphene; and b. an antiprogestin selected from the group consisting of mifepristone, ulipristal acetate, alepristone, lilopristone and onapristone.

14. Use of a VRAC modulator of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for enhancing the therapeutic effect of an anti-hormone agent Wherein the anti-hormone agent is selected from: a. an anti-estrogen selected from the group consisting of fulvestrant, tamoxifen, toremifene and clomiphene; and b. an antiprogestin selected from the group consisting of mifepristone, ulipristal acetate, alepristone, lilopristone and onapristone.

15. Use of a VRAC modulator of formula I or a pharmaceutically acceptable salt thereof and an anti-hormone agent in the preparation of a kit for treating cancer, Wherein the anti-hormone agent is selected from: a. an anti-estrogen selected from the group consisting of fulvestrant, tamoxifen, toremifene and clomiphene; and b. an antiprogestin selected from mifepristone, ulipristal acetate, alepristone, lilopristone and onapristone; The VRAC modulator and the anti-hormonal agent are formulated for simultaneous, sequential or separate administration.

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