USE OF AN INHIBITOR OF AN ENT FAMILY TRANSPORTER IN THE TREATMENT OF CANCER AND COMBINATION THEREOF WITH AN ADENOSINE RECEPTOR ANTAGONIST

Abstract

The present invention relates to the use of an inhibitor of an ENT family transporter for the treatment of cancer. The invention further relates to the combined use of such inhibitor of an ENT family transporter with an adenosine receptor antagonist, for the treatment of cancer. The invention further relates to a pharmaceutical composition and a kit of parts comprising such combination.

Claims

1. An inhibitor of an ENT family transporter for use in the treatment of cancer in a human subject.

2. The inhibitor of an ENT family transporter for use according to claim 1, wherein the ENT family transporter is ENT1, and wherein the inhibitor is selected from the group consisting of a small molecule, a nucleic acid, a peptide, and an antibody.

3. The inhibitor of an ENT family transporter for use according to any one of claim 1 or claim 2, wherein the subject is treated with an additional therapeutic agent in combination with the inhibitor of the ENT family transporter, or has received the additional therapeutic agent within about fourteen days of administration of the inhibitor of the ENT family transporter.

4. The inhibitor of an ENT family transporter for use according to claim 3, wherein the additional therapeutic agent comprises an adenosine receptor antagonist.

5. The inhibitor of an ENT family transporter for use according to any one of claims 1-4, wherein the subject has previously received at least one prior therapeutic treatment, and has progressed subsequent to the administration of the at least one prior therapeutic treatment and prior to administration of the inhibitor of an ENT family transporter.

6. The inhibitor of an ENT family transporter for use according to claim 5, wherein the prior therapeutic treatment is selected from the group consisting of chemotherapy, immunotherapy, radiation therapy, stem cell transplant, hormone therapy, and surgery.

7. The inhibitor of an ENT family transporter for use according to claim 4, wherein the ENT family transporter inhibitor is administered prior to, concomitant with, or subsequent to administration of the additional therapeutic agent comprising an adenosine receptor antagonist.

8. A combination comprising (a) an effective amount of an inhibitor of an ENT family transporter; and (b) an effective amount of an adenosine receptor antagonist.

9. A pharmaceutical composition comprising: (a) an effective amount of an inhibitor of an ENT family transporter; (b) an effective amount of an adenosine receptor antagonist; and (c) at least one pharmaceutically acceptable excipient.

10. A kit of parts comprising: (a) a first part comprising an effective amount of an inhibitor of an ENT family transporter; and (b) a second part comprising an effective amount of an adenosine receptor antagonist.

11. The combination, the pharmaceutical composition or the kit of parts according to any one of claim 8 to 10, wherein the adenosine receptor antagonist is an A2A or A2B receptor antagonist.

12. The combination, the pharmaceutical composition or the kit of parts according to any one of claim 8 to 11, wherein the adenosine receptor antagonist is selected from: 5-bromo-2,6-di-(1H-pyrazol-1-yl)pyrimidin-4-amine; (S)-7-(5-methylfuran-2-yl)-3-((6-(([tetrahydrofuran-3-yl]oxy)methyl)pyridin-2-yl)methyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine; 6-(2-chloro-6-methylpyridin-4-yl)-5-(4-fluorophenyl)-1,2,4-triazin-3-amine; 3-(2-amino-6-(1-((6-(2-hydroxypropan-2-yl)pyridin-2-yl)methyl)-1H-1,2,3-triazol-4-yl)pyrimidin-4-yl)-2-methylbenzonitrile; 2-(2-furanyl)-7-(2-(4-(4-(2-methoxyethoxy)phenyl)-1-piperazinyl)ethyl)-7H-pyrazolo(4,3-e)(1,2,4)triazolo(1,5-c)pyrimidine-5-amine; 3-(4-amino-3-methylbenzyl)-7-(2-furyl)-3H-(1,2,3)triazolo(4,5-d)pyrimidine-5-amine; and 4-hydroxy-N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-methylpiperidine-1-carboxamide.

13. The combination, the pharmaceutical composition or the kit of parts according to any one of claim 8 to 11, wherein the adenosine receptor antagonist is a compound of Formula (I): ##STR00152## or a pharmaceutically acceptable salt or solvate thereof, wherein: R.sup.1 represents 5- or 6-membered heteroaryl or 5- or 6-membered aryl, wherein heteroaryl or aryl groups are optionally substituted by one or more substituent selected from C1-C6 alkyl (preferably methyl) and halo (preferably fluoro or chloro); preferably R.sup.1 represents 5-membered heteroaryl; more preferably R.sup.1 represents furyl; R.sup.2 represents 6-membered aryl or 6-membered heteroaryl, wherein heteroaryl or aryl groups are optionally substituted by one or more substituent selected from halo, alkyl, heterocyclyl, alkoxy, cycloalkyloxy, heterocyclyloxy, carbonyl, alkylcarbonyl, aminocarbonyl, hydroxycarbonyl, heterocyclylcarbonyl, alkylsulfoxide, alkylsulfonyl, aminosulfonyl, heterocyclylsulfonyl, alkylsulfonimidoyl, carbonylamino, sulfonylamino and alkylsulfonealkyl; said substituents being optionally substituted by one or more substituent selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkyne, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl alkylsulfonyl and alkylsulfonealkyl; or the heteroaryl or aryl groups are optionally substituted with two substituents that form together with the atoms to which they are attached a 5- or 6-membered aryl ring, a 5- or 6-membered heteroaryl ring, a 5- or 6-membered cycloalkyl ring or a 5- or 6-membered heterocyclyl ring; optionally substituted by one or more substituent selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkyne, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl and alkylsulfonealkyl.

14. The combination, the pharmaceutical composition or the kit of parts according to any one of claim 8 to 13, wherein the ENT family transporter is ENT1.

15. The combination, the pharmaceutical composition or the kit of parts according to any one of claim 8 to 14, further comprising an additional therapeutic agent.

16. The combination, the pharmaceutical composition or the kit of parts according to any one of claim 8 to 15, for medical use.

17. The combination, the pharmaceutical composition or the kit of parts according to any one of claim 8 to 15, for use in the treatment of cancer.

18. The combination, the pharmaceutical composition or the kit of parts according to any one of claim 8 to 15, wherein the ENT family transporter inhibitor is administered prior to, concomitant with, or subsequent to administration of an adenosine receptor antagonist.

19. A pharmaceutical formulation for use in the treatment of a cancer, wherein the pharmaceutical formulation is administered to a human subject in an amount effective to treat the cancer and wherein the formulation comprises: (a) an inhibitor of an ENT family transporter; and (b) optionally one or more of a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

20. The pharmaceutical formulation for use according to claim 19, wherein the ENT family transporter is ENT1, and wherein the inhibitor is selected from the group consisting of a small molecule, a nucleic acid, a peptide, and an antibody.

21. The pharmaceutical formulation for use according to any one of claim 19 or claim 20, wherein the pharmaceutical formulation further comprises an additional therapeutic agent.

22. The pharmaceutical formulation for use according to claim 21, wherein the additional therapeutic agent comprises an adenosine receptor antagonist.

23. The pharmaceutical formulation for use according to any one of claims 19 to 22, wherein the subject has previously received at least one prior therapeutic treatment.

24. The pharmaceutical formulation for use according to claim 23, wherein the prior therapeutic treatment is selected from the group consisting of chemotherapy, immunotherapy, radiation therapy, stem cell transplant, hormone therapy, and surgery.

25. The pharmaceutical formulation for use according to any one of claims 21 to 24, wherein the pharmaceutical formulation is administered prior to, concomitant with, or subsequent to administration of the additional therapeutic agent comprising an adenosine receptor antagonist.

26. A pharmaceutical formulation for use in the treatment of a cancer, wherein the pharmaceutical formulation is administered to a human subject in an amount effective to treat the cancer, and wherein the formulation comprises: (a) an inhibitor of an ENT family transporter (b) an adenosine receptor antagonist; and (c) optionally one or more of a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

27. The pharmaceutical formulation for use according to any one of claims 22 to 26, wherein the adenosine receptor antagonist comprises an A2A or A2B receptor antagonist.

28. The pharmaceutical formulation for use according to any one of claims 22 to 27, wherein the adenosine receptor antagonist is selected from: 5-bromo-2,6-di-(1H-pyrazol-1-yl)pyrimidin-4-amine; (S)-7-(5-methylfuran-2-yl)-3-((6-(([tetrahydrofuran-3-yl]oxy)methyl)pyridin-2-yl)methyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine; 6-(2-chloro-6-methylpyridin-4-yl)-5-(4-fluorophenyl)-1,2,4-triazin-3-amine; 3-(2-amino-6-(1-((6-(2-hydroxypropan-2-yl)pyridin-2-yl)methyl)-1H-1,2,3-triazol-4-yl)pyrimidin-4-yl)-2-methylbenzonitrile; 2-(2-furanyl)-7-(2-(4-(4-(2-methoxyethoxy)phenyl)-1-piperazinyl)ethyl)-7H-pyrazolo(4,3-e)(1,2,4)triazolo(1,5-c)pyrimidine-5-amine; 3-(4-amino-3-methylbenzyl)-7-(2-furyl)-3H-(1,2,3)triazolo(4,5-d)pyrimidine-5-amine; and 4-hydroxy-N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-methylpiperidine-1-carboxamide.

29. The pharmaceutical formulation for use according to any one of claims 22 to 27, wherein the adenosine receptor antagonist comprises a compound of formula (I) as defined in claim 13.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0424] FIGS. 1A, 1B and 1C are graphs representing the expression of ENT1, ENT2 and ENT4, respectively, in human primary lymphocytes. FPKMs: Fragments Per Kilobase Million. Source: Bonnal R J P, Nature 2015.

[0425] FIGS. 2A and 2B are graphs representing viability of CD3+ T cells under different treatments and show the reversal of Adenosine/ATP-mediated reduction in T cell viability by ENT inhibitors. Human primary CD3+ T cells were stimulated with antiCD3/antiCD28-coated microbeads, and treated with 25 μM adenosine (FIG. 2A) or 200 μM ATP (FIG. 2B). T cell viability was analysed by flow cytometry after a 3 days incubation period. Mean+/−SD of biological replicates are shown. Representative data for 1 healthy donor are shown. Similar data were obtained in 2 healthy donors.

[0426] FIGS. 3A, 3B, 3C and 3D are graphs representing proliferation of CD4+ T cells and CD8+ T cells under different treatments and show rescue of Adenosine/ATP-mediated suppression of T cell proliferation by ENT inhibitors. Human primary CD3+ T cells were stimulated with antiCD3/antiCD28-coated microbeads, and treated with 25 μM adenosine (FIGS. 3A and 3C) or 200 μM ATP (FIGS. 3B and 3D). Proliferation of CD4+(FIGS. 3A and 3B) and CD8+(FIGS. 3C and 3D) T cells was analysed by flow cytometry after a 3 days incubation period. Mean+/−SD of biological replicates are shown. Representative data for 1 healthy donor are shown. Similar data were obtained in 2 healthy donors.

[0427] FIGS. 4A and 4B are graphs representing IL-2 secretion by CD3+ T cells under different treatments and show rescue of Adenosine/ATP-mediated suppression of T cell cytokine secretion by a combination treatment with ENT inhibitors and A.sub.2AR antagonist Compound 8a. CD3+ T cells were stimulated with antiCD3/antiCD28-coated microbeads, and treated with 25 μM adenosine (FIG. 4A) or 200 μM ATP (FIG. 4B). IL-2 secretion in the supernatant was analysed by AlphaLISA after a 3 days incubation period. Mean+/−SD of biological replicates are shown. Representative data for 1 healthy donor are shown. Similar data were obtained in 2 healthy donors.

[0428] FIG. 5A shows the viability of CD3+ T cells as determined by staining with a fixable viability dye. FIG. 5B shows the proportion of proliferating CD8+ T cells as determined by CFSE dilution. FIG. 5C shows the concentration of TNFα present in the cell culture supernatants. Human primary CD3+ T cells were stimulated with antiCD3/antiCD28-coated microbeads, and treated with 100 μM ATP or control medium. Viability of CD3+ T cells (FIG. 5A) and proliferation of CD8+ T cells (FIG. 5B) were analysed by flow cytometry after a 3 days incubation period. TNFα secretion in the supernatant was analysed by AlphaLISA. Grey shaded bars show A2AR antagonist treated wells, whilst clear bars denote concentration-matched DMSO treated wells. Results are shown as mean value from sextuplicate (DMSO) or duplicate (A2AR antagonists) wells±standard deviation. Data shown are representative of data obtained in three healthy individuals in 4 independent experiments.

[0429] FIG. 6 shows in vivo LPS induced TNFalpha production in mouse, upon prior treatment with Compound 8b (A2AR antagonist) or NBMPR (ENT1 inhibitor) or the combination thereof. BALB/c mice were pretreated for 1 h before LPS (1 mg/kg I.V) injection with NBMPR (20 mg/kg p.o.) or vehicle. Thirty minutes prior mice were treated with a single dose of Compound 8b (3 mg/kg p.o.) or vehicle. One hour after LPS treatment, blood serum was collected from the mice. TNFalpha levels were determined by ELISA (n=5). P values are indicated using Mann-Whitney test, ns=not significant.

[0430] FIG. 7 shows the MCA205 tumor growth upon treatment with Compound 8b (A2AR antagonist) used in combination with NBMPR (ENT1 inhibitor). FIG. 7A is a graph representing the median tumor volume over time after subcutaneous inoculation of tumor cells. FIG. 7B-D are the individual tumor growth volumes of mice treated with Vehicle (FIG. 7B) and NBMPR at 20 mg/kg BIDx22 (day 11) in standalone (FIG. 7C) or in combination with Compound 8b at 3 mg/kg BIDx22 (FIG. 7D). (n=10, *P=0.0043, **P=0.0015, ***P<0.0001, linear mixed model statistical analysis).

EXAMPLES

[0431] The present invention will be better understood with reference to the following examples. These examples are intended to representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.

[0432] The following abbreviations are used:

ATP: adenosine triphosphate
BSA: bovine serum albumin
CFSE: carboxyfluorescein succinimidyl ester
DMSO: dimethylsulfoxide
EDTA: ethylenediaminetetraacetic acid
FACS: fluorescence-activated cell sorting,
FBS: fetal bovine serum
mL: milliliter
μL: microliter
mM: millimolar
nM: nanomolar
pM: micromolar
PBS: phosphate buffered saline
rpm: rounds per minute
RPMI: Roswell Park Memorial Institute medium

Example 1. ENT1 Inhibitor in Combination with A2AR Antagonist Restores TNFalpha Production in T Cells

Materials

[0433] Reagents and compounds used in the following assays have the following sources:

TABLE-US-00002 Reagent Source SepMate-50 tubes Stemcell Technologies Lymphoprep Elitechgroup EasySep Human T Cell Isolation Kit Stemcell Technologies Fetal bovine serum (FBS) Life Technologies Dimethyl sulfoxide (DMSO) Sigma-Aldrich RPMI 1640 Westburg (Lonza) 1x non-essential amino acids Westburg (Lonza) Sodium Pyruvate Westburg (Lonza) Dynabeads human T-activator CD3/ Life Technologies/ CD28 Thermo Fisher X-Vivo 15 Westburg (Lonza) CFSE 1 Life Technologies PBS Gibco/Lonza BSA Miltenyi biotec EDTA Westburg (Lonza) Dipyridamole Tocris Dilazep dihydrochloride Tocris Ticagrelor Sigma-Aldrich Adenosine Sigma-Aldrich Adenosine 5′-triphosphate disodium salt Sigma-Aldrich hydrate (ATP) Compound 8a prepared as described in PCT/EP2018/058301 AlphaLISA Human IL2 Biotin Free Promega Detection kit AlphaLISA Human TNFα Biotin Free Perkin-Elmer Detection kit Anti-human CD3-APC Biolegend (Imtec) Anti-human CD4-BV786 BD biosciences Anti-human CD8-APC-Cy7 Biolegend (Imtec) Anti-human CD25-PE Biolegend (Imtec) Fixable Viability Dye eFluor506 Life Technologies CPI-444 Prepared using a method adapted from patent WO2009/156737 (Example 1) NIR178 Prepared using a method adapted from patent WO2011/121418 (Example 1) AZD4635 Prepared using a method adapted from patent WO2011/095625 (compound number cxiv) AB928 Prepared using a method adapted from WO2019/161054 (Example 1)

Methods

[0434] PBMC and CD3+ T cell isolation. Venous blood from healthy volunteers, all of whom signed an informed consent approved by the Ethics Committee (FOR-UIC-BV-050-01-01 ICF_HBS_HD Version 5.0), was obtained via Unité d'Investigation Clinique (Centre Hospitalier Universitaire de Tivoli, La Louviere, Belgium). Mononuclear cells were collected by density gradient centrifugation, using SepMate-50 tubes and Lymphoprep according to the manufacturer's instructions. CD3+ T cells were isolated by immunomagnetic negative selection, using the EasySep Human T Cell Isolation Kit as per manufacturer's instructions. CD3+ T cells were stored in heat inactivated FBS and 10% DMSO in liquid nitrogen.

[0435] Human primary T cell culture. Human purified CD3+ T cells were thawed and washed twice with RPMI1640 medium, UltraGlutamine, supplemented with 1× non-essential amino acids (Lonza), and 1 mM Sodium Pyruvate (Gibco) (complete media), containing 10% hiFBS. After the final wash, cells were resuspended in PBS containing 10% FBS, and labeled using 3 μM CFSE during 5 min at room temperature, followed by two wash steps in complete media. After the last wash, cells were suspended at 1.6×10.sup.6 cells/mL in X-Vivol5 medium. 50 μL of cell suspension (8×10.sup.4 T cells) was added to wells of sterile round-bottom 96-well plates. Cells were activated by adding 50 μL of anti-CD3 anti-CD28 coated microbeads (Dynabeads human T-activator CD3/CD28), suspended in X-Vivo15 medium, at a ratio of one microbead per two cells. Adenosine (stock solution of 75 mM in DMSO) and ATP (powder) were diluted in X-Vivol5 medium, and 25 or 50 μL was added to the wells to reach final assay concentrations of 25 μM Adenosine or 200 μM ATP. Serial dilutions of the ENT inhibitors dilazep dihydrochloride, dipyridamole and ticagrelor were prepared in X-Vivol5 medium from 100 mM stock solutions (in DMSO or water), and 25 μL was added to the wells. The A.sub.2AR antagonist Compound 8a (stock 10 mM in DMSO) was diluted in X-Vivol5 medium, and 25 μL was added to the wells to reach a final assay concentration of 300 nM. For studies including the different A2AR antagonists, wells received either the A2AR antagonist Compound 8a at a final concentration of 300 nM, NIR178 at a final concentration of 5 μM, CPI-444 at a final concentration of 5 μM, AZD4635 at a final concentration of 1 μM, or AB928 at a final concentration of 1 μM, or a matched concentration of DMSO. In addition, some wells received the ENT-1 inhibitor dipyridamole at a final concentration of 1 μM, or a combination of dipyridamole and A2AR antagonist. The final concentration of DMSO in the assay was 0.05%, and the final assay volume was 200 μL. Experiments were performed in biological duplicate, and DMSO control was added in quadruplicate. The cells were placed in a 37° C. humidified tissue culture incubator with 5% CO.sub.2 for 72 hours. After 72 hours, the cells were pelleted by centrifugation at 1600 rpm for 5 minutes, and supernatants were transferred to V-bottom 96-well plates for cytokine quantification. Cell pellets were resuspended in FACS buffer (see below) for flow cytometry analysis.

[0436] Cytokine quantification. Supernatants were centrifuged at 4000 rpm for 10 minutes, and IL-2 was quantified using the IL-2 (human) AlphaLISA Biotin-Free Detection Kit, and TNFα was quantified using the AlphaLISA Human TNFα Biotin-Free Detection Kit, according to the manufacturer's instructions.

[0437] Flow cytometry. Cells were washed with FACS buffer (PBS containing 2 mM EDTA and 0.1% BSA) and stained with surface marker antibodies and viability dye for 20 min on ice. Cells were washed twice with FACS buffer and data acquired using an LSRFortessa (BD biosciences). Analysis was performed using FlowJo software (Treestar).

Results

[0438] To evaluate effects of adenosine on human primary T cells, CD3+ T cells were stimulated with anti-CD3/CD28-coated microbeads, and treated with adenosine or ATP. Adenosine as well as ATP profoundly suppressed T cell proliferation and cytokine secretion (IL-2), and strongly reduced T cell viability (FIGS. 2-4).

[0439] Adenosine- and ATP-mediated suppression of T cell viability, proliferation and IL-2 secretion were not restored by Compound 8a, a potent and selective A2AR antagonist, suggesting an A2AR-independent mechanism of T cell suppression (FIGS. 2-4).

[0440] Extracellular adenosine is taken up by cells by Equilibrative Nucleoside Transporters (ENTs).

[0441] Primary T cells were treated with adenosine or ATP, and incubated with three different, structurally unrelated, ENT inhibitors: dipyridamole, dilazep hydrochloride and ticagrelor. All ENT inhibitors tested restored T cell proliferation and viability (FIGS. 2 and 3), thus illustrating the toxic effects of cellular uptake of adenosine by ENTs on T cell metabolism. However, none of the ENT inhibitors rescued T cell-derived IL-2 secretion as a single agent.

[0442] Complete rescue of adenosine- and ATP-mediated suppression of T cell cytokine secretion was achieved upon combined treatment with the A2A receptor antagonist Compound 8a and ENT inhibitors (FIGS. 4A and 4B). These results show that adenosine suppresses T cell function and vitality by both extracellular as well as intracellular mechanisms, which can be reversed by combined treatment with A2AR antagonists (not limited to Compound 8a—as shown further below) and ENT inhibitors.

[0443] FIG. 5A-C illustrates that the inhibition of T cells in the presence of ATP may be reversed by a combination of the ENT-1 inhibitor dipyridamole and the A2A receptor antagonists Compound 8a, AB928, CPI-444, NIR178 and AZD4635. More specifically, ATP significantly inhibited CD8+ T cell proliferation and reduced T cell viability which was completely reversed by the ENT-1 inhibitor dipyridamole. ATP also significantly inhibited the production of TNFα. Both the A2AR and the ENT-1 inhibitors individually could partially restore cytokine production. The combination of both inhibitors further increased the restoration of cytokine secretion compared to either treatment alone. Overall, these data illustrate the value of combining Compound 8a, AB928, AZD4635, NIR178 or CPI-444 with an ENT-1 inhibitor for cancer therapy, aiming at full restoration of T cell function and viability in the ATP/adenosine-rich tumor microenvironment.

Example 2. ENT1 Inhibitor in Combination with A2AR Antagonist Restores TNFalpha Production in an LPS Model of Endotoxemia

[0444] The most prominent target of adenosine transport is equilibrative nucleoside transporters, ENT1, 2, 3 and 4. ENT inhibitors regulate the import and export of nucleosides for example, adenosine across cell membranes such that inhibition of the equilibrative nucleoside transport may decrease the concentration of adenosine inside the cell and allow accumulation of adenosine extracellularly. High intracellular adenosine has been shown to lead to suppression of proliferation, survival and function of immune cells. On the other hand, extracellular adenosine by binding to the adenosine receptor 2A (A2AR) decreases activation and function, such as for example pro-inflammatory cytokine production and cytotoxicity of immune cells. Inhibition of equilibrative transporters therefore aims to decrease intracellular adenosine and thereby improving immune cell survival, proliferation and function, while A2AR inhibition aims to restore pro-inflammatory activity, cytotoxicity and immune cell activation by inhibiting extracellular adenosine.

[0445] NBMPR (6-S-[(4-Nitrophenyl)methyl]-6-thioinosine) is a specific ENT1 inhibitor and has similar activity in humans and mice. NBMPR is therefore used in mouse models to specifically inhibit the activity ENT1.

[0446] Using a Lipopolysaccharides (LPS) model, known to induce secretion of pro-inflammatory cytokine TNFalpha, it was assessed if TNFalpha-mediated suppression observed in presence of ENT1 inhibitor NBMPR, could be rescued by addition of A2AR antagonist, Compound 8b.

[0447] BALB/c mice were treated p.o. with Vehicle (2.5% DMSO, 10% Solutol HS15 in dH.sub.2O pH3) as control or Compound 8b at 3 mg/kg. After 30 minutes, mice were treated with NBMPR p.o. at 20 mg/kg. 60 minutes later, mice were treated with LPS and blood was drawn for TNFalpha measurement in the serum, by ELISA.

[0448] LPS treatment significantly elevated TNFalpha levels in the serum. A2AR antagonist, Compound 8b had no effect on TNFalpha levels after LPS treatment, while NBMPR treatment on its own, significantly suppressed TNFalpha production by almost 2 fold (p=0.008). Pretreatment of mice with Compound 8b, reversed the effects of NBMPR and rescued TNFalpha production by more than 2 fold (p=0.016) (FIG. 6).

Example 3. ENT1 Inhibitor in Combination with A2AR Antagonist Demonstrates Antitumor Efficacy in Mouse Syngeneic MCA205 Experimental Fibrosarcoma Model

[0449] The anti-tumor efficacy of A2AR antagonist, Compound 8b, was assessed in combination with ENT1 inhibitor, NBMPR, in an established murine syngeneic MCA205 fibrosarcoma tumor model.

[0450] MCA205 tumor cells were inoculated subcutaneously into the right flank of C57BL/6 mice. When tumors reached an average size of about 50 mm, mice were randomly allocated into groups. Mice were administered vehicle p.o. (2.5% DMSO, 10% Solutol HS15 in dH.sub.2O pH3) as control or NBMPR at 20 mg/kg p.o. BIDx22 (day7-29) as single agent or in combination with Compound 8b p.o. at 3 mg/kg QDx22 (day7-29).

[0451] NBMPR, given as a single agent at 20 mg/kg p.o. twice daily (BID) for 22 consecutive days, demonstrated a significant delay in tumor growth (p=0.0043) compared to the Vehicle (FIGS. 7A, 7B and 7C).

[0452] Compound 8b, administered p.o. at 3 mg/kg in combination with NBMPR at 20 mg/kg, both twice daily (BID) for 22 consecutive days, demonstrated significant tumor growth delay compared to Vehicle (p<0.0001) and also significant tumor growth delay when compared to NBMPR single agent therapy (p=0.0015) (FIGS. 7A-7D).