Combination therapy for the treatment or prevention of tumours

Abstract

The present invention relates to a combination therapy for tumours comprising the administration of an epoxytigliane compound and an immune checkpoint inhibitor. In particular embodiments, there is a method of treating a tumour and/or treating or preventing one or more bystander tumours with the therapy. Pharmaceutical compositions and kits containing epoxytigliane compounds and immune checkpoint inhibitors are also described.

Claims

1. A method of treating a tumour comprising administering to a subject in need thereof, an epoxytigliane compound or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor; wherein the epoxytigliane compound is administered locally to the tumour; wherein the epoxytigliane compound is a compound of formula (I): ##STR00007## or a geometric isomer or stereoisomer or a pharmaceutically acceptable salt thereof; wherein R.sub.1 is hydrogen or C.sub.1-6alkyl; R.sub.2 is —OH or —OR.sub.9; R.sub.3 is —OH or —OR.sub.9; R.sub.4 and R.sub.5 are independently selected from hydrogen and C.sub.1-6alkyl; R.sub.6 is hydrogen or —R.sub.10; R.sub.7 is hydroxy or OR.sub.10; R.sub.8 is hydrogen or C.sub.1-6alkyl; R.sub.9 is —C.sub.1-20alkyl, —C.sub.2-20alkenyl, —C.sub.2-20alkynyl, —C(O)C.sub.1-20alkyl, —C(O)C.sub.2-20alkenyl, —C(O)C.sub.2-20alkynyl, —C(O)cycloalkyl, —C(O)C.sub.1-10alkyl-cycloalkyl; —C(O)C.sub.2-10alkenylcycloalkyl, —C(O)C.sub.2-10alkynylcycloalkyl, —C(O)aryl, —C(O)C.sub.1-10alkylaryl, —C(O)C.sub.2-10alkenylaryl, —C(O)C.sub.2-10alkynylaryl, —C(O)C.sub.1-10alkylC(O)R.sub.11, —C(O)C.sub.2-10alkenylC(O)R.sub.11, —C(O)C.sub.2-10alkynylC(O)R.sub.11, —C(O)C.sub.1-10alkylCH(OR.sub.11)(OR.sub.11), —C(O)C.sub.2-10alkenyl-CH(OR.sub.11)(OR.sub.11), —C(O)C.sub.2-10alkynylCH(OR.sub.11)(OR.sub.11), —C(O)C.sub.1-10alkylSR.sub.11, —C(O)C.sub.2-10alkenylSR.sub.11, —C(O)C.sub.2-10alkynylSR.sub.11, —C(O)C.sub.1-10alkylC(O)OR.sub.11, —C(O)C.sub.2-10alkenylC(O)OR.sub.11, —C(O)C.sub.2-10alkynylC(O)OR.sub.11, —C(O)C.sub.1-10alkyl-C(O)SR.sub.11, —C(O)C.sub.2-10alkenylC(O)SR.sub.11, —C(O)C.sub.2-10alkynylC(O)SR.sub.11, ##STR00008## R.sub.10 is —C.sub.1-6alkyl, —C.sub.2-6alkenyl, —C.sub.2-6alkynyl, —C(O)C.sub.1-6alkyl, —C(O)C.sub.2-6alkenyl, —C(O)C.sub.2-6alkynyl, —C(O)aryl, —C(O)C.sub.1-6alkylaryl, —C(O)C.sub.2-6alkenylaryl, or —C(O)C.sub.2-6alkynylaryl; and R.sub.11 is hydrogen, —C.sub.1-10alkyl, —C.sub.2-10alkenyl, —C.sub.2-10alkynyl, cycloalkyl or aryl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl or aryl group is optionally substituted with one or more optional substituents selected from the group consisting of C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.3-6cycloalkyl, oxo (═O), —OH, —SH, C.sub.1-6alkylO-, C.sub.2-6alkenylO-, C.sub.3-6cycloalkylO-, C.sub.1-6alkylS-, C.sub.2-6alkenylS-, C.sub.3-6cycloalkylS-, —CO.sub.2H, —CO.sub.2C.sub.1-6alkyl, —NH.sub.2, —NH(C.sub.1-6alkyl), —N(C.sub.1-6alkyl).sub.2, —NH(phenyl), —N(phenyl).sub.2, —CN, —NO.sub.2, -halogen, —CF.sub.3, —OCF.sub.3, —SCF.sub.3, —CHF.sub.2, —OCHF.sub.2, —SCHF.sub.2, -phenyl, —C.sub.1-6alkylphenyl, —Ophenyl, —C(O)phenyl, and —C(O)C.sub.1-6alkyl; wherein the immune checkpoint inhibitor is selected from an antagonist of Programmed Death 1 (PD-1) receptor or its ligand PD-L1, and an antagonist of Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4); and wherein the tumour is one that has acquired or has intrinsic resistance to monotherapy with the immune checkpoint inhibitor.

2. A method of treating one or more bystander tumours in a subject comprising administering to a subject in need thereof, an epoxytigliane compound or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor; wherein the epoxytigliane compound is administered locally to a tumour other than the one or more bystander tumours; wherein the epoxytigliane compound is a compound of formula (I): ##STR00009## or a geometric isomer or stereoisomer or a pharmaceutically acceptable salt thereof; wherein R.sub.1 is hydrogen or C.sub.1-6alkyl; R.sub.2 is —OH or —OR.sub.9; R.sub.3 is —OH or —OR.sub.9; R.sub.4 and R.sub.5 are independently selected from hydrogen and C.sub.1-6alkyl; R.sub.6 is hydrogen or —R.sub.10; R.sub.7 is hydroxy or OR.sub.10; R.sub.5 is hydrogen or C.sub.1-6alkyl; R.sub.9 is —C.sub.1-20alkyl, —C.sub.2-20alkenyl, —C.sub.2-20alkynyl, —C(O)C.sub.1-20alkyl, —C(O)C.sub.2-20alkenyl, —C(O)C.sub.2-20alkynyl, —C(O)cycloalkyl, —C(O)C.sub.1-10alkyl-cycloalkyl; —C(O)C.sub.2-10alkenylcycloalkyl, —C(O)C.sub.2-10alkynylcycloalkyl, —C(O)aryl, —C(O)C.sub.1-10alkylaryl, —C(O)C.sub.2-10alkenylaryl, —C(O)C.sub.2-10alkynylaryl, —C(O)C.sub.1-10alkylC(O)R.sub.11, —C(O)C.sub.2-10alkenylC(O)R.sub.11, —C(O)C.sub.2-10alkynylC(O)R.sub.11, —C(O)C.sub.1-10alkylCH(OR.sub.11)(OR.sub.11), —C(O)C.sub.2-10alkenyl-CH(OR.sub.11)(OR.sub.11), —C(O)C.sub.2-10alkynylCH(OR.sub.11)(OR.sub.11), —C(O)C.sub.1-10alkylSR.sub.11, —C(O)C.sub.2-10alkenylSR.sub.11, —C(O)C.sub.2-10alkynylSR.sub.11, —C(O)C.sub.1-10alkylC(O)OR.sub.11, —C(O)C.sub.2-10alkenylC(O)OR.sub.11, —C(O)C.sub.2-10alkynylC(O)OR.sub.11, —C(O)C.sub.1-10alkyl-C(O)SR.sub.11, —C(O)C.sub.2-10alkenylC(O)SR.sub.11, —C(O)C.sub.2-10alkynylC(O)SR.sub.11, ##STR00010## R.sub.10 is —C.sub.1-6alkyl, —C.sub.2-6alkenyl, —C.sub.2-6alkynyl, —C(O)C.sub.1-6alkyl, —C(O)C.sub.2-6alkenyl, —C(O)C.sub.2-6alkynyl, —C(O)aryl, —C(O)C.sub.1-6alkylaryl, —C(O)C.sub.2-6alkenylaryl, or —C(O)C.sub.2-6alkynylaryl; and R.sub.11 is hydrogen, —C.sub.1-10alkyl, —C.sub.2-10alkenyl, —C.sub.2-10alkynyl, cycloalkyl or aryl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl or aryl group is optionally substituted with one or more optional substituents selected from the group consisting of C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.3-6cycloalkyl, oxo (═O), —OH, —SH, C.sub.1-6alkylO-, C.sub.2-6alkenylO-, C.sub.3-6cycloalkylO-, C.sub.1-6alkylS-, C.sub.2-6alkenylS-, C.sub.3-6cycloalkylS-, —CO.sub.2H, —CO.sub.2C.sub.1-6alkyl, —NH.sub.2, —NH(C.sub.1-6alkyl), —N(C.sub.1-6alkyl).sub.2, —NH(phenyl), —N(phenyl).sub.2, —CN, —NO.sub.2, -halogen, —CF.sub.3, —OCF.sub.3, —SCF.sub.3, —CHF.sub.2, —OCHF.sub.2, —SCHF.sub.2, -phenyl, —C.sub.1-6alkylphenyl, —Ophenyl, —C(O)phenyl, and —C(O)C.sub.1-6alkyl; wherein the immune checkpoint inhibitor is selected from an antagonist of Programmed Death 1 (PD-1) receptor or its ligand PD-L1, or an antagonist of Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4), and wherein the tumour to which the epoxytigliane is administered is one that has acquired or has intrinsic resistance to monotherapy with the immune checkpoint inhibitor.

3. The method according to claim 1 wherein the epoxytigliane compound is administered by intra-tumoural injection.

4. The method according to claim 1 wherein the immune checkpoint inhibitor is administered systemically.

5. The method according to claim 4 wherein the immune checkpoint inhibitor is administered by parenteral injection.

6. The method according to claim 1 wherein R.sub.1 is —CH.sub.3.

7. The method according to claim 1 wherein R.sub.2 and R.sub.3 are independently selected from —OC(O)C.sub.1-20alkyl, —OC(O)C.sub.2-20alkenyl, —OC(O)C.sub.2-20alkynyl, —OC(O)cycloalkyl, —OC(O)C.sub.1-10alkylcycloalkyl; —OC(O)C.sub.2-10alkenylcycloalkyl, —OC(O)C.sub.2-10alkynylcycloalkyl, —OC(O)aryl, —OC(O)C.sub.1-10alkylaryl, —OC(O)C.sub.2-10alkenylaryl, —OC(O)C.sub.2-10alkynylaryl, —OC(O)C.sub.1-10alkylC(O)R.sub.11, —OC(O)C.sub.2-10alkenylC(O)R.sub.11, —OC(O)C.sub.2-10alkynylC(O)R.sub.11, —OC(O)C.sub.1-10alkylCH(OR.sub.11)(OR.sub.11), —OC(O)C.sub.2-10alkenylCH(OR.sub.11)(OR.sub.11), —OC(O)C.sub.2-10alkynylCH(OR.sub.11)(OR.sub.11), —OC(O)C.sub.1-10alkylSR.sub.11, —OC(O)C.sub.2-10alkenylSR.sub.11, —OC(O)C.sub.2-10alkynylSR.sub.11, —OC(O)C.sub.1-10alkylC(O)OR.sub.11, —OC(O)C.sub.2-10alkenylC(O)OR.sub.11, —OC(O)C.sub.2-10alkynylC(O)OR.sub.11, —OC(O)C.sub.1-10alkylC(O)SR.sub.11, —OC(O)C.sub.2-10alkenylC(O)SR.sub.11 and —OC(O)C.sub.2-10alkynylC(O)SR.sub.11.

8. The method according to claim 1 wherein R.sub.4 and R.sub.5 are each independently selected from H and —CH.sub.3.

9. The method according to claim 1 wherein R.sub.6 is hydrogen, —C(O)C.sub.1-6alkyl, —C(O)C.sub.2-6alkenyl, —C(O)C.sub.2-6alkynyl or —C(O)aryl.

10. The method according to claim 1 wherein R.sub.7 is hydroxyl, —OC(O)C.sub.1-6alkyl, —OC(O)C.sub.2-6alkenyl or —OC(O)C.sub.2-6alkynyl.

11. The method according to claim 1 wherein R.sub.5 is H or —CH.sub.3.

12. The method according to claim 1 wherein the epoxytigliane compound of formula (I) is selected from the following compounds: 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; 12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; 12-hexanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; 12-myristoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13-pentahydroxy-20-acetyloxy-1-tiglien-3-one; 12-myristoyl-13-acetyloxy-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; 12-propanoyl-13-2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; 12,13-ditigloyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; and 12-(2-methylbutanoyl)-13-tigloyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; or a pharmaceutically acceptable salt thereof.

13. The method according to claim 1 wherein the immune checkpoint inhibitor is an antagonist of Programmed Death 1 (PD-1) receptor or Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4).

14. The method according to claim 1 wherein the immune checkpoint inhibitor is administered in multiple doses.

15. The method according to claim 14 wherein the multiple doses are administered prior to, simultaneously with and/or subsequent to the administration of the epoxytigliane compound.

16. A pharmaceutical composition comprising an epoxytigliane compound or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor and optionally a pharmaceutically acceptable carrier; wherein the epoxytigliane compound is a compound of formula (I): ##STR00011## or a geometric isomer or stereoisomer or a pharmaceutically acceptable salt thereof, wherein R.sub.1 is hydrogen or C.sub.1-6alkyl, R.sub.2 is —OH or —OR.sub.9; R.sub.3 is —OH or —OR.sub.9; R.sub.4 and R.sub.5 are independently selected from hydrogen and C.sub.1-6alkyl, R.sub.6 is hydrogen or -R.sub.10; R.sub.7 is hydroxy or OR.sub.10; R.sub.8 is hydrogen or C.sub.1-6alkyl; R.sub.9 is —C.sub.1-20alkyl, —C.sub.2-20alkenyl, —C.sub.2-20alkynyl, —C(O)C.sub.1-20alkyl, —C(O)C.sub.2-20alkenyl, —C(O)C.sub.2-20alkynyl, —C(O)cycloalkyl, —C(O)C.sub.1-10alkyl-cycloalkyl, —C(O)C.sub.2-10alkenylcycloalkyl, —C(O)C.sub.2-10alkynylcycloalkyl, —C(O)aryl, —C(O)C.sub.1-10alkylaryl, —C(O)C.sub.2-10alkenylaryl, —C(O)C.sub.2-10alkynylaryl, —C(O)C.sub.1-10alkylC(O)R.sub.11, —C(O)C.sub.2-10alkenylC(O)R.sub.11, —C(O)C.sub.2-10alkynylC(O)R.sub.11, —C(O)C.sub.1-10alkylCH(OR.sub.11)(OR.sub.11), —C(O)C.sub.2-10alkenylCH(OR.sub.11)(OR.sub.11), —C(O)C.sub.2-10alkynylCH(OR.sub.11)(OR.sub.11), —C(O)C.sub.1-10alkylSR.sub.11, —C(O)C.sub.2-10alkenylSR.sub.11, —C(O)C.sub.2-10alkynylSR.sub.11, —C(O)C.sub.1-10alkylC(O)OR.sub.11, —C(O)C.sub.2-10alkenylC(O)OR.sub.11, —C(O)C.sub.2-10alkynylC(O)OR.sub.11, —C(O)C.sub.1-10alkyl-C(O)SR.sub.11, —C(O)C.sub.2-10alkenylC(O)SR.sub.11, —C(O)C.sub.2-10alkynylC(O) SR.sub.11, ##STR00012## R.sub.10 is —C.sub.1-6alkyl, —C.sub.2-6alkenyl, —C.sub.2-6alkynyl, —C(O)C.sup.1-6alkyl, —C(O)C.sub.2-6alkenyl, —C(O)C.sub.2-6alkynyl, —C(O)aryl, —C(O)C.sub.1-6alkylaryl, —C(O)C.sub.2-6alkenylaryl, or —C(O)C.sub.2-6alkynylaryl, and R.sub.11 is hydrogen, —C.sub.1-10alkyl, cycloalkyl or aryl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl or aryl group is optionally substituted with one or more optional substituents selected from the group consisting of C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.3-6cycloalkyl, oxo (═O), —OH, —SH, C.sub.1-6alkylO-, C.sub.2-6alkenylO-, C.sub.3-6cycloalkylO-, C.sub.1-6alkylS-, C.sub.2-6alkenylS-, C.sub.3-6cycloalkylS-, —CO.sub.2H, —CO.sub.2C.sub.1-6alkyl, —NH.sub.2, —NH(C.sub.1-6alkyl), —N(C.sub.1-6alkyl).sub.2, —NH(phenyl), —N(phenyl).sub.2, —CN, —NO.sub.2, -halogen, —CF.sub.3, —OCF.sub.3, —SCF.sub.3, —CHF.sub.2, —OCHF.sub.2, —SCHF.sub.2—, -phenyl, —C.sub.1-6alkylphenyl, —Ophenyl, —C(O)phenyl, and —C(O)C.sub.1-6alkyl; and wherein the immune checkpoint inhibitor is selected from an antagonist of Programmed Death 1 (PD-1) receptor or its ligand PD-L1, or an antagonist of Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4).

17. The kit comprising one or more doses of epoxytigliane compound and one or more doses of immune checkpoint inhibitor; wherein the epoxytigliane compound is a compound of formula (I): ##STR00013## or a geometric isomer or stereoisomer or a pharmaceutically acceptable salt thereof; wherein R.sub.1 is hydrogen or C.sub.1-6alkyl; R.sub.2 is —OH or —OR.sub.9; R.sub.3 is —OH or —OR.sub.9; R.sub.4 and R.sub.5 are independently selected from hydrogen and C.sub.1-6alkyl; R.sub.6 is hydrogen or -R.sub.10; R.sub.7 is hydroxy or OR.sub.10; R.sub.5 is hydrogen or C.sub.1-6alkyl; R.sub.9 is —C.sub.1-20alkyl, —C.sub.2-20alkenyl, —C.sub.2-20alkynyl, —C(O)C.sub.1-20alkyl, —C(O)C.sub.2-20alkenyl, —C(O)C.sub.2-20alkynyl, —C(O)cycloalkyl, —C(O)C.sub.1-10alkylcycloalkyl; —C(O)C.sub.2-10alkenyl-cycloalkyl, —C(O)C.sub.2-10alkynylcycloalkyl, —C(O)aryl, —C(O)C.sub.1-10alkylaryl, —C(O)C.sub.2-10alkenylaryl, —C(O)C.sub.2-10alkynylaryl, —C(O)C.sub.1-10alkylC(O)R.sub.11, —C(O)C.sub.2-10alkenylC(O)R.sub.11, —C(O)C.sub.2-10alkynylC(O)R.sub.11, —C(O)C.sub.1-10alkylCH(OR.sub.11)(OR.sub.11), —C(O)C.sub.2-10alkenylCH(OR.sub.11)(OR.sub.11), —C(O)C.sub.2-10alkynylCH(OR.sub.11)(OR.sub.11), —C(O)C.sub.1-10alkylSR.sub.11, —C(O)C.sub.2-10alkenylSR.sub.11, —C(O)C.sub.2-10alkynylSR.sub.11, —C(O)C.sub.1-10alkylC(O)OR.sub.11, —C(O)C.sub.2-10alkenylC(O)OR.sub.11, —C(O)C.sub.2-10alkynylC(O)OR.sub.11, —C(O)C.sub.1-10alkyl-C(O) SR.sub.11, —C(O)C.sub.2-10alkenylC(O) SR.sub.11, —C(O)C.sub.2-10alkynylC(O)SR.sub.11, ##STR00014## R.sub.10 is —C.sub.1-6alkyl, —C.sub.2-6alkenyl, —C.sub.2-6alkynyl, —C(O)C.sub.1-6alkyl, —C(O)C.sub.2-6alkenyl, —C(O)C.sub.2-6alkynyl, —C(O)aryl, —C(O)C.sub.1-6alkylaryl, —C(O)C.sub.2-6alkenylaryl, or —C(O)C.sub.2-6alkynylaryl; and R.sub.11 is hydrogen, —C.sub.1-10alkyl, —C.sub.2-10alkenyl, —C.sub.2-10alkynyl, cycloalkyl or aryl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl or aryl group is optionally substituted with one or more optional substituents selected from the group consisting of C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.3-6cycloalkyl, oxo (═O), —OH, —SH, C.sub.1-6alkylO-, C.sub.2-6alkenylO-, C.sub.3-6cycloalkylO-, C.sub.1-6alkylS-, C.sub.2-6alkenylS-, C.sub.3-6cycloalkylS-, —CO.sub.2H, —CO.sub.2C.sub.1-6alkyl, —NH.sub.2, —NH(C.sub.1-6alkyl), —N(C.sub.1-6alkyl), —NH(phenyl), —N(phenyl).sub.2, —CN, —NO, -halogen, —CF, —OCHF.sub.2, —SCHF.sub.2, -phenyl, —C.sub.1-6alkylphenyl, —Ophenyl, —C(O)phenyl, and —C(O)C.sub.1-6alkyl; and wherein the immune checkpoint inhibitor is selected from an antagonist of Programmed Death 1 (PD-1) receptor or its ligand PD-L1, or an antagonist of Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4).

18. The kit according to claim 17 comprising one or more doses of epoxytigliane compound formulated for topical administration and one or more doses of immune checkpoint inhibitor formulated for administration by injection.

19. The kit according to claim 17 comprising one dose of epoxytigliane compound formulated for intra-tumoural injection and one or more doses of immune checkpoint inhibitor formulated for administration by injection.

20. The method according to claim 2 wherein the epoxytigliane compound is administered by intra-tumoural injection.

21. The method according to claim 2 wherein the immune checkpoint inhibitor is administered systemically.

22. The method according to claim 21 wherein the immune checkpoint inhibitor is administered by parenteral injection.

23. The method according to claim 2 wherein one or more of the following applies: i) R.sub.1 is —CH.sub.3; ii) R.sub.2 and R.sub.3 are independently selected from —OC(O)C.sub.1-20alkyl, —OC(O)C.sub.2-20alkenyl, —OC(O)C.sub.2-20alkynyl, —OC(O)cycloalkyl, —OC(O)C.sub.1-10alkylcycloalkyl; —OC(O)C.sub.2-10 alkenylcycloalkyl, —OC(O)C.sub.2-10alkynylcycloalkyl, —OC(O)aryl, —OC(O)C.sub.1-10alkylaryl, —OC(O)C.sub.2-10alkenylaryl, —OC(O)C.sub.2-10alkynylaryl, —OC(O)C.sub.1-10alkylC(O)R.sub.11, —OC(O)C.sub.2-10alkenylC(O)R.sub.11, —OC(O)C.sub.2-10alkynylC(O)R.sub.11, —OC(O)C.sub.1-10alkylCH(OR.sub.11)(OR.sub.11), —OC(O)C.sub.2-10alkenylCH(OR.sub.11) (OR.sub.11), —OC(O)C.sub.2-10alkynylCH(OR.sub.11)(OR.sub.11), —OC(O)C.sub.1-10alkylSR.sub.11, —OC(O)C.sub.2-10alkenylSR.sub.11, —OC(O)C.sub.2-10alkynylSR.sub.11, —OC(O)C.sub.1-10alkylC(O)OR.sub.11, —OC(O)C.sub.2-10alkenylC(O)OR.sub.11, —OC(O) C.sub.2-10alkynylC(O)OR.sub.11, —OC(O)C.sub.1-10alkylC(O)SR.sub.11, —OC(O)C.sub.2-10alkenylC(O)SR.sub.11 and —OC(O)C.sub.2-10alkynylC(O)SR.sub.11; iii) R.sub.4 and R.sub.5 are each independently selected from H and —CH.sub.3; iv) R.sub.6 is hydrogen, —C(O)C.sub.1-6alkyl, —C(O)C.sub.2-6alkenyl, —C(O)C.sub.2-6alkynyl or —C(O)aryl; v) R.sub.7 is hydroxyl, —OC(O)C.sub.1-6alkyl, —OC(O)C.sub.2-6alkenyl or —OC(O)C.sub.2-6alkynyl; and vi) R.sub.5 is H or —CH.sub.3.

24. The method according to claim 2 wherein the epoxytigliane compound of formula (I) is selected from the following compounds: 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; 12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; 12-hexanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; 12-myristoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13-pentahydroxy-20-acetyloxy-1-tiglien-3-one; 12-myristoyl-13-acetyloxy-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; 12-propanoyl-13-2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; 12,13-ditigloyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; and 12-(2-methylbutanoyl)-13-tigloyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; or a pharmaceutically acceptable salt thereof.

25. The method according to claim 2 wherein the immune checkpoint inhibitor is an antagonist of Programmed Death 1 (PD-1) receptor or Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4).

26. The method according to claim 25 wherein the immune checkpoint inhibitor is selected from an anti-PD-1 antibody and an anti-CTLA4 antibody.

27. The method according to claim 2 wherein the immune checkpoint inhibitor is administered in multiple doses.

28. The method according to claim 27 wherein the multiple doses are administered prior to, simultaneously with and/or subsequent to the administration of the epoxytigliane compound.

29. The method according to claim 13 wherein the immune checkpoint inhibitor is selected from an anti-PD-1 antibody and an anti-CTLA4 antibody.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1A provides: images of PKC-α, -βII, -γ, -δ and -θ translocation after treatment with vehicle (Vehc.) or 500 nM Compound 1 (Comp 1), Compound 3 (Comp 3) and PMA. These images were also used for the quantitation shown in FIG. 1B.

(2) FIG. 1B: Heatmap depicting PKC isoform translocation profile (-α, -βI, βII, -γ, -δ, -θ, -η and mean -ζ—of cells showing EGFP translocation to the plasma membrane) in response to 500, 50 and 5 nM Compound 1 and the indicated analogues. >150 cells counted per biological replicate. n=3.

(3) FIG. 2A provides: Schematic of the experimental design.

(4) FIG. 2B: Select host cytokines/chemokines with roles in leukocyte recruitment are induced by Compound 1 treatment (30 μg). Fold changes in gene expression of the indicated cytokines/chemokines are shown.

(5) FIG. 2C: A heat map was generated after comparison of intensity data from both Vehicle and Compound 1 gene expression profiles at t=8 h. Subsequent gene lists were analysed by Ingenuity Pathway Analysis (IPA; Qiagen) to identify pathways affected by Compound 1 treatment.

(6) FIG. 3A provides: LDH release from cancer cell lines treated with Compound 1. A431, MM649 and B16-OVA were treated with Compound 1 at the indicated concentrations or vehicle only (Vehc.) and LDH released assayed over time using a Pierce LDH cytotoxicity assay kit. n=3.

(7) FIG. 3B: Compound 1 induces significant reduction of intracellular ATP levels. Again, cells were treated with Compound 1 or Vehicle (Vehc.) and intracellular ATP levels were assayed at the indicated timepoints using a CellTiter-Glo 2® assay kit. n=3.

(8) FIG. 4A provides: Determination of ATP release from cancer cell lines in response to treatment with Compound 1. Cells were treated with Compound 1 or Vehicle (Vehc.) for the indicated times and ATP release from cell culture supernatants was assayed using a bioluminecsence based ATP assay kit. Mean RLU values+/−S.D. were determined and plotted vs. time. n=4.

(9) FIG. 4B: Additional epoxytigliane analogues (Compound 2: Bi, Compound 3: Bii, and Compound 4: Biii) also promote ATP release from A431, MM649 and B16-OVA cell lines. n=4.

(10) FIG. 4C: Determination of HMGB1 release from cancer cell lines treated with the Compound 1 and epoxytigliane analogues. Cell culture supernatants from epoxytigliane or vehicle (Vehc.) treated A431 (Ci), MM649 (Cii) or B16-OVA (Ciii) were analysed for HMGB1 release via ELISA. Values from Compound 1 treated cells were normalised to vehicle treated cells to determine fold increase in HMGB1 release. n=2 for A431 and MM649, whilst n=3 for B16-OVA. (Civ) HMGB1 release also occurred in response to treatment with Compound 2, 3 and 4. n=3.

(11) FIG. 4D: Compound 1 promotes calreticulin externalisation in a range of cancer cell lines. A431 (Di), MM649 (Dii) and B16-OVA (Diii) were treated with Compound 1 or Vehicle (Vehc.) only for the indicated times and then stained with anti-Calreticulin/anti-rabbit Alexa 488 prior to flow cytometry. Mean fluorescence intensity values (Ex:488 nm, Em: 530/530 nm)+/−S.D. are indicated for each timepoint.

(12) FIGS. 5A and 5B provide Epoxytigliane treated cells are processed by CD11c.sup.+ BMDCs in vitro. CMFDA labelled B16-OVA cells treated with Compound 1 or vehicle (Vehc.) were incubated with immature BMDCs for 4 h and then stained with anti-CD11c-APC prior to flow cytometry. Solid grey box—CD11c.sup.+ BMDC cells. Dashed black box—CD11c.sup.+ BMDCs that have taken up dying B16-OVA cell fragments (CD11c.sup.+ CMFDA.sub.mid). The percentage of CD11c.sup.+ CMFDA.sub.mid cells is indicated on FIG. 5A and data from 4 biological replicates is shown in FIG. 5B. n=4.

(13) FIG. 5C provides Epoxytigliane analogues also induce uptake of B16-OVA cells by CD11c.sup.+ BMDCs. Data acquired from the use of Compound 2, 3 and 4 are depicted. n=3.

(14) FIG. 6A provides: Schematic of the experimental approach to Compound 1 and ICI combinations. All tumours were injected with either 15/30 μg Compound 1 or Vehicle (Vehc.). Dosage regime of ICI treatment is also depicted.

(15) FIGS. 6B and 6C: Kaplan-Meier plots indicating the % of tumours treated that remain below 100 mm.sup.3 in size under the distinct treatment conditions with ICIs, with either anti-PD-1 (FIG. 6B) or anti-CTLA-4 (FIG. 6C). Mouse survival in response to the combination is also depicted.

(16) FIG. 7A provides: A schematic diagram of the combination therapy approach: index and bystander tumour are shown together with dosage regime; and

(17) FIG. 7B: Graphical representations of the combination therapy results (tumour volumes). Isotype antibody+Compound 1 (dot line), Anti-PD-1 antibody or Anti-CTLA-4 antibody+vehicle (dash line) and Anti-PD-1 antibody or Anti-CTLA-4 antibody+Compound 1 (solid line) in treated (white circles) and bystander (black circles) tumours.

(18) FIG. 8 provides both volume (left panel) and Kaplan-Meier (right panel) based analyses of tumours (% tumours <100 mm.sup.3) injected with a sub-optimal dose of Compound 1 (15 μg) in wt and GCSF knockout backgrounds.

EXAMPLES

(19) The compounds of the present invention may be obtained by isolation from a plant or plant part, or by derivatisation of the isolated compound, or by derivatisation of a related compound. Isolation procedures and derivatisation procedures may be found in WO 2007/070985 and WO2014/169356.

(20) Compound 6, the 20-acetyl derivative of compound 1 may be produced from Compound 1 by acetylation with acetic anhydride (1 equiv) in the presence of triethylamine in dichloromethane. These conditions allow selective acetylation of the C-20 hydroxy group without acetylation of the secondary hydroxy groups.

(21) Compound 10, although not specifically synthesized in WO2014/169356, may be prepared using the general method for obtaining unsymmetrical esters set out in Example 1 of WO2014/169356, pages 64 to 70.

Example 1: Epoxytigliane Analogues Activate PKC Isoforms

(22) Protein kinase C are a family of key enzymes involved in signalling pathways that specifically phosphorylate substrates at serine/threonine residues. Phosphorylation by PKC is important in regulation a variety of cellular events such as proliferation and regulation of gene expression. PKC isoforms (-θ, -η, -α, -β, -δ, -ε) are directly implicated in immune cell responses and can also promote the expression of key immune genes. However, the expression pattern and levels of each of these PKC isoforms are cell-type and context specific (Lim et al. 2015; Anel et al. 2012; Pfeifhofer et al. 2006).

(23) To identify specific PKC isoform activation profiles (specificity and potency) of epoxytigliane compounds, HeLa cells were transiently transfected with a selection of PKC-EGFP vectors (PKC-α, PKC-βI, PKC-βII, PKC-γ, PKC-δ, PK-Cθ, PKC-η, PKC-ζ—generated in house) using Lipofectamine 2000 (Invitrogen) following methods described in Boyle et al. 2014. A volume corresponding to 0.16 μg PKC-EGFP and 0.48 μL Lipofectamine was mixed with 25 μl Opti-MEM medium (Invitrogen) and incubated for 5 min at RT. The solutions were combined and incubated for another 20 min at RT (1:3 ratio of DNA:Lipofectamine 2000). The complexes (50 μl) were added to each well and after 3 h incubation at 37° C., another 50 μl RPMI-1640, 10% FCS was added to a total volume of 100 μl per well. After 24 h incubation at 37° C., cells were washed with phosphate-buffered saline (PBS) and treated with 500, 50 and 5 nM of epoxytigliane. Three compounds could be tested per 96-well plate. Five 96-well plates were required per experimental run. After 1 h treatment, the cells were washed twice with 100 μl PBS and fixed with 50 μl of 2% formaldehyde/0.2% gluteraldehyde in PBS for 10 min. The fixed cells were subsequently washed twice with 100 μl PBS. To stain nuclei, Hoechst 3342 was used at a 1:10000 dilution. After 7 min of incubation in the dark, Hoechst was removed and cells were washed with PBS. Finally the cells were overlaid with 100 μl PBS and stored at 4° C. in the dark until imaging. Imaging was performed using a GE InCell Analyzer 2000. The translocation of PKC to plasma membrane or other subcellular positions was counted manually using Adobe Photoshop CS6.

(24) The images showing translocation of selected PKC isoforms-α, -βII, -γ, -δ and -θ to the cell membrane in the presence of the epoxytigliane compounds Compound 1, Compound 3 as well as PMA (phorbol-12-myristate-13-acetate) are shown in FIG. 1A. These images indicate that different epoxytigliane compounds can activate different PKC isoforms.

(25) The percentage of cells showing plasma membrane translocation of Compounds 1 to 10 as well as positive control PMA were converted to a Heatmap depicting the PKC isoform translocation profile in response to 500, 50 and 5 nM of each compound. The Heatmap is shown in FIG. 1B.

(26) The results show that Compounds 1 to 7 all activate PKC-θ (to differing extents), a PKC isoform known to be involved in T- and NK-cell activation and the suppression of Treg development (Brezar et al., 2015; Anel et al., 2012). All epoxy-tigliane compounds activate PKC-β, which is critical in B cell receptor signalling and antigen presentation (e.g. Kang et al. 2001; Lim et al. 2015). Three other PKC isoforms (-η, -α, -δ) which were more weakly activated by some or all of the epoxytiglianes have also been directly implicated in immune cell responses (Lim et al. 2015; Pfeifhofer et al. 2006)

Example 2: Gene Expression Changes in Mouse Tumour Stroma Consistent with Immune Cell Recruitment and the Induction of a Th-1/M1-Like Anti-Tumour Immune Response

(27) The Th1/M-1 Like Response in Anti-Tumour Immunity.

(28) A Th1/M1-like immune response has been associated with induction of anti-tumour cellular immunity through a range of mechanisms including direct tumouricidal activity, modification of anti-tumour cytokine responses and potentiation of long-term immunologic memory. For example, several lines of evidence show that CD4.sup.+ T helper type 1 (Th1) cells, the drivers of Th1 immunity, can help support the clearance of tumour cells via the secretion of various cytokines, including interferon-γ (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α) (Knutson et al. 2005; DeNardo et al. 2010; Burkholder et al. 2014). These cytokines promote the activities of several cell types, including antigen presenting cells (APCs), cytotoxic T cells, NK cells, and various innate immune cell subtypes (e.g. Cohen et al. 2000; Bos & Sherman 2010). IFN-γ and TNF are also known to have direct effects on tumour cell survival (Sugarman et al. 1985; Bayaert et al. 1994). Although IL-1 and IL-6 (produced by M1 macrophages) have been associated with tumour development, more recent evidence suggests that they are in fact crucial components of acute anti-tumour immune responses (Haabeth et al. 2011; Gabrilovich et al. 2012; Haabeth et al. 2016). They have been shown to enhance B cell proliferation and antibody production, increase the activity of antigen-presentation cells (APC), stimulate the proliferation of antigen specific cytotoxic cell types and promote Th1 cell differentiation (Haabeth et al. 2011; Burkholder et al. 2014). Combinations between Th1 and M1 cytokines have also been shown to be important in tumour immunosurveillance. For example, both IL-1 and IFN-γ synergise to activate the tumouricidal activity of macrophages (Hori et al. 1989; Haabeth et al. 2016). Importantly, the occurrence of M1 macrophages and Th1 lymphocytes in tumours has been positively associated with improved prognosis and survival times in many cancers (Pages et al. 2010; Fridman et al. 2012; Senovilla et al. 2012). Indeed, inducing Th1/M1-type inflammation has been proposed to significantly improve anti-cancer immunotherapy based approaches (Haabeth et al. 2012). Below the effect of Compound 1 in promoting a Th1/M1 like anti-tumour immune response in the stroma of a xenograft mouse model is described.

(29) Mouse Stroma in Human Tumour Xenografts from Mice.

(30) The SK-MEL-28 human melanoma cell line was injected subcutaneously (s.c) into 2 sites on the flanks of each BALB/c Foxn1nu mouse (2 million cells/site) and allowed to grow to approximately 7 mm diameter. Each tumour was then injected with 50 μl of 20% propylene glycol containing 30 μg Compound 1 or with 50 μl of 20% propylene glycol. At different times after injection a mouse was euthanased and the tumours harvested, the skin covering removed, and the intact tumours stored at −80° C.

(31) RNA Extraction.

(32) RNA was extracted from 30 mg of frozen tumour using the Qiagen RNeasy Plus Mini Kit, according to manufacturer's instructions, then quantitated with a NanoDrop instrument and integrity confirmed on denaturing agarose gels bearing a 1 kb DNA marker and stained with ethidium bromide.

(33) RNA Amplification and Labelling.

(34) Approximately 500 ng of total unlabelled RNA was adjusted to a final volume of 11 μl with nuclease-free water. The RNA was incubated with 9 μl of the reverse transcriptase master mix (1 μl of T7 Oligo (dT) Primer, 2 μl of 10× first strand buffer, 4 μl of dNTP mix, 1 μl of RNase inhibitor and 1 μl of ArrayScript) at 42° C. for 2 h. This was followed by the second strand cDNA synthesis step which involved a further incubation at 16° C. for 2 hr with 80 μl of the second strand master mix (63 μL nuclease-free water, 10 μl 10× second strand buffer, 4 μl dNTP mix, 2 μl DNA polymerase and 1 μl RNase H). The cDNA was purified by filtering through a cDNA Filter Cartridge with 250 μl of cDNA binding buffer and washing with 500 μl of the wash buffer provided in the kit. Purified cDNA was eluted with 20 μl of 55° C. nuclease-free water. Each cDNA sample was incubated with 7.5 μl of the IVT master mix (2.5 μl of T7 10× reaction buffer, 2.5 μl of T7 enzyme mix and 2.5 μl biotin-NTP mix) at 37° C. for 16 h. The reaction was stopped with the addition of 75 μl of nuclease-free water to each cRNA sample. The biotinylated, amplified RNA was purified by filtering the cRNA samples through cRNA Filter Cartridges with 350 μL of cRNA binding buffer and 250 μl of 100% ethanol mixed together prior to loading onto the filters. The cRNA filter cartridges with attached RNA were then washed with 650 μl of wash buffer before eluting purified cRNA with 200 μl of 55° C. nuclease-free water.

(35) Illumina Expression BeadChip Hybridization.

(36) The cRNA samples were heated at 65° C. for 5 min and collected by pulse centrifugation. After heating at 65° C. for 5 min, approximately 750 ng of the cRNA sample was aliquoted into separate tubes to which were added ˜5 μl of RNase-free water and 10 μl of Hyb Mix. Approximately 15 μl of the prepared cRNA mix was loaded onto the Illumina Expression BeadChips. Subsequent steps of hybridisation and washing were carried out according to the Whole-Genome Gene Expression Direct Hybridization Assay Guide supplied by Illumina. The HumanHT-12 v4 Expression BeadChips cover more than 47,000 transcripts and known splice variants across the human transcriptome. The MouseRef-8 v2.0 Expression BeadChips cover approximately 25,600 well-annotated RefSeq (Reference Sequence) transcripts, comprising over 19,000 unique genes.

(37) Data Analysis.

(38) BeadChips were read by the iScan System, and transferred via GenomeStudio into GeneSpring GX v12.5 (Agilent Technologies, Santa Clara, Calif., USA). The expression values were normalized using quantile normalization with default settings. The entities were filtered based on the detection score calculated by GenomeStudio where p≤0.05 was considered significant.

(39) The results are shown in FIG. 2. FIG. 2A shows that several host cytokines/chemokines which are important for the recruitment/activation of immune cells are upregulated at the tumour site in response to Compound 1 treatment. Of note, Cxcl1, which is heavily upregulated by Compound 1 is known to promote neutrophil recruitment and subsequent killing of residual cancer cells (Garg et al. 2017). Furthermore, FIG. 2B shows that Compound 1 induces gene expression changes in the host which are associated with the development of a Th-1/M-like response i.e. IFN-γ, TNF, IL-δ and IL-1β induction. The data also suggest that there may be a downregulation of TGF beta signalling (FIG. 2C), which is a known immunosuppressive signalling pathway (Neuzillet et al. 2015).

Example 3: Demonstration that Therapeutic Concentrations of Compound 1 and Other Epoxytiglianes Induce Cellular Oncosis

(40) Oncosis is a form of necrotic cell death, characterised by the swelling and rupture of subcellular organelles and subsequent permeabilisation of the plasma membrane, due in part to loss of ATP-driven ion pump activity that maintains osmotic balance. Oncosis has been shown to be immunogenic in nature and is associated with the efficacy of some oncolytic viruses and small molecules developed as anti-cancer agents (e.g. Dyer et al. 2016).

(41) Cell Lines, Reagents and Media.

(42) A431 (human epidermoid carcinoma), MM649 (human melanoma), B16-OVA (B16-F10 mouse melanoma cell line stably transfected with chicken ovalbumin), MM415 (human melanoma) and FaDu (human hypopharyngeal carcinoma) cells were cultured in RPMI-1640, 10% FCS (complete medium) at 37° C., 5% CO.sub.2 in a humidified incubator. All cell lines used in this study were confirmed as mycoplasma negative using MycoAlert (Lonza). STR profiling was also performed to confirm the identity of the human cell lines used.

(43) IncuCyte Cytotoxicity Assays.

(44) Four cancer cell lines (A431, MM649 (human melanoma), FaDu (HNSCC) and MM415 (human melanoma) were assessed for their response to epoxytigliane treatment. Cells were plated at a density of 10,000 cells per well (100 μl of complete media) into clear bottom black 96-well plates (Corning, #3603). Following 24 h incubation, the media within each well was aspirated and replaced with 50 μl of fresh media containing 1 μg/ml propidium iodide (PI). Stock solutions (20 mg/ml in ethanol) of four epoxytiglianes (Compounds 1, 2, 3 and 4) were diluted to 2× final assay concentration (1 mM) in identical media and inserted into a U-bottom 96 well plate in preparation for transfer. 50 μl of dilution was added to the required wells and the resultant plates inserted into an Essen Biosciences IncuCyte in preparation for imaging. Images were acquired at various time points (30 min, 1 h and at hourly intervals) for a total of 24 h.

(45) Lactate Dehydrogenase (LDH) Release Assays.

(46) Measuring the release of LDH is a well-recognised assay to assess plasma membrane permeabilisation and detect cell death by necrosis (Chan et al. 2013). Three cell lines (A431, MM649 and B16-OVA) were used in these assays. Cells were plated at a density of 10,000 cells per well into clear 96-well plates (Corning, #3595; 100 μl of complete media) and the resultant plates incubated overnight as previously detailed. The following day, media was aspirated from each well and 50 μl of fresh medium was inserted. Stock solutions of Compound 1 were diluted to 2× final assay concentrations (1 mM and 600 μM) and 50 μl of these dilutions added to the required wells. Ethanol only control solutions were also compiled and administered. At the indicated time points, 50 μl of media was removed and assayed for LDH release using a Pierce LDH cytotoxicity assay kit (ThermoFisher Scientific). OD490 nm and OD690 nm readings were recorded for each sample using a Hybrid Synergy H4 plate reader. Absorbance readings from drug treated samples were normalized to detergent treated controls to determine the % LDH release per well.

(47) Intracellular ATP Assay.

(48) CellTiter-Glo® 2.0 assay kits (Promega Corporation), a luminescence based assay quantitating the amount of ATP present in cells, were used to determine intracellular ATP levels in cultures of A431 and MM649 exposed to two concentrations (300 μM and 500 μM) of Compound 1. Again, cells were plated at a density of 10,000 cells per well into a clear bottomed black 96-well plates (in 100 μl of complete medium) and incubated at 37° C., 5% CO.sub.2 overnight. At the time of assay, media was aspirated from each well and 50 μl of fresh medium was inserted. Stock solutions of Compound 1 were diluted to 2× final assay concentrations (1 mM and 600 μM) and 50 μl of these dilutions added to the required wells. Ethanol only control solutions were also compiled and administered. At the indicated time points, media was gently removed making sure not to disturb the cells and 100 μl of CellTiter-Glo® 2.0 Reagent added. The resultant plate was mixed on an orbital shaker for 2 mins to induce cell lysis after which it was incubated in the dark for 10 mins. Following this, the luminescent signal in each well was determined. Luminescent signals from compound treated wells were normalised to Vehicle treated wells and expressed as % intracellular ATP vs. time.

(49) Images acquired from the IncuCyte showed strong red staining after 120 minutes in the majority of cells of all four cell lines treated with 500 μM of Compound 1, 2, 3 and 4 (A431, FaDu, MM649 and MM415). There was no staining of cells treated with the vehicle only, confirming loss of plasma membrane integrity was confined to cells treated with Compound 1. In addition, significant cytoplasmic swelling and ‘blistering’ was also observed in all cells treated with Compound 1, 2, 3 and 4, although to differing extents and with different kinetics of onset.

(50) Results from assays for assessing the release of LDH and for evaluating intracellular ATP levels are shown in FIGS. 3A and 3B, respectively. Compared to the controls, there was significant release of LDH from all three cancer cell lines treated at both concentrations of Compound 1 that were tested (300 μM and 500 μM) (FIG. 3A). Intracellular ATP levels declined very rapidly following treatment with 500 μM of Compound 1 and were almost undetectable after 60 minutes (FIG. 3B). At 300 μM of Compound 1, intracellular ATP declined more slowly, and less catastrophically, than for the 500 μM concentration of Compound 1, being approximately 50% of the pre-treatment level after 120 minutes (FIG. 3B). ATP depletion has been previously associated with the induction of oncosis (Kim et al. 2003).

(51) These results demonstrate that the epoxytigliane compounds induce oncosis at therapeutically relevant concentrations (i.e. those concentrations which are effective in inducing haemorrhagic necrosis in vivo).

Example 4: The Oncosis Induced by Compound 1 and Other Epoxytiglianes Displays Characteristics of Immunogenic Cell Death

(52) Immunogenic cell death (ICD) is a specific type of regulated cell death which results in the release or extemalisation of mediators called damage associated molecular patterns (DAMPs), which interact with receptors expressed by dendritic cells/macrophages to promote their recruitment and stimulate the uptake/presentation of tumour antigens to T cells. ICD is a prominent pathway for the activation of the immune system against cancer. Biochemical hallmarks of ICD include the exposure of calreticulin (CALR) and other endoplasmic reticulum/mitochondrial proteins on the surface of dying cells, and the release of large amounts of ATP and high-mobility group box 1 (HMGB1) into the extracellular environment (Kroemer et al. 2013). These parameters have been used to make accurate predictions about the capacity of chemotherapeutic drugs (including doxorubicin, mitoxantrone, oxaliplatin and bortezomib) to induce ICD (Kroemer et al. 2013; Galluzzi et al. 2017). The ability of the epoxytiglianes to induce these characteristics is detailed below.

(53) Cell Lines, Reagents and Media.

(54) A431 (human epidermoid carcinoma), MM649 (human melanoma) and B16-OVA (B16-F10 mouse melanoma cell line stably transfected with chicken ovalbumin) were cultured in RPMI-1640, 10% FCS (complete medium) at 37° C., 5% CO.sub.2 in a humidified incubator. BMDCs were cultured in R10 media (RPMI, 10% FCS, 2 mM glutamine, 50 μM beta-mercaptoethanol, Pen/Strep). All cell lines used in this study were confirmed as mycoplasma negative using MycoAlert (Lonza). STR profiling was also performed to confirm the identity of the human cell lines used.

(55) ATP Release Assays.

(56) Cell lines were plated at a density of 10,000 cells per well into clear 96-well plates (Corning, #3595; 100 μl of complete media) and the resultant plates incubated overnight as previously detailed. The following day, media was aspirated from each well and 50 μl of fresh medium was inserted. Stock solutions of Compounds 1, 2, 3 and 4 were diluted to 2× final assay concentration (1 mM and 600 μM) and 50 μl of these dilutions added to the required wells. Ethanol only control solutions were also compiled and administered. At the indicated timepoints, 80 μl of media was removed, centrifuged at 1,200 rpm for 4 mins to pellet cell debris and 50 μl assayed for ATP using a bioluminescence based ATP assay kit (FLAA, Sigma-Aldrich). Relative luminescence units were recorded for each sample using a Hybrid Synergy H4 plate reader (BioTek).

(57) HMGB1 Release Assays.

(58) Cell lines (A431, MM649 and B16-OVA) were plated into T75 cm.sup.2 flasks (Nunc) in 10 ml of complete media and cultured at 37° C., 5% CO.sub.2 until they reached 90% confluency. Compounds 1, 2, 3 and 4 were diluted in 5 ml of identical media to a final concentration of 500 and 300 μM and then administered to the cells. Several flasks were prepared such that a kinetic curve of HMGB1 release in response to drug treatment could be established. Ethanol only controls were also generated. At the required timepoint, media was removed from the flask into a 10 ml polypropylene tube that was placed on ice for 5 mins. Cell culture supernatants were centrifuged at 1,200 rpm for 4 mins to remove cellular debris, after which 4.5 ml of supernatant was inserted into a concentrator spin column (Amicon® Ultra 50 kDa cut-off membrane, Merck) to remove FCS. The flow through from this column was then inserted into another concentrator column (Amicon® Ultra 10 kDa cut-off membrane, Merck) and centrifuged at 3,500 rpm to concentrate HMGB1 prior to assay via ELISA (SEA399Hu/SEA399Mu, Cloud-Clone Corp). OD450 nm values were determined using a Hybrid Synergy H4 plate reader (BioTek). Absorbance values from drug treated samples were normalized to vehicle treated samples to determine the fold increase in HMGB1 release in response to epoxytigliane treatment.

(59) Calreticulin Externalization Assays.

(60) Calreticulin externalization was determined as previously detailed (Gomez-Cadena et al. 2016). Briefly, cells (A431, MM649, B16-OVA) cultured in complete medium at 37° C., 5% CO.sub.2 were detached via trypsinisation, centrifuged at 1,200 rpm and washed ×2 with fresh medium. After an additional round of centrifugation, the resultant cell pellet was resuspended at a concentration of 1×10.sup.6 cells/ml, after which epoxytigliane was added to a final concentration of 500 or 300 μM. Ethanol only controls were also performed. Cell suspensions were incubated at 37° C., 5% CO.sub.2 and at the indicated timepoints, 200 μl of sample was removed and incubated with LIVE/DEAD fixable far red stain for 5 mins on ice. Following this, cells were pelleted at 1,200 rpm for 4 mins and washed ×1 with PBS. 500 μl of PBS, 0.25% formaldehyde was then added to each pellet to perform a light fixation without compromising plasma membrane integrity. Following this, the cells were washed ×1 with PBS and 100 μl of PBS, 1% BSA, 2 mM EDTA (FACs buffer) containing anti-Calreticulin (ab2907, Abcam; 1:50 dilution) added. After 1 h incubation at room temperature, cells were centrifuged at 1,200 rpm for 4 mins and washed ×1 with FACs buffer prior to incubation with 100 μl of FACs buffer containing anti-rabbit Alexa488 at 1:750 for a further hour at room temperature. Cells were again pelleted and then resuspended in 500 μl of FACs buffer in preparation for flow cytometry.

(61) Samples were gated by FSC-H v FSC-A first to identify single cells, after which the LIVE/DEAD stain (Ex: 640 nm, Em: 670/14) negative (intact cell) population was analysed for green fluorescence (Ex: 488 nm, Em: 530/30; calreticulin externalization). Mean fluorescence intensity values were subsequently determined and graphed versus time.

(62) The results showed that the rapid oncosis induced by epoxytigliane compounds also induced characteristics of immunogenic cell death, with the release/externalisation of critical DAMPs including ATP (FIGS. 4A and 4B), HMBG1 (FIG. 4C) and calreticulin (FIG. 4D). There was significant release of ATP within 60 minutes in all three cancer cell lines treated with both concentrations of Compound 1 (FIG. 4A) and of Compounds 2, 3 and 4 (FIG. 4B). These compounds also promoted release of HMGB1 from cancer cell lines. After ELISA assay values for treated cells were normalised to vehicle treated cells to determine fold increase, Compound 1 was shown to increase HMBG1 release within 120 minutes by up to 40% for A341 and MM649 cells (FIGS. 4Ci and 4Cii), and by greater than 3-fold for B16-OVA cells (FIG. 4Ciii). Compounds 2, 3 and 4 which were also assayed on B16-OVA cells and showed a minimum of 2-fold increase in HMBGlrelease from this cell line after 120 minutes (FIG. 4Civ). Data for calreticulin externalisation following treatment of the 3 cell-lines with Compound 1 showed significant increases in mean fluorescence intensity within 60 minutes of treatment (FIG. 4D). Such DAMP release/externalisation is known to promote the recruitment of antigen presenting cells, stimulate the efficient uptake of cancer cell associated antigens and stimulate presentation to T cell subsets (Kolaczkowska & Kubes, 2013)

Example 5: Fragments from Epoxytigliane Treated Cancer Cells are Ingested by CD11c.SUP.+ Bone Marrow Derived Cell Populations

(63) CD11c is a well described marker of DC/macrophages (Merad et al. 2013). One way to investigate the potential of anti-cancer agents (and the associated death processes they induce) to promote the development of immunogenic responses is to determine whether they lead to the uptake of cellular components by CD11c.sup.+ dendritic cells/macrophages and their subsequent maturation into bone fide antigen presenting cells (APCs) (Guermonprez et al. 2002). Here we demonstrate that the oncosis induced by the Compound 1 and related epoxytiglianes leads to uptake of dying cancer cell components by such cells.

(64) Isolation and Culture of Bone Marrow Derived Cell (BMDC).

(65) The tibias and fibias from four 7-8 week old C57Bl/6 mice were first surgically removed under sterile conditions. The marrow was flushed from the bone cavity with 10 ml of ice cold R10 media (using a 27G needle/syringe into a 50 ml polypropylene tube). Cells were pelleted at 1,500 rpm for 5 mins and washed ×2 with ice cold R10 media. After resuspension of the pellet into 10 ml of ice cold R10 medium, cells were counted using an improved haemocytometer and plated at a density of 2×10.sup.6 cells per plate (petri dish) in 10 ml of R10 supplemented with 20 ng/ml murine GM-CSF. All plates were incubated at 37° C., 5% CO.sub.2 and at day 3 an additional 10 ml R10 with 20 ng/ml GM-CSF was added. Cells were again fed at day 6 and used for downstream assays on day 7.

(66) BMDC Uptake Experiments.

(67) Prior to co-incubation with BMDCs, B16-OVA cells were trypsinised, centrifuged at 1,200 rpm for 4 mins to pellet cells and washed ×2 with complete medium. Cells were then stained with 2 μM Cell Tracker Green in complete medium for 45 mins, after which they were pelleted and washed ×2 as above. Labelled B16-OVA cells were subsequently treated with Compounds 1, 2, 3 and 4 at two concentrations (500 or 300 μM) in media at a density of 1×10.sup.6 cells/ml for 30 and 60 mins, after which cells were pelleted via centrifugation, supernatant removed via aspiration and the cell pellet washed ×1 with complete media. After the wash, the treated cells were resuspended in R10 media, inserted into the wells of a 6-well plate (Corning, #3471. Ultra low attachment surface) and BMDCs added. Co-cultures were incubated for 4 h after which cell suspensions were transferred to a microfuge tube and centrifuged at 1,500 rpm for 5 mins. Pellets were resuspended in 100 μl FACs buffer containing anti-CD11c-APC and incubated for 10 mins at 4° C. Cells were again pelleted, washed ×1 with FACs buffer and then fixed using PBS, 1% formaldehyde for 10 mins. After centrifugation and removal of the supernatant, cells were resuspended in 500 μl FACs buffer in preparation for flow cytometry.

(68) Samples were first gated by FSC-H v FSC-A, then SSC-H v SS-A to identify single cells. The proportion of CD11c.sup.+ cells (Ex: 640 nm, Em: 670/14) with CMFDA.sub.mid (Ex: 488 nm, Em: 530/30) were then determined for treated and untreated cells as a percentage of all CD11c.sup.+ cells.

(69) The results (FIGS. 5A and 5B) show that the oncosis induced by Compound 1 promotes the uptake (i.e. effective ingestion) of dying cancer cell fragments by CD11c.sup.+ dendritic cells/macrophages. This ingestion appears to be dependent on both Compound 1 concentration and treatment time, such that at 500 μM of Compound 1 antigen uptake occurs to a greater extent after a shorter treatment time compared to the use of 300 M. This is consistent with the kinetics of oncosis observed in Example 3. FIG. 5C demonstrates that Compounds 2, 3 and 4 are also capable of inducing a cell death response that is immunogenic in vitro i.e. promotes uptake by CD1c.sup.+ BMDCs.

Example 6: Combination of Compound 1 with Immune Checkpoint Inhibitors

(70) Immune checkpoint inhibitor therapies, especially those targeting receptors involved in T cell immunosuppression (e.g. cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or programmed death 1 (PD-1) and its ligand PD-L1), have greatly improved patient outcomes in several types of late stage cancer, including melanoma, renal cell carcinoma, bladder cancer and head and neck squamous cell carcinoma (HNSCC) (Msaouel & Massarelli 2016; Sharma & Allison 2015). However, primary and de novo resistance to these ICI drugs is a significant clinical problem. Furthermore, extensive clinical follow-up has shown that some melanoma patients are developing resistance to treatment and are undergoing disease relapse (O'Donnell et al., 2017). Therapies combining ICIs with compounds which may act as adjuvants by promoting immunogenic cell death (especially by resulting in increased immune cell infiltration into the tumour paracheyma and antigen uptake/presentation) hold promise to overcome some limitations of ICIs and improve overall clinical outcomes (Workenhe et al. 2014; Ribas et al. 2017). Here, and in Example 7, the data examines combinations of ICIs and intralesional injection of Compound 1.

(71) A schematic of the experimental approach can be seen in FIG. 6A. Briefly, 6-7 week old C57BL/6 mice were injected subcutaneously (s.c.) on both flanks with B16-F10-OVA mouse melanoma cells (2×10.sup.5 cells per site in 100 μl). Tumours were allowed to develop to approximately 5-50 mm.sup.3, after which 200 μg of anti-PD-1 (RMPI-14, BioXCell), anti-CTLA-4 (9H10, BioXCell) or isotype control antibody (2A3 and Syrian Hamster IgG, BioXCell) was injected i.p. per mouse (day −2). On day 0, both tumours were injected I.T. with either Compound 1 (15 μg in 50 μL) or vehicle (Vehc.) only. Each mouse received an additional i.p. injection of the same antibody that was administered on day −2 (again, 200 μg). Antibody was administered for a further 3 times via i.p. injection every 2 days. The volume of treated tumours was measured using calipers as previously detailed (Boyle et al. 2014) and mouse survival was determined over time.

(72) The results displayed in FIGS. 6B and 6C show that Compound 1 can combine with anti-PD-1 and anti-CTLA-4 to restrict tumour growth and improve mouse survival to a greater extent than single agent treatment. This effect appears to be concentration dependent for each Compound1/ICI combination. For example, the injection of 30 μg of Compound 1 together with anti-PD-1 leads to improved survival/reduced tumour growth when compared to the use of 30 μg of Compound 1 alone (FIG. 6Biii, iv). This is not observed when 15 μg of Compound 1 is used in the same combinations i.e. no improvement in survival or tumour growth (FIG. 6Bi, ii). The situation is reversed when anti-CTLA4 is used, where 15 μg of Compound 1 in the combination treatment (FIG. 6Ci, ii) gives the optimal response and 30 μg of Compound 1 does not (FIG. 6Ciii, iv).

Example 7: Observation of Abscopal Effects when Using Compound 1 in Combination with Immune Checkpoint Inhibitors

(73) A schematic of the experimental approach can be seen in FIG. 7A. Briefly, 6-7 week old C57BL/6 mice were injected s.c. on both flanks with B16-F10-OVA mouse melanoma cells (2×10.sup.5 cells per site in 100 μl). Tumours were allowed to develop to approximately 5-50 mm.sup.3, after which 200 μg of anti-PD-1 (RMP1-14, BioXCell), anti-CTLA-4 (9H10, BioXCell) or isotype control antibody (2A3, BioXCell) was injected i.p. per mouse (day −2). On day 0, the largest of the two tumours (approx. 50-75 mm.sup.3) was injected I.T. with either Compound 1 (15 μg in 50 μL) or vehicle (Vehc.) only. The remaining tumour was left untreated and each mouse received an additional i.p. injection of the same antibody that was administered on day −2 (again, 200 μg). Antibody was administered for a further 3 times via i.p. injection every 2 days. The volume of both treated and untreated tumours was measured during the course of the experiment as previously detailed.

(74) The results are shown in FIG. 7B. The results show that not only were the tumours treated with a combination of the antibodies and Compound 1 effectively ablated, but that some untreated adjacent tumours also showed a response to the combination therapy that was not observed with either agent alone.

Example 8: Myeloid Derived Suppressor Cells May Affect Low Dose Efficacy of Compound 1

(75) Myeloid derived suppressor cells (MDSCs) are immature myeloid cells that can suppress host immune response to tumours via multiple pathways (Qu et al. 2016). We have used granulocyte colony-stimulating factor (GCSF) knock-out C57BL/6 mice to provide a model for assessing the future potential for possible combination therapies involving Compound 1 with molecules that inhibit or block proteins (e.g. indoleamine 2,3-dioxygenase (IDO)) associated with MDSC recruitment and function. GCSF, an essential regulator of neutrophil production and trafficking, also plays a critical role in migration and proliferation of MDSCs (Li et al. 2016). Using our GCSFR knock-out model the possible role of granulocyte derived MSDCs in limiting the efficacy of monotherapy (at a sub-optimal dose) of Compound 1 against MC38 colorectal adenocarincomas was examined.

(76) Briefly, 6-7 week old wt C57BL/6 and gcsfr.sup.−/− mice were injected s.c. on both flanks with MC38 mouse colorectal cancer cells (1×10.sup.6 cells per site in 100 μl). Tumours were allowed to develop to approximately 5-50 mm.sup.3, after which 15 μg of Compound 1 or vehicle (Vehc.) was injected I.T. (50 μl). The volume of the treated tumours were followed as previously detailed in Examples 6 and 7. Tumour size and survival comparisons between Compound 1 injected tumours in wild type (wt) and GCSFR knockout mice can be seen in FIG. 8.

(77) Given the role of GCSFR in granulocyte recruitment, these data suggest that such immune cell infiltration may be limiting the anti-cancer efficacy of Compound 1 at a known suboptimal dose (15 μg). Combinations of Compound 1 or analogues with compounds that prevent the development of such immunosuppressive cell populations (e.g. IDO inhibitors) may thus lead to better anti-cancer efficacy and improve patient survival.

(78) It will be understood to persons skilled in the art of the invention that many modifications may be made without departing from the spirit and scope of the invention.

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