COMBINATION THERAPIES COMPRISING PANOBINOSTAT FOR THE TREATMENT OF CHOLANGIOCARCINOMA

Abstract

The present invention relates to compositions and methods for treatment of cholangiocarcinoma and in particular to combination therapies comprising panobinostat compositions in combination with other cytotoxic agents, e.g. agents that potentiate the effects of panobinostat, for use in the treatment of cholangiocarcinoma. Pharmaceutical compositions comprising panobinostat and other cytotoxic agents are also provided.

Claims

1. A method of treating cholangiocarcinoma in a subject comprising administering to the subject a therapeutically effective amount of Panobinostat or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of a cytotoxic agent, wherein the cytotoxic agent is selected from any one or more of carboplatin, BI 2536, cisplatin, combretastatin A4, dactolisib, daporinad, dasatanib, doxorubicin, docetaxel, elesclomol, ispinesib, luminespib, methotrexate, molibresib, obatoclax, pelitinib, SB-743921, topotecan, trametinib, triptolide or a pharmaceutically acceptable salt, solvate or hydrate thereof.

2. The method of claim 1, wherein the panobinostat or a pharmaceutically acceptable salt thereof and cytotoxic agent are administered separately, simultaneously or sequentially.

3. The method of claim 1, wherein the cytotoxic agent is selected from any one or more of carboplatin, cisplatin, dasatinib, doxorubicin, docetaxel, methotrexate, topotecan, trametinib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

4. The method of claim 1, wherein said cholangiocarcinoma is intrahepatic cholangiocarcinoma.

5. The method of claim 1, wherein said cholangiocarcinoma is extrahepatic cholangiocarcinoma.

6. The method of claim 1, wherein said panobinostat or a pharmaceutically acceptable salt thereof is provided in a pharmaceutical composition together with a pharmacologically acceptable excipient.

7. The method of claim 6, wherein the pharmaceutical composition is formulated for oral administration.

8. The method of claim 6, wherein the pharmaceutical composition is in the form of a tablet or capsule.

9. The method of claim 6, wherein the pharmaceutical composition comprises a cytotoxic agent selected from any one or more of dasatinib, methotrexate, topotecan, trametinib, BI 2536, combretastatin A4, dactolisib, daporinad, elesclomol, ispinesib, luminespib, molibresib, obatoclax, pelitinib, triptolide or a pharmaceutically acceptable salt thereof.

10. (canceled)

11. The method of claim 1, wherein the cytotoxic agent is formulated for parenteral administration.

12. The method of claim 11, wherein the cytotoxic agent is formulated for administration by injection or infusion, preferably intravenous injection or infusion.

13. The method of claim 11, wherein the cytotoxic agent is selected from any one or more of carboplatin, BI 2536, cisplatin, combretastatin A4, dactolisib, daporinad, doxorubicin, docetaxel, elesclomol, ispinesib, luminespib, methotrexate, molibresib, obatoclax, pelitinib, SB-743921, topotecan, triptolide or a pharmaceutically acceptable salt, solvate or hydrate thereof.

14. A kit comprising panobinostat or a pharmaceutically acceptable salt thereof and a cytotoxic agent selected from any one or more of carboplatin, BI 2536, cisplatin, combretastatin A4, dactolisib, daporinad, dasatanib, doxorubicin, docetaxel, elesclomol, ispinesib, luminespib, methotrexate, molibresib, obatoclax, pelitinib, SB-743921, topotecan, triptolide or a pharmaceutically acceptable salt, solvate or hydrate thereof.

15. (canceled)

16. The kit of claim 14, wherein: (a) said panobinostat or pharmaceutically acceptable salt thereof is provided in a pharmaceutical composition together with a pharmacologically acceptable excipient, or (b) said cytotoxic agent is selected from one or more of: (i) dasatinib, methotrexate, topotecan, trametinib, BI 2536, combretastatin A4, dactolisib, daporinad, elesclomol, ispinesib, luminespib, molibresib, obatoclax, pelitinib, triptolide or a pharmaceutically acceptable salt thereof or iii) carboplatin, BI 2536, cisplatin, combretastatin A4, dactolisib, daporinad, doxorubicin, docetaxel, elesclomol, ispinesib, luminespib, methotrexate, molibresib, obatoclax, pelitinib, SB-743921, topotecan, triptolide or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

17. A pharmaceutical composition comprising panobinostat or a pharmaceutically acceptable salt thereof and a cytotoxic agent selected from: (i) dasatinib or a pharmaceutically acceptable salt thereof; (ii) methotrexate or a pharmaceutically acceptable salt thereof; (iii) topotecan or a pharmaceutically acceptable salt thereof; (iv) BI 2536 or a pharmaceutically acceptable salt thereof; (v) combretastatin A4 or a pharmaceutically acceptable salt thereof; (vi) dactolisib or a pharmaceutically acceptable salt thereof; (vii) daporinad or a pharmaceutically acceptable salt thereof; (viii) elesclomol or a pharmaceutically acceptable salt thereof; (ix) ispinesib or a pharmaceutically acceptable salt thereof; (x) luminespib or a pharmaceutically acceptable salt thereof; (xi) molibresib or a pharmaceutically acceptable salt thereof; (xii) obatoclax or a pharmaceutically acceptable salt thereof; (xiii) pelitinib or a pharmaceutically acceptable salt thereof; (xiv) triptolide or a pharmaceutically acceptable salt thereof; and (xv) a combination of any one of (i) to (xiv).

18. The pharmaceutical composition of claim 17, wherein the composition is formulated for oral administration.

19. The pharmaceutical composition of claim 17, wherein the composition is in the form of a tablet or capsule.

20. The method of claim 6, wherein the pharmaceutical composition comprises panobinostat or a pharmaceutically acceptable salt thereof and a cytotoxic agent selected from: (i) dasatinib or a pharmaceutically acceptable salt thereof; (ii) methotrexate or a pharmaceutically acceptable salt thereof; (iii) topotecan or a pharmaceutically acceptable salt thereof; (iv) BI 2536 or a pharmaceutically acceptable salt thereof; (v) combretastatin A4 or a pharmaceutically acceptable salt thereof; (vi) dactolisib or a pharmaceutically acceptable salt thereof; (vii) daporinad or a pharmaceutically acceptable salt thereof; (viii) elesclomol or a pharmaceutically acceptable salt thereof; (ix) ispinesib or a pharmaceutically acceptable salt thereof; (x) luminespib or a pharmaceutically acceptable salt thereof; (xi) molibresib or a pharmaceutically acceptable salt thereof; (xii) obatoclax or a pharmaceutically acceptable salt thereof; (xiii) pelitinib or a pharmaceutically acceptable salt thereof; (xiv) triptolide or a pharmaceutically acceptable salt thereof; and (xv) a combination of any one of (i) to (xiv).

21-23. (canceled)

Description

[0478] The invention will now be described in more detail in the following non-limiting Examples with reference to the following drawings in which:

[0479] FIG. 1 shows concentration-response-curves for panobinostat of seven cholangiocarcinoma cell lines. A) Intrahepatic cholangiocarcinoma cell lines. B) Extrahepatic cholangiocarcinoma cell lines and KMCH-1 as combined cholangio- and hepatocarcinoma cell line. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0480] FIG. 2 shows the effects of panobinostat (squares) and bortezomib (triangles) as single substance treatments and the combination of panobinostat with 1.3 nM bortezomib (circles) in the cell line HuCC-T1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0481] FIG. 3 shows the effects of panobinostat (squares) and carboplatin (triangles) as single substance treatments and the combination of panobinostat with 1000 nM carboplatin (circles) in the cell line TFK-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0482] FIG. 4 shows the effects of panobinostat (squares) and cisplatin (triangles) as single substance treatments and the combination of panobinostat with 100 nM cisplatin (circles) in the cell line TFK-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0483] FIG. 5 shows the effects of panobinostat (squares) and dasatinib (triangles) as single substance treatments and the combination of panobinostat with 5 nM dasatinib (circles) in the cell line TFK-1. Cell viability was measured 48 h post drug-addition. IC50 values are highlighted in vertical lines.

[0484] FIG. 6 shows the effects of panobinostat (squares) and doxorubicin (triangles) as single substance treatments and the combination of panobinostat with 87 or 100 nM doxorubicin (circles) in the cell lines: (A) HuCCT-1 and (B) TFK-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0485] FIG. 7 shows the effects of panobinostat (squares) and gemcitabine (triangles) as single substance treatments and the combination of panobinostat with 12 or 1000 nM gemcitabine (circles) in the cell lines (A)CC-SW-1 and (B) TFK-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0486] FIG. 8 shows the effects of panobinostat (squares) and methotrexate (triangles) as single substance treatments and the combination of panobinostat with 24 nM methotrexate (circles) in the cell line TFK-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0487] FIG. 9 shows the effects of panobinostat (squares) and trametinib (triangles) as single substance treatments and the combination of panobinostat with 8.8 or 1000 nM trametinib (circles) in the cell line (A) HuCCT-1 and (B) TFK-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0488] FIG. 10 shows the effects of panobinostat (squares) and topotecan (triangles) as single substance treatments and the combination of panobinostat with 24 or 120 nM topotecan (circles) in the cell line (A)CC-SW-1 and (C) TFK-1. (B) show the effects of topotecan (squares) and panobinostat (triangles) as single substance treatments and the combination of topotecan with 5.3 nM panobinostat (circles) in the cell line TFK-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0489] FIG. 11 shows the effects of panobinostat (squares) and BI 2536 (triangles) as single substance treatments and the combination of panobinostat with 490 nM BI 2536 (circles) in the cell line TFK-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0490] FIG. 12 shows the effects of panobinostat (squares) and triptolide (triangles) as single substance treatments and the combination of panobinostat with 30 nM triptolide (circles) in the cell line TFK-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0491] FIG. 13 shows the effects of panobinostat (squares) and dactolisib (triangles) as single substance treatments and the combination of panobinostat with 80 or 2 nM dactolisib (circles) in the cell line (A) EGI-1 and (B) TFK-1, respectively. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0492] FIG. 14 shows (A) the effects of panobinostat (squares) and daporinad (triangles) as single substance treatments and the combination of panobinostat with 23 nM daporinad (circles) in the cell line TFK-1; and (B) the effects of daporinad (squares) and panobinostat (triangles) as single substance treatments and the combination of daporinad with 5.3 nM panobinostat (circles) in the cell line CC-SW-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0493] FIG. 15 shows the effects of panobinostat (squares) and obatoclax mesylate (triangles) as single substance treatments and the combination of panobinostat with 16 nM obatoclax mesylate (circles) in the cell line TFK-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0494] FIG. 16 shows the effects of panobinostat (squares) and SB-743921 (triangles) as single substance treatments and the combination of panobinostat with 6.4 nM SB-743921 (circles) in the cell line TFK-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0495] FIG. 17 shows the effects of panobinostat (squares) and combretastatin A4 (triangles) as single substance treatments and the combination of panobinostat with 1000 or 100 nM combretastatin A4 (circles) in the cell lines (A) EGI-1 and (B) HuCC-T1, respectively. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0496] FIG. 18 shows the effects of panobinostat (squares) and ispinesib (triangles) as single substance treatments and the combination of panobinostat with 75 nM ispinesib (circles) in the cell line TFK-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0497] FIG. 19 shows the effects of panobinostat (squares) and molibresib (triangles) as single substance treatments and the combination of panobinostat with 1000 nM molibresib (circles) in the cell line TFK-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0498] FIG. 20 shows the effects of panobinostat (squares) and luminespib (triangles) as single substance treatments and the combination of panobinostat with 56 nM luminespib (circles) in the cell line TFK-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0499] FIG. 21 shows the effects of panobinostat (squares) and pelitinib (triangles) as single substance treatments and the combination of panobinostat with nM pelitinib (circles) in the cell line TFK-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

[0500] FIGS. 22 (A) and (B) show the effects of elesclomol (squares) and panobinostat (triangles) as single substance treatments and the combination of elesclomol with 5.3 or 14 nM panobinostat (circles) in the cell lines CC-SW-1 and EGI-1, respectively. (C) and (D) show the effects of panobinostat (squares) and elesclomol (triangles) as single substance treatments and the combination of panobinostat with 7 or 70 nM elesclomol (circles) in the cell lines HuCC-T1 and TFK-1, respectively. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

EXAMPLES

[0501] Experimental Procedure and Analysis Description for Drug Combinations

[0502] Various cholangiocarcinoma (CCA) cell lines were used for this drug screening. Table 1 provides details of culture medium and cell numbers used for the experiments.

TABLE-US-00001 TABLE 1 Cell Culture Conditions for Cholangiocarcinoma Cell Lines Cell line Cell culture medium Cells/well EGI-1 DMEM high glucose, 10% FCS, antibiotics 750 HuCC-T1 RPMI GlutaMAX, 10% FCS, antibiotics 750 TFK-1 RPMI GlutaMAX, 10% FCS, antibiotics 500 CC-SW-1 RPMI GlutaMAX, 10% FCS, antibiotics 500 KMBC DMEM high glucose, 10% FCS, antibiotics 750 CC-LP-1 RPMI GlutaMAX, 10% FCS, antibiotics 750 KMCH-1 RPMI GlutaMAX, 10% FCS, antibiotics 500

[0503] After trypsinization and counting, single cells were seeded into Greier 384-well tissue culture treated polystyrene plates (#781098) in 10 μL of appropriate media (see Table 1 for exact cell numbers). Seeded cells were allowed to attach to plates over a period of 24 hours, then appropriate volumes of compounds were added using an acoustic liquid dispenser (Labcyte Echo 550) in order to get concentrations of a single drug between 0.1 nM to 1000 nM (“dose response”) in 25 μL total volume. The wells were filled with 15 μL of appropriate media and incubated as above for 48 hours. After 48 hours of incubation, the cells were treated with 25 μL of 0.5× Cell TitreGlo luminescence viability reagent. The cells were incubated in the dark for 10 minutes and then read on a Synergy Neo2 plate reader for luminescence read from the top with autogain.

[0504] For panobinostat as single substance treatment seven cholangiocarcinoma cell lines were used. Panobinostat was then tested in combination with 23 other drugs on four cholangiocarcinoma cell lines. A single concentration of the combination drug was added based on a previously determined IC20 value for the combination drug tested alone on each of the four cell lines. The cell line TFK-1 was insensitive to some tested drugs. If IC20 values could not be calculated, a dose between 100 to 1000 nM was selected for combination testing (applies to carboplatin, cisplatin, gemcitabine, trametinib). For HuCCT-1 cells, trametinib was added in a dose of IC44=8.8 nM, instead of 1020.

[0505] Data Analysis

[0506] Cell viability from the Cell TitreGlo assay resulted in 576 dose response curves (24 monotherapies with DMSO and 552 combinations with drugs at IC20) for each cell line tested.

Example 1: Effect of Panobinostat on Seven Cholangiocarcinoma Cell Lines

[0507] Panobinostat was tested on seven different cholangiocarcinoma cell lines as single substance treatment in comparison to DMSO as described in detail above in method section. These revealed that all analysed cell lines show a response to panobinostat (see FIG. 1): five cell lines show an IC50 of around 100 nM and two cell lines (CC-SW-1 and EGI-1) show a higher sensitivity to Panobinostat with an IC50 of 12 nM or 36 nM (see Table 2 for IC50 values and FIG. 1 for dose-response curves).

[0508] Although being effective as single substance, panobinostat efficacy is increased by combination with other cancer drugs (Example 2-22) as described below.

TABLE-US-00002 TABLE 2 IC50 values for panobinostat in seven cholangiocarcinoma cell lines Cell Line Cell Type IC50 (nM) CC-SW-1 intrahepatic cholangiocarcinoma 12 HuCC-T1 intrahepatic cholangiocarcinoma 90 EGI-1 intrahepatic cholangiocarcinoma 36 CC-LP-1 intrahepatic cholangiocarcinoma 81 TFK-1 extrahepatic cholangiocarcinoma 95 KMBC extrahepatic cholangiocarcinoma 88 KMCH-1 combined cholangio- and hepatocarcinoma 99

Example 2: Combination of Panobinostat and Bortezomib in HuCCT-1 Cholangiocarcinoma Cells

[0509] The cell line HuCCT-1 was treated with the combination of panobinostat and low-dose bortezomib (1.3 nM, the IC20 dose of single substance curve), see FIG. 2. For experimental details on combined drug testing, please refer to the above method section.

[0510] Indeed, the combination of panobinostat and bortezomib is more efficient as highlighted by the lowering of the IC50 value from 90 nM for panobinostat as single substance treatment down to 51 nM in combination with 1.3 nM bortezomib.

[0511] Therefore, it can be concluded that panobinostat shows higher efficacy in combination with bortezomib compared to single substance treatment.

Example 3: Combination of Panobinostat and Carboplatin on TFK-1 Cholangiocarcinoma Cells

[0512] For experimental details on combined drug testing, please refer to the above method section. Interestingly, carboplatin had no effect on the cell viability of TFK-1 cells as single substance treatment (FIG. 3, triangles). However, when carboplatin was added in a dose of 1000 nM to panobinostat (FIG. 3, circles), the efficacy of panobinostat was increased, as indicated by the lowering of IC50 value from 70 nM to 26 nM (FIG. 3). Therefore, the combination of panobinostat with carboplatin is expected to show better efficiency for cholangiocarcinoma treatment than panobinostat alone.

Example 4: Combination of Panobinostat and Cisplatin on TFK-1 Cholangiocarcinoma Cells

[0513] Cisplatin had no effect on TFK-1 as single substance treatment (FIG. 4, triangles). However, the addition of 100 nM cisplatin to panobinostat (1050=42 nM; FIG. 4, circles), showed higher efficacy than panobinostat alone (1050=70 nM; squares), see FIG. 4. For experimental details on combined drug testing, please refer to the above method section. Hence, it is expected that the combination of panobinstat with cisplatin will have higher efficacy for treatment of cholangiocarcinoma.

Example 5: Combination of Panobinostat and Dasatinib on TFK-1 Cholangiocarcinoma Cell Line

[0514] For experimental details on combined drug testing, please refer to the above method section. Dasatinib as single substance showed only limited efficacy in TFK-1 cells (FIG. 5, triangles). Interestingly, the addition of low-dose dasatinib (5 nM, the 1020 dose of single substance curve) increased the effects of panobinostat. This is illustrated by shifting the IC50 value from 70 nM for panobinostat alone (FIG. 5, squares) down to 39 nM for the combination (FIG. 5, circles). Hence, it is expected from these results that the combination of panobinostat and dasatinib has higher efficacy than panobinostat alone for cholangiocarcinoma therapy.

Example 6: Combination of Panobinostat and Doxorubicin on HuCCT-1 and TFK-1 Cholangiocarcinoma Cells

[0515] For experimental details on combined drug testing, please refer to the above method section. The cell line HuCCT-1 was sensitive to panobinostat and doxorubicin with IC50 values of 90 nM and 158 nM, respectively. The combination of panobinostat with doxorubicin (FIG. 6A, circles) showed better response with an IC50 value of 44 nM compared to panobinostat (squares) or doxorubicin (triangles) alone, see FIG. 6A.

[0516] The cell line TFK-1 was sensitive to panobinostat and doxorubicin with IC50 values of 70 nM and 123 nM, respectively. The combination of panobinostat with doxorubicin (FIG. 6B, circles) showed better response with an IC50 value of 40 nM compared to panobinostat (squares) or doxorubicin (triangles) alone, see FIG. 6B.

[0517] Therefore, the combination of panobinostat and doxorubicin is expected to be beneficial for treatment of cholangiocarcinoma.

Example 7: Combination of Panobinostat and Gemcitabine in CC-SW-1 and TFK-1 Cholangiocarcinoma Cell Lines

[0518] For experimental details on combined drug testing, please refer to the above method section. The cell line CC-SW-1 is sensitive for both panobinostat (FIG. 7A, squares) and gemcitabine (FIG. 7A, triangles), but especially for the combination of panobinostat and 12 nM gemcitabine (FIG. 7A, circles). The combination showed a lower IC50 value with 3 nM compared to panobinostat alone with 12 nM.

[0519] Interestingly, the cell line TFK-1 was not sensitive to gemcitabine as single substance treatment. Nevertheless, the addition of gemcitabine to panobinostat (1050=46 nM) increased the efficacy compared to only panobinostat (1050=70 nM see FIG. 7B).

[0520] Therefore, addition of gemcitabine to panobinostat leads to increased effects on cell viability during cholangiocarcinoma treatment.

Example 8: Combination of Panobinostat and Methotrexate in TFK-1 Cholangiocarcinoma Cells

[0521] For experimental details on combined drug testing, please refer to the above method section. Methotrexate had only minor effects on the cell viability in TFK-1 cells (FIG. 8, triangles). The addition of 24 nM methotrexate to panobinostat (FIG. 8, circles) nevertheless showed increased efficacy with an IC50 value of 37 nM compared to panobinostat alone (FIG. 8, squares) with an IC50 of 70 nM (see FIG. 8). Similar effects are expected for cholangiocarcinoma treatment.

Example 9: Combination of Panobinostat and Trametinib in HuCCT-1 and TFK-1 Cholangiocarcinoma Cell Lines

[0522] For experimental details on combined drug testing, please refer to the above method section. Trametinib showed only minor efficacy in both HuCCT-1 and TFK-1 cells (triangles, FIGS. 9A and B). However, addition of low-dose trametinib (8.8 nM) to panobinostat in HuCCT-1 cells increased the effect of panobinostat on cell viability. This is indicated by shifted IC50 values from 90 nM for panobinostat alone (FIG. 9A, squares) to 42 nM for the combined treatment (circles, FIG. 9A).

[0523] Similarly, combined trametinib and panobinostat treatment also showed higher effects than panobinostat alone in TFK-1 cells by lowering the IC50 value from 70 nM to 31 nM (FIG. 9B).

[0524] Therefore, combined treatment with panobinostat and trametinib is predicted to show higher effects in cholangiocarcinoma therapy.

Example 10: Combination of Panobinostat and Topotecan in CC-SW-1 and TFK-1 Cholangiocarcinoma Cell Lines

[0525] For experimental details on combined drug testing, please refer to the above method section. The cell line CC-SW-1 was sensitive to combinations of panobinostat and topotecan with IC50 values of 12 nM and 54 nM, respectively. The combination of panobinostat with fixed dose topotecan (FIG. 10A, circles) in CC-SW-1 cells showed better response with an IC50 value of 5.0 nM compared to panobinostat (squares) alone, see FIG. 10A. Additionally, the combination of topotecan with fixed dose panobinostat (FIG. 10B, circles) in CC-SW-1 cells also showed better response with an IC50 value of 37 nM compared to topotecan (squares) alone, see FIG. 10B.

[0526] The cell line TFK-1 was also sensitive to combinations of panobinostat and topotecan with IC50 values of 70 nM and 373 nM, respectively. The combination of panobinostat with fixed dose topotecan (FIG. 10C, circles) in TFK-1 cells showed better response with an IC50 value of 28 nM compared to panobinostat (squares) alone, see FIG. 10C.

[0527] Therefore, the combination of panobinostat and topotecan is expected to be beneficial for treatment of cholangiocarcinoma.

Example 11: Combination of Panobinostat and BI 2536 in TFK-1 Cholangiocarcinoma Cells

[0528] For experimental details on combined drug testing, please refer to the above method section. The cell line TFK-1 was sensitive to panobinostat and BI 2536 with IC50 values of 70 nM and 80 nM, respectively. The combination of panobinostat with BI 2536 (FIG. 11, circles) in TFK-1 cells showed better response with an IC50 value of 38 nM compared to panobinostat (squares) alone, see FIG. 11. Therefore, the combination of panobinostat and BI 2536 is expected to be beneficial for treatment of cholangiocarcinoma.

Example 12: Combination of Panobinostat and Triptolide in TFK-1 Cholangiocarcinoma Cells

[0529] For experimental details on combined drug testing, please refer to the above method section. The cell line TFK-1 was sensitive to panobinostat and triptolide with IC50 values of 70 nM and 15 nM, respectively. The combination of panobinostat with tripolide (FIG. 12, circles) in TFK-1 cells showed better response with an IC50 value of 64 nM compared to panobinostat (squares), see FIG. 12. Therefore, the combination of panobinostat and tripolide is expected to be beneficial for treatment of cholangiocarcinoma.

Example 13: Combination of Panobinostat and Dactolisib in EGI-1 and TFK-1 Cholangiocarcinoma Cell Lines

[0530] For experimental details on combined drug testing, please refer to the above method section. The cell line EGI-1 was sensitive to panobinostat and dactolisib with IC50 values of 99 nM and 99 nM, respectively. The combination of panobinostat with fixed dose dactolisib (FIG. 13A, circles) in EGI-1 cells showed better response with an IC50 value of 34 nM compared to panobinostat (squares) alone, see FIG. 13A.

[0531] The cell line TFK-1 was also sensitive to panobinostat and dactolisib with IC50 values of 70 nM and 93 nM, respectively. The combination of panobinostat with fixed dose dactolisib (FIG. 13B, circles) in TFK-1 cells showed better response with an IC50 value of 30 nM compared to panobinostat (squares) alone, see FIG. 13B. Therefore, the combination of panobinostat and dactolisib is expected to be beneficial for treatment of cholangiocarcinoma.

Example 14: Combination of Panobinostat and Daporinad in TFK-1 and CC-SW-1 Cholangiocarcinoma Cell Lines

[0532] For experimental details on combined drug testing, please refer to the above method section. The cell line TFK-1 was sensitive to panobinostat and dactolisib with IC50 values of 70 nM and 73 nM, respectively. The combination of panobinostat with fixed dose dactolisib (FIG. 14A, circles) in TFK-1 cells showed better response with an IC50 value of 48 nM compared to panobinostat (squares) alone, see FIG. 14A.

[0533] Additionally, the cell line CC-SW-1 was sensitive to panobinostat and daporinad with IC50 values of 12 nM and 8 nM, respectively. The combination of daporinad with fixed dose panobinostat (FIG. 14B, circles) in TFK-1 cells showed better response with a drug sensitivity score (DSS) of 34 for the combination compared to a DSS of 17 for daporinad (squares) alone, see FIG. 14B. Therefore, the combination of panobinostat and daporinad is expected to be beneficial for treatment of cholangiocarcinoma.

Example 15: Combination of Panobinostat and Obatoclax Mesylate in TFK-1 Cholangiocarcinoma Cells

[0534] For experimental details on combined drug testing, please refer to the above method section. The cell line TFK-1 was sensitive to panobinostat and obatoclax mesylate with IC50 values of 70 nM and 1523 nM, respectively. The combination of panobinostat with fixed dose obatoclax mesylate (FIG. 15, circles) in TFK-1 cells showed better response with an IC50 value of 52 nM compared to panobinostat (squares) alone, see FIG. 15. Therefore, the combination of panobinostat and obatoclax mesylate is expected to be beneficial for treatment of cholangiocarcinoma.

Example 16: Combination of Panobinostat and SB-743921 in TFK-1 Cholangiocarcinoma Cell Lines

[0535] For experimental details on combined drug testing, please refer to the above method section.

[0536] The cell line TFK-1 was also sensitive to panobinostat and SB-743921 with IC50 values of 70 nM and 8 nM, respectively. The combination of panobinostat with fixed dose SB-743921 (FIG. 16, circles) in TFK-1 cells showed better response with an IC50 value of 37 nM compared to panobinostat (squares) alone, see FIG. 16.

[0537] Therefore, the combination of panobinostat and SB-743921 is expected to be beneficial for treatment of cholangiocarcinoma.

Example 17: Combination of Panobinostat and Combretastatin A4 in EGI-1 and HuCC-T1 Cholangiocarcinoma Cells

[0538] For experimental details on combined drug testing, please refer to the above method section. The cell line EGI-1 was sensitive to panobinostat but not to combretastatin A4 with IC50 values of 99 nM and >1000 nM, respectively. The combination of panobinostat with fixed dose combretastatin A4 (FIG. 17A, circles) in EGI-1 cells showed better response with an IC50 value of 52 nM compared to panobinostat (squares) alone, see FIG. 17A.

[0539] The cell line HuCC-T1 was also sensitive to panobinostat and combretastatin A4 with IC50 values of 90 nM and 11 nM, respectively. The combination of panobinostat with fixed dose combretastatin A4 (FIG. 17B, circles) in HuCC-T1 cells showed better response with an IC50 value of 53 nM compared to panobinostat (squares) alone, see FIG. 17B.

[0540] Therefore, the combination of panobinostat and combretastatin A4 is expected to be beneficial for treatment of cholangiocarcinoma.

Example 18: Combination of Panobinostat and Ispinesib in TFK-1 Cholangiocarcinoma Cells

[0541] For experimental details on combined drug testing, please refer to the above method section. The cell line TFK-1 was sensitive to panobinostat and ispinesib with IC50 values of 70 nM and 11 nM, respectively. The combination of panobinostat with fixed dose ispinesib (FIG. 18, circles) in TFK-1 cells showed better response with an IC50 value of 45 nM compared to panobinostat (squares) alone, see FIG. 18. Therefore, the combination of panobinostat and ispinesib is expected to be beneficial for treatment of cholangiocarcinoma.

Example 19: Combination of Panobinostat and Molibresib in TFK-1 Cholangiocarcinoma Cells

[0542] For experimental details on combined drug testing, please refer to the above method section. The cell line TFK-1 was sensitive to panobinostat and molibresib with IC50 values of 70 nM and 181 nM, respectively. The combination of panobinostat with fixed dose molibresib (FIG. 19, circles) in HuCC-T1 cells showed better response with an IC50 value of 26 nM compared to panobinostat (squares) alone, see FIG. 19. Therefore, the combination of panobinostat and molibresib is expected to be beneficial for treatment of cholangiocarcinoma.

Example 20: Combination of Panobinostat and Luminespib in TFK-1 Cholangiocarcinoma Cells

[0543] For experimental details on combined drug testing, please refer to the above method section. The cell line TFK-1 was sensitive to panobinostat and luminespib with IC50 values of 70 nM and 21 nM, respectively. The combination of panobinostat with fixed dose luminespib (FIG. 20, circles) in TFK-1 cells showed better response with an IC50 value of 11 nM compared to panobinostat (squares) alone, see FIG. 20. Therefore, the combination of panobinostat and luminespib is expected to be beneficial for treatment of cholangiocarcinoma.

Example 21: Combination of Panobinostat and Pelitinib in TFK-1 Cholangiocarcinoma Cells

[0544] For experimental details on combined drug testing, please refer to the above method section. The cell line TFK-1 was sensitive to panobinostat and pelitinib with IC50 values of 70 nM and 0.08 nM, respectively. The combination of panobinostat with fixed dose pelitinib (FIG. 21, circles) in TFK-1 cells showed better response with an IC50 value of 34 nM compared to panobinostat (squares) alone, see FIG. 21. Therefore, the combination of panobinostat and pelitinib is expected to be beneficial for treatment of cholangiocarcinoma.

Example 22: Combination of Panobinostat and Elesclomol in CC-SW-1, EGI-1, HuCC-T1, and TFK-1 Cholangiocarcinoma Cell Lines

[0545] For experimental details on combined drug testing, please refer to the above method section. The cell line CC-SW-1 was sensitive to panobinostat and elesclomol with IC50 values of 12 nM and 50 nM, respectively. The combination of elesclomol with fixed dose panobinostat (FIG. 22A, circles) in CC-SW-1 cells showed better response with an IC50 value of 19 nM compared to elesclomol (squares) alone, see FIG. 22A.

[0546] The cell line EGI-1 was also sensitive to panobinostat and elesclomol with IC50 values of 99 nM and 34 nM, respectively. The combination of elesclomol with fixed dose panobinostat (FIG. 22B, circles) in EGI-1 cells showed better response with an IC50 value of 19 nM compared to elesclomol (squares) alone, see FIG. 22B.

[0547] The cell line HuCC-T1 was also sensitive to panobinostat and elesclomol with IC50 values of 90 nM and 9 nM, respectively. The combination of panobinostat with fixed dose elesclomol (FIG. 22C, circles) in HuCC-T1 cells showed better response with an IC50 value of 46 nM compared to panobinostat (squares) alone, see FIG. 22C.

[0548] The cell line TFK-1 was also sensitive to panobinostat and elesclomol with IC50 values of 70 nM and 35 nM, respectively. The combination of panobinostat with fixed dose elesclomol (FIG. 22D, circles) in TFK-1 cells showed better response with an IC50 value of 24 nM compared to panobinostat (squares) alone, see FIG. 22D. Therefore, the combination of panobinostat and elesclomol is expected to be beneficial for treatment of cholangiocarcinoma.

Example 23: Capsules Comprising Panobinostat and Dasatinib

[0549] Panobinostat (99% purity) may be bought from Shandong Sunrise Technology Co., Ltd. in China. Alternatively, panobinostat lactate may be produced from panobinostat and lactic acid according to WO2007146716 (incorporated herein by reference). Dasatinib monohydrate (99.0% purity) may be bought from Beijing Yibai Biotechnology Co., Ltd. in China.

[0550] Capsules comprising panobinostat and dasatinib were prepared as described below:

[0551] Components

TABLE-US-00003   Panobinostat lactate (equivalent to 15 g panobinostat) Dasatinib monohydrate (equivalent to 50 g dasatinib) Magnesium stearate 1 g Mannitol 50 g Microcrystalline cellulose q.s to 500 g

[0552] The components were volumetrically mixed in a mixer and filled in 1000 hard gelatin capsules size 0. Each capsule comprises 15 mg panobinostat and 50 mg dasatinib.

Example 24: Drug Product Comprising Two Different Drug Formulations

[0553] Panobinostat (99% purity) may be bought from Shandong Sunrise Technology Co., Ltd. in China. Alternatively, panobinostat lactate may be produced from panobinostat and lactic acid according to WO2007146716 (incorporated herein by reference).

[0554] Capsules similar to Farydak 15 mg (Novartis) are prepared.

[0555] The capsules are packed in blisters (6 capsules per blister).

[0556] Dasatinib monohydrate (≥99.0% purity) may be bought from Beijing Yibai Biotechnology Co., Ltd. in China.

[0557] Tablets similar to Sprycel 50 mg (Bristol-Myers Squibb) are prepared.

[0558] The tablets are packed in blisters (6 tablets per blister)

[0559] The blisters (5 tablet blisters and 5 capsule blisters) are packed together with a packet insert in a drug product package.

Example 25: Reduction of Toxicity of Panobinostat in Combination with Cytotoxic Agents Compared to Panobinostat Monotherapy in Normal Cholangiocytes

[0560] The effects of various cytotoxic agents on the toxicity of panobinostat in normal cholangiocytes was examined. The cell line H69 (CVCL_8121) was used in experimental procedures described above to determine the IC50 value for panobinostat (the primary drug) on these cells when used in combination with cytotoxic agents (secondary drug) added to the cells at their IC20 concentrations.

[0561] The effect of the secondary drug was quantified using the delta IC50 measurement, which is calculated as the IC50 for panobinostat alone minus the IC50 for the panobinostat combination. A positive figure shows that the combination is more toxic than panobinostat alone. The larger difference the more toxic the combination. A negative delta IC50 shows that the combination is less toxic than the monotherapy. The results are set out below:

TABLE-US-00004 Secondary drug Delta LD50 (nM) Cell line Daporinad −73.1 H69 Dasatinib −14.0 H69 Gemcitabine −23.1 H69 Luminespib −248 H69 Pelitinib −25.8 H69 Topotecan −27.4 H69 Trametinib −16.4 H69

[0562] The results show that the tested secondary drugs reduce the toxicity of panobinostat in normal cholangiocytes.

Example 26: Therapeutic Index for Panobinostat in Combination with Other Cytotoxic Agents Compared to Panobinostat Monotherapy

[0563] The therapeutic index of various panobinostat combination therapies was determined by comparing the effects of the combinations and monotherapy (i.e. panobinostat alone) in normal cholangiocytes (cell line H-69) and various CCA cell lines as described above. The experimental procedure described above was used to determine the IC50 value for panobinostat (the primary drug) on the cells when used in alone or combination with cytotoxic agents (secondary drug) added to the cells at their 1020 concentrations.

[0564] The therapeutic index (TI) refers to the ratio of the IC50 in normal cells to the IC50 in the CCA cell line. A TI above 1 indicates that the therapy is effective at reducing the viability of the CCA cells relative to normal cells. A high TI, e.g. 1.5 or higher, indicates that there is a large difference in potency between normal cells and cancer cells, i.e. the therapy shows high selectivity against cancer cells versus normal cells. A TI that is higher for the combination therapy than the monotherapy indicates that the combination therapy is more selective for cancer cells than the monotherapy. The results are shown below (the IC50 values are in nM):

TABLE-US-00005 H69 cell line CC-SW-1 cell line Therapeutic Secondary Combination Combination index drug IC50 Mono IC50 Mono Combination Mono Carboplatin 41.7 56.8 7.1 18.4 5.9 3.1 Cisplatin 45.5 56.8 12.7 18.4 3.6 3.1 Dasatinib 70.8 56.8 9.7 18.4 7.2 3.1 Docetaxel 49.7 56.8 8.4 18.4 5.9 3.1 Doxorubicin 36.7 56.8 3.6 18.4 10.2 3.1 Methotrexate 48.9 56.8 14.3 18.4 3.4 3.1 Topotecan 84.2 56.8 16.5 18.4 5.1 3.1 Trametinib 73.2 56.8 7.1 18.4 10.3 3.1 Gemcitabine 79.9 56.8 1.2 18.4 66.6 3.1 Bortezomib 22.9 56.8 1.0 18.4 22.9 3.1

TABLE-US-00006 H69 cell line EGI-1 cell line Therapeutic Secondary Combination Combination index drug IC50 Mono IC50 Mono Combination Mono Carboplatin 41.7 56.8 34.8 62.9 1.2 0.9 Cisplatin 45.5 56.8 64.6 62.9 0.7 0.9 Dasatinib 70.8 56.8 49.4 62.9 1.4 0.9 Docetaxel 49.7 56.8 69.2 62.9 0.7 0.9 Doxorubicin 36.7 56.8 48.8 62.9 0.8 0.9 Methotrexate 48.9 56.8 77.9 62.9 0.6 0.9 Topotecan 84.2 56.8 29.8 62.9 2.8 0.9 Trametinib 73.2 56.8 33.6 62.9 2.2 0.9 Gemcitabine 79.9 56.8 58.5 62.9 1.4 0.9 Bortezomib 22.9 56.8 35.1 62.9 0.7 0.9

TABLE-US-00007 H69 cell line HuCCT-1 cell line Therapeutic Secondary Combination Combination index drug IC50 Mono IC50 Mono Combination Mono Carboplatin 41.7 56.8 13.3 42.4 3.1 0.9 Cisplatin 45.5 56.8 55.9 42.4 0.8 0.9 Dasatinib 70.8 56.8 54.2 42.4 1.3 0.9 Docetaxel 49.7 56.8 64.6 42.4 0.8 0.9 Doxorubicin 36.7 56.8 61.1 42.4 0.6 0.9 Methotrexate 48.9 56.8 40.3 42.4 1.2 0.9 Topotecan 84.2 56.8 57.4 42.4 1.5 0.9 Trametinib 73.2 56.8 37.9 42.4 1.9 0.9 Gemcitabine 79.9 56.8 25.3 42.4 3.2 0.9 Bortezomib 22.9 56.8 48.3 42.4 0.5 0.9

TABLE-US-00008 H69 cell line TFK-1 cell line Therapeutic Secondary Combination Combination index drug IC50 Mono IC50 Mono Combination Mono Carboplatin 41.7 56.8 39.0 51.8 1.1 1.1 Cisplatin 45.5 56.8 43.2 51.8 1.1 1.1 Dactosilib 16.3 56.8 5.7 51.8 2.9 1.1 Dasatinib 70.8 56.8 16.2 51.8 4.3 1.1 Docetaxel 49.7 56.8 44.3 51.8 1.1 1.1 Doxorubicin 36.7 56.8 25.1 51.8 1.4 1.1 Methotrexate 48.9 56.8 10.6 51.8 4.6 1.1 Topotecan 84.2 56.8 29.5 51.8 2.9 1.1 Trametinib 73.2 56.8 32.9 51.8 2.2 1.1 Gemcitabine 79.9 56.8 68.6 51.8 1.2 1.1 Bortezomib 22.9 56.8 35.5 51.8 0.6 1.1

[0565] These results indicate that the tested panobinostat combination therapies may be particularly effective against CCA tumours which share characteristics with the CC-SW-1 cell line. However, the data identifies combination therapies that are effective in other cell lines. For instance, combination therapies with trametinib and doxorubicin are particularly effective in the CC-SW-1 cell line. Combination therapies with trametinib and topotecan are particularly effective in the EGI-1 cell line. Combination therapies with trametinib and carboplatin are particularly effective in the HuCCT-1 cell line. Combination therapies with dasatinib, methotrexate, dactolisib, topotecan and trametinib are particularly effective in the TFK-1 cell line.

Example 27: Panobinostat Combination Therapies that Modulate the IC50 of Panobinostat in CCA Cell Lines

[0566] The experimental data described herein was used to identify cytotoxic agents that are particularly effective at potentiating the effects of panobinostat in CCA cells. The combinations were identified by determining the delta IC50, wherein a higher positive delta IC50 represents a more effective combination. The table below shows the absolute delta IC50 (nM) and the relative change as a percentage.

TABLE-US-00009 Delta IC50 Secondary drug absolute value and (%) Cell line Bortezomib 17.3(95) CC-SW-1 Carboplatin 11.2(61) CC-SW-1 Dactolisib 14.5(79) CC-SW-1 Doxorubicin 14.8(81) CC-SW-1 Gemcitabin 17.2(94) CC-SW-1 Ispinesib 11.3(62) CC-SW-1 SB-743921 17.1(93) CC-SW-1 Trametinib 11.3(62) CC-SW-1 Bortezomib 27.8(44) EGI1 Carboplatin 28.1(45) EGI1 Dactolisib 37.8(60) EGI1 Dasatinib 13.5(21) EGI1 Doxorubicin 14.1(22) EGI1 Ispinesib 21.6(34) EGI1 Luminesib 54.7(87) EGI1 Molibresib 14.8(24) EGI1 Obatoclax 19.6(31) EGI1 SB-743921 18.8(30) EGI1 Topotecan 33.1(53) EGI1 Trametinib 29.4(47) EGI1 Carboplatin 28.6(69) HuCCT-1 Combrestatin A4 10.9(27) HuCCT-1 Dactolisib 19.2(46) HuCCT-1 Gemcitabine 16.6(40) HuCCT-1 SB-743921 10.8(27) HuCCT-1 Bortezomib 16.3(31) TFK-1 Carboplatin 12.8(25) TFK-1 Dactolisib 46.1(84) TFK-1 Daporinad 22.3(43) TFK-1 Doxorubicin 26.7(84) TFK-1 Elesclomol 10.5(20) TFK-1 Ispinesib 27.2(53) TFK-1 Luminespib 23.5(45) TFK-1 Methotrexate 41.2(80) TFK-1 Molibresib 17.7(34) TFK-1 Obitoclax 14.4(28) TFK-1 Pelitinib 22.6(44) TFK-1 SB-743921 28.7(55) TFK-1 Topotecan 22.3(43) TFK-1 Trametinib 18.9(36) TFK-1

[0567] The Table below demonstrates that not all combinations are effective in all cell lines, i.e. some combinations show a negative effect on the toxicity of panobinostat in some cell lines, as shown by the negative delta IC50 values.

TABLE-US-00010 Secondary drug Delta IC50 (nM) Cell line BI 2536 −15.8 CC-SW-1 Molibresib −20.5 CC-SW-1 Pelitinib −17.5 CC-SW-1 BI-2536 −22.9 EGI-1 Methotrexate −15.0 EGI-1 Pelitinib −16.5 EGI-1 Cisplatin −13.5 HuCCT Daporinad −11.2 HuCCT Dasatinib −11.8 HuCCT Docetaxel −22.2 HuCCT

Example 28: Determination of Combination Index for Panobinostat and Elesclomol

[0568] Data Normalization and Curve Fitting

[0569] Cell viability from the CTG assay resulted in X dose response curves (Y monotherapies with DMSO and Z combinations with drugs at IC20) for each cell line tested. The viability data for each plate were normalized to the average of eight replicates of DMSO at 0.1% and seven replicates of Benzethonium Chloride (BzCl) at 100 uM. BzCl serves as a cell killing control that accounts for background signal from dead cells in the CTG luminescence assay. Raw luminescence data were normalized according to the following equation:

[00001]$\mathrm{Sample}\mathrm{Viability}\%=\frac{\mathrm{Sample}-{\mu }_{BzCl}}{{\mu }_{DMSO}-{\mu }_{BzCl}}\times 100\%$

[0570] The normalized data for each dose response curve were then fit using the function drm from the R package drc. This function uses a four parameter log-logistic curve to fit the dose response data, resulting in values for curve minimum, maximum, IC50, and slope. In the case where a log-logistic curve could not be fit to the data, a logistic curve was used instead. The IC50 corresponds to the concentration at 50% response between the calculated curve maximum and minimum, and is therefore a relative IC50 value.

[00002]$\mathrm{Log}-\mathrm{logistic}\mathrm{function}:y=\mathrm{minimum}+\frac{\mathrm{maximum}-\mathrm{minimum}}{1+e^{\mathrm{slope}\left(\log \left(x\right)-\log \left(\mathrm{IC}50\right)\right)}}$$\mathrm{Logistic}\mathrm{function}:y=\mathrm{minimum}+\frac{\mathrm{maximum}-\mathrm{minimum}}{1+e^{\mathrm{slope}\left(x-\mathrm{IC}50\right)}}$

[0571] Synergy Score Calculations

[0572] Synergy scores were calculated for monotherapy dose responses versus combination dose responses for each drug combination following the Loewe additivity, Bliss independence, and Zero-Interaction Potential (ZIP) methods. These methods calculate a predicted response based on the monotherapy responses of the drugs used in the combination. The measured responses for the combinations are then subtracted from these predicted responses to generate a synergy score for each tested concentration; a positive score indicates synergy while a negative score indicates antagonism.

[0573] Loewe Synergy

[0574] Loewe synergy values were calculated using the explicit method. For each drug in the combination, the predicted response calculated from the response of that drug alone at a dose equivalent to the sum of the doses of the two drugs in combination. The predicted responses for each drug are then averaged and the observed values at the measured concentrations are subtracted from this average to generate synergy scores.

[00003]${\mathrm{LOEWE}}_{\left(x1,x2\right)}=\frac{y_{1_{\left(x1+x2\right)}}+y_{2_{\left(x1+x2\right)}}}{2}-y_{c_{\left(x1,x2\right)}}$

[0575] where y.sub.1.sub.(x1+x2) is the calculated response using the curve fit of drug 1 monotherapy at the sum of concentrations of drug 1 at concentration x.sub.1 and drug 2 at concentration x.sub.2, y.sub.2.sub.(x1+x2) is the calculated response using the curve fit of drug 2 monotherapy at the same sum of concentrations of drugs 1 and 2, and y.sub.c.sub.(x1,x2) is the measured response for the combination of drug 1 and drug 2 at their respective doses x.sub.1 and x.sub.2. In the case that the terms y.sub.1.sub.(x1+x2), y.sub.2.sub.(x1+x2) or y.sub.c.sub.(x1,x2) were >100 or <0, they were set to 100 and 0, respectively.

[0576] The Loewe additivity model is preferred when the drugs used in combination target the same pathways, as they are expected to have additive effects.

[0577] Bliss Synergy

[0578] Predicted responses generated using the Bliss model were calculated by multiplying the monotherapy responses of each drug at the respective concentrations tested in the combination. The measured responses at these concentrations were then subtracted from these predicted values to generate synergy scores.

[00004]${\mathrm{BLISS}}_{\left(x1,x2\right)}=y_{1_{\left(x1\right)}}\times \frac{y_{2_{\left(x2\right)}}}{100}-y_{c\left(x1,x2\right)}$

[0579] where y.sub.1.sub.(x1) is the response from the curve fit of drug 1 monotherapy at dose x.sub.1, y.sub.2.sub.(x2) is the response from the curve fit of drug 2 monotherapy at dose x.sub.2, and y.sub.c.sub.(x1,x2) is the measured response for the combination of drug 1 and drug 2 at their respective doses x.sub.1 and x.sub.2. In the case that either or both of the terms

[00005]$y_{1_{\left(x1\right)}}\times \frac{{y_2}_{\left(x2\right)}}{100}\mathrm{or}{y}_{c_{\left(x1,x2\right)}}$

were >100 or <0, they were set to 100 and 0, respectively.

[0580] The Bliss independence model is preferred when the drugs used in combination target different pathways, as they are expected to have independent effects.

[0581] ZIP Synergy

[0582] Predicted responses generated using the ZIP model were calculated according to the Bliss method described above. The observed responses for the combinations were fitted to a log-logistic function, setting the curve maximum to the corresponding response of the IC20 drug monotherapy at the relevant dose. These fitted combination values were then subtracted from the predicted responses to generate synergy scores.

[00006]${\mathrm{ZIP}}_{\left(x1,x2\right)}=y_{1_{\left(x1\right)}}\times \frac{y_{2_{\left(x2\right)}}}{100}-y_{c_{\left(x1,x2\right)}}^{\prime }$

[0583] where y.sub.1.sub.(x1) is the response from the curve fit of drug 1 monotherapy at dose x.sub.1, y.sub.2.sub.(x2) is the response from the curve fit of drug 2 monotherapy at the dose x.sub.2, and y′.sub.c.sub.(x1,x2) is the calculated response using the curve fit of drug 1 with fixed dose drug 2, with the upper limit parameter set to the response of drug 2 monotherapy at dose x.sub.2. In the case that either or both of the terms

[00007]$y_{1_{\left(x1\right)}}\times \frac{{y_2}_{\left(x2\right)}}{100}\mathrm{or}{y}_{c_{\left(x1,x2\right)}}^{\prime }$

were >100 or <0, they were set to 100 and 0, respectively.

[0584] The ZIP model was created to integrate the Bliss and Loewe models.

[0585] The combination index for panobinostat and elesclomol was determined as described herein. The table below shows that this combination shows synergy in three cell lines, i.e. a combination index of less than 1.

TABLE-US-00011 Panobinostat Elesdomol Concentration Concentration Combination (nM) (nM) Cell Line Index 0.1 20 CCSW1 0.3 1 20 CCSW1 0.6 3 20 CCSW1 0.7 10 20 CCSW1 0.7 0.1 20 EGI1 0.5 1 20 EGI1 0.5 3 20 EGI1 0.5 10 20 EGI1 0.7 30 20 EGI1 0.7 100 20 EGI1 0.6 0.1 7 HuCCT 0.4 1 7 HuCCT 0.5 3 7 HuCCT 0.6 10 7 HuCCT 0.7

Example 29: Genome Sequence of Cell Lines

[0586] Whole genome sequencing was performed on the cell lines used herein to identify genetic markers (mutations) that are specific to the cell lines and may be expected to occur in CCA tumours. The table below shows markers in the cell lines that are linked to predictive, prognostic, diagnostic, and predisposition biomarkers in the CIViC database and the Cancer Biomarkers Database, coding variants that are found in known cancer mutation hotspots, predicted as cancer driver mutations, or curated as disease-causing and coding variants found in oncogenes or tumor suppressor genes.

TABLE-US-00012 Gene abbreviation Type of marker/mutation Protein change CELL_LINE GENE_NAME KRAS missense_variant p.Gly12Asp EGI-1 KRAS proto-oncogene, GTPase TP53 missense_variant p.Arg273His EGI-1 tumor protein p53 additional sex combs like 1, transcriptional ASXL1 missense_variant p.Glu865Lys EGI-1 regulator platelet derived growth factor receptor PDGFRA missense_variant p.Leu97Phe EGI-1 alpha MYH11 missense_variant p.Leu903Pro EGI-1 myosin heavy chain 11 E2F1 missense_variant p.Thr195Ile EGI-1 E2F transcription factor 1 AHNAK missense_variant p.Asn3518His EGI-1 AHNAK nucleoprotein AHNAK inframe_deletion p.Glu1270_Glu1273del EGI-1 AHNAK nucleoprotein SAFB2 missense_variant p.Ala895Glu EGI-1 scaffold attachment factor B2 NOTCH1 missense_variant p.Arg1984Gln EGI-1 notch 1 PEG3 splice_acceptor_variant EGI-1 paternally expressed 3 CADM3 missense_variant p.Asp254His EGI-1 cell adhesion molecule 3 SPI1 inframe_deletion p.Lys170del EGI-1 Spi-1 proto-oncogene AR inframe_deletion p.Gly472_Gly473del EGI-1 androgen receptor HCAR2 missense_variant p.Arg228Ile EGI-1 hydroxycarboxylic acid receptor 2 protein phosphatase 1 regulatory inhibitor PPP1R1B missense_variant p.Ile93Met EGI-1 subunit 1B BAP1 stop_gained p.Gln456Ter TFK1 BRCA1 associated protein 1 PBRM1 missense_variant p.Pro407Ala TFK1 polybromo 1 PBRM1 splice_acceptor_variant, coding_ TFK1 sequence_variant, intron_variant IKZF3 stop_lost p.Ter510SerextTer19 TFK1 IKAROS family zinc finger 3 PAWR missense_variant p.Pro39Ser TFK1 pro-apoptotic WT1 regulator FGFR3 missense_variant p.Gly145Val TFK1 fibroblast growth factor receptor 3 STIL missense_variant p.Arg216Lys TFK1 STIL, centriolar assembly protein SEMA3F missense_variant p.Glu192Lys TFK1 semaphorin 3F PCM1 missense_variant p.Gln289His TFK1 pericentriolar material 1 FGF5 missense_variant p.Gly201Arg TFK1 fibroblast growth factor 5 nuclear receptor binding SET domain WHSC1 missense_variant p.Asp69Gly TFK1 protein 2 KRAS missense_variant p.Gly12Asp HUCC1 KRAS proto-oncogene, GTPase TP53 missense_variant p.Arg175His HUCC1 tumor protein p53 FBXW7 stop_gained p.Ser294Ter HUCC1 F-box and WD repeat domain containing 7 LETMD1 missense_variant p.Pro283Ala HUCC1 LETM1 domain containing 1 SETD2 stop_gained p.Gln2285Ter HUCC1 SET domain containing 2 KDM5A missense_variant p.Pro60Thr HUCC1 lysine demethylase 5A MYO18B missense_variant p.Val1341Ile HUCC1 myosin XVIIIB RB1 missense_variant p.Ala106Glu HUCC1 RB transcriptional corepressor 1 DnaJ heat shock protein family (Hsp40) DNAJA3 missense_variant p.Gln153Glu HUCC1 member A3 chromatin licensing and DNA replication CDT1 missense_variant p.Glu122Asp HUCC1 factor 1 ZFP36L2 frameshift_variant p.Phe200ProfsTer276 HUCC1 ZFP36 ring finger protein like 2 MAF inframe_deletion p.Gly236_Gly238del HUCC1 MAF bZIP transcription factor GMPS missense_variant p.Arg435Thr HUCC1 guanine monophosphate synthase NPAS2 missense_variant p.Ile505Met HUCC1 neuronal PAS domain protein 2 CNTNAP2 frameshift_variant p.Leu695PhefsTer49 HUCC1 contactin associated protein like 2 platelet derived growth factor receptor PDGFRA missense_variant p.Tyr731Phe CCSW1 alpha CCAR2 missense_variant p.Gln137His CCSW1 cell cycle and apoptosis regulator 2 reversion inducing cysteine rich protein with RECK missense_variant p.Arg778Pro CCSW1 kazal motifs ZNF292 missense_variant p.Ala1318Glu CCSW1 zinc finger protein 292 PYHINI missense_variant p.His240Gln CCSW1 pyrin and HIN domain family member 1 DSP missense_variant p.Glu1740Lys CCSW1 desmoplakin

Example 30: Xenograft Studies in Mice

[0587] Cell Cultures

[0588] The normal human biliary cell line (H69) and various human cholangiocarcinoma cell lines (HuCCT, CC-SW1 EGI-1 and TFK-1) are cultured according to standard conditions.

[0589] Mice Experiments

[0590] All mice experiments are performed according to protocols approved by Ethical Committee for use of animals in research in Norway. The animals are maintained in cages with temperature controlled environment. The animals get free access to standard feed and water. The light/dark cycle is 12 h/12 h. Suspensions of cells are injected subcutaneously into the nude mice.

[0591] Tumor growth is confirmed 10 days after administration of the cell suspensions. The mice are divided into 5 groups (10 animals in each group); the first group gets no active treatment, the second group gets drug A, the third group gets drug B, the fourth group gets the combination drug A plus drug B and the fifth group gets a gemcitabine based combination therapy. All animals get free access to feed and water.

[0592] If the drugs are regulatory approved drugs, the drugs are administered in the same way, with the same dose (per kg) and dose frequency as it is used in the clinic for treatment of other cancer diseases. The highest and most frequently administration is used. If the drug is an experimental drug (i.e. not currently approved), the drug is administered in the same way, with the same dose (per kg) and dose frequency as it is used in prior art documents for treatment of cancer.

[0593] Tumor volume is determined weekly throughout the treatment period. Some of the mice undergo an ultrasound examination and/or an MRI examination to follow tumor growth during the treatment period. The mice are anesthetized and scarified according to standard procedure after 50 days. The tumors are removed, weighed and kept in the freezer for further analysis.

[0594] The results are expected to show that some drug combinations are very potent for the treatment of human cholangiocarcinoma in a xenograft nude mice model. The in vivo efficacy is anticipated to correlates well with in vitro cell line efficacy.

Example 31: Clinical Protocol for a Combined Therapy Using Drug a and Drug B as a Second Line Therapy in Patients with Cholangiocarcinoma

[0595] Single arm, open label, non-randomized, exploratory, multi-center pilot study. Drug A and drug B are regulatory approved drugs for other cancer indications.

[0596] 30 participants

Inclusion Criteria:

[0597] Patients with histologically or cytologically confirmed diagnosed cholangiocarcinoma [0598] Radiographically measurable disease (per RECIST v1.1) [0599] Patients previously treated with gemcitabine based First Line Therapy [0600] Age: 18 to 80 years, male or female [0601] Female on contraceptives if relevant

Exclusion Criteria

[0602] Lactating or pregnant females [0603] Severe cardiac dysfunction [0604] High blood pressure (systolic ≥150 mmHg or diastolic ≥100 mmHg) [0605] Positive Hepatitis C and/or Human immunodeficiency virus (HIV) and/or Covid-19 [0606] Primary Sclerosing Cholangitis and/or Inflammatory Bowel Disease and/or autoimmune diseases [0607] Active drug treatment of systemic infections. [0608] History of allergy or severe adverse events to drugs in the combination or drugs with same mechanism of action as in the drug combination. [0609] History of substance abuse including alcohol abuse and/or drug abuse. [0610] Insufficient organ function [0611] Absolutely Neutrophil Count (ANC)<1,000/mm3 [1.0×109/L] [0612] Platelets <75,000/mm3 [75×109/L] [0613] Hemoglobin <109.0 g/dL [0614] Total bilirubin >1.5×ULN [0615] Aspartate aminotransferase/glutamic oxaloacetic transaminase/GOT (AST/SGOT) and Alanine aminotransferase/glutamic pyruvic transaminase/GPT (ALT/SGPT)>2.5×ULN (AST and ALT)>5× upper limit of normal (ULN) in the presence of liver metastases) [0616] Serum creatinine >1.5×ULN and a calculated or measured creatinine clearance <45 mL/min [0617] Inorganic phosphorus outside of normal limits [0618] Total and ionized serum calcium outside of normal limits

[0619] Other protocol-defined inclusion/exclusion criteria may apply

Dosing

[0620] The drugs are individually dosed at 50% of the highest approved acceptable dose used for treatment of other cancer forms. The individual drugs are administered the same way and with the same frequency as the drugs are used for other indications. The two drugs are preferably administered together.

Duration

[0621] Up to 24 months for each patient.

Outcome Measures

Primary:

[0622] Objective response rate (ORR) [Time Frame: up to 24 months] [0623] Defined as the proportion of participants in each cohort who achieve a complete response (CR) or partial response (PR) based on Response Evaluation Criteria in Solid Tumors Version 1.1 (RECIST v1.1).

Secondary:

[0624] Progression-free survival (PFS) [Time Frame: up to 24 months] [0625] Defined as the time from first dose until progressive disease (per RECIST v1.1) or death (whichever is first) in each cohort. Median progression free survival. [0626] Duration of response (DOR) [Time Frame: up to 24 months] [0627] Defined as the time from the date of first assessment of CR or PR until the date of the first progressive disease (per RECIST v1.1) or death (whichever is first) in each cohort. [0628] Best overall response [Time Frame: up to 24 months] [0629] The best overall response will be summarized by the proportion of patients having a best overall response of PR, CR, stable disease (SD) or PD. [0630] Disease control rate (DCR) [Time Frame: up to 24 months] [0631] Defined as the proportion of participants who achieved best overall response of CR, PR, or stable disease per RECIST v1.1. [0632] Overall survival (OS) [Time Frame: up to 24 months] [0633] Defined as the time from first dose of study drug to death of any cause in each cohort. [0634] Median overall survival. [0635] Number of treatment-related adverse events [Time Frame: up to 24 months] [0636] Adverse events and Severe Adverse Events, type and frequency reported for the first time or worsening of a pre-existing event after first dose of study drug/treatment. [0637] Quality of life—Analysis of quality of life. Form: (https://www.eortc.org/app/uploads/sites/2/2018/08/Specimen-QLQ-C30-English.pdf

Example 32: Clinical Protocol for Combined Therapy Using Drug a and Drug B Versus Gemcitabine and Cisplatin Treatment in Patients with Cholangiocarcinoma

[0638] Two arms, double blinded randomized, clinical phase III study, multi-center. Drug X and drug Y are regulatory approved drugs for other cancer indications. 80 participants.

Arm A: Combined Therapy Using Drug A and drug B (40 participants)
Arm B: Gemcitabine (1000 mg/m2) administered according to regulatory accepted dosing in combination with cisplatin (25 mg/m2) according to regulatory accepted dosing.

Inclusion Criteria:

[0639] Patients with histologically or cytologically confirmed diagnosed cholangiocarcinoma diagnosed cholangiocarcinoma [0640] Radiographically measurable disease (per RECIST v1.1) [0641] Age: 18 to 80 years, male or female [0642] Female on contraceptives if relevant

Exclusion Criteria

[0643] Lactating or pregnant females [0644] Severe cardiac dysfunction [0645] High blood pressure (systolic ≥150 mmHg or diastolic ≥100 mmHg) [0646] Positive Hepatitis C and/or Human immunodeficiency virus (HIV) and/or Covid-19 [0647] Primary Sclerosing Cholangitis and/or Inflammatory Bowel Disease and/or autoimmune diseases [0648] Active drug treatment of systemic infections. [0649] History of allergy or severe adverse events to drugs in the combination or drugs with same mechanism of action as in the drug combination. [0650] History of substance abuse including alcohol abuse and drug abuse. [0651] Insufficient organ function [0652] Absolutely Neutrophil Count (ANC)<1,000/mm3 [1.0×109/L] [0653] Platelets <75,000/mm3 [75×109/L] [0654] Hemoglobin <109.0 g/dL [0655] Total bilirubin >1.5×ULN [0656] Aspartate aminotransferase/glutamic oxaloacetic transaminase/GOT (AST/SGOT) and Alanine aminotransferase/glutamic pyruvic transaminase/GPT (ALT/SGPT)>2.5×ULN (AST and ALT)>5× upper limit of normal (ULN) in the presence of liver metastases) [0657] Serum creatinine >1.5×ULN and a calculated or measured creatinine clearance <45 mL/min [0658] Inorganic phosphorus outside of normal limits [0659] Total and ionized serum calcium outside of normal limits

[0660] Other protocol-defined inclusion/exclusion criteria may apply

Dosing

[0661] The results from the explorative study form basis for the dosing of the drugs in the combination. Without relevant guiding. The drugs in the combination are individually dosed at 50% of the highest approved acceptable dose used for treatment of other cancer forms. The individual drugs are administered the same way and with the same frequency as the drugs are used for other indications. The two drugs are preferably administered together.

Duration

[0662] 24 months for each patient.

Outcome Measures

Primary:

[0663] Objective response rate (ORR) [Time Frame: up to 24 months] [0664] Defined as the proportion of participants in each cohort who achieve a complete response (CR) or partial response (PR) based on Response Evaluation Criteria in Solid Tumors Version 1.1 (RECIST v1.1).

Secondary:

[0665] Progression-free survival (PFS) [Time Frame: up to 24 months] [0666] Defined as the time from first dose until progressive disease (per RECIST v1.1) or death (whichever is first) in each cohort. Median progression free survival. [0667] Duration of response (DOR) [Time Frame: up to 24 months] [0668] Defined as the time from the date of first assessment of CR or PR until the date of the first progressive disease (per RECIST v1.1) or death (whichever is first) in each cohort. [0669] Best overall response [Time Frame: up to 24 months] [0670] The best overall response will be summarized by the proportion of patients having a best overall response of PR, CR, stable disease (SD) or PD. [0671] Disease control rate (DCR) [Time Frame: up to 24 months] [0672] Defined as the proportion of participants who achieved best overall response of CR, PR, or stable disease per RECIST v1.1. [0673] Overall survival (OS) [Time Frame: up to 24 months] [0674] Defined as the time from first dose of study drug to death of any cause in each cohort. [0675] Median overall survival. [0676] Number of treatment-related adverse events [Time Frame: up to 24 months] [0677] Adverse events and Severe Adverse Events, type and frequency reported for the first time or worsening of a pre-existing event after first dose of study drug/treatment. [0678] Quality of life—Analysis of quality of life. Form: [0679] (https://www.eortc.org/app/uploads/sites/2/2018/08/Specimen-QLQ-C30-English.pdf

Example 33: Reference Example—Analysis of the Effects of a Combination Therapy Comprising Gemcitabine and Cisplatin

[0680] A combination of gemcitabine and cisplatin is currently a common treatment of cholangiocarcinoma (see Legemiddelh{dot over (a)}ndboka, https://www.legemiddelhandboka.no/T2.2.1.4/Galleveiscancer and Juan Valle et al, Annals of Oncology 25: 391-398, 2014).

[0681] This combination was tested using the experimental procedures described above. The therapeutic index results for the combination using gemcitabine as the primary drug are set out below.

TABLE-US-00013 H69 cell line CC-SW-1 cell line Therapeutic Secondary Combination Combination index drug IC50 Mono IC50 Mono Combination Mono Cisplatin 10.4 6.5 14.7 17.1 0.7 0.4 Therapeutic Secondary H69 cell line EGI-1 cell line index drug Combination Mono Combination Mono Combination Mono Cisplatin 10.4 6.5 3121 16.6. 0.0 0.4 Therapeutic Secondary H69 cell line HuCCT-1 cell line index drug Combination Mono Combination Mono Combination Mono Cisplatin 10.4 6.5 25.3 15.9 0.4 0.4 Therapeutic Secondary H69 cell line TFK-1 cell line index drug Combination Mono Combination Mono Combination Mono Cisplatin 10.4 6.5 10.7 32.9 1,0 0.2

[0682] Gemcitabine monotherapy is one preferred treatment for cholangiocarcinoma. The monotherapy data for gemcitabine in CC-SW-1, EGI-1, HuCCT and TFK-1 shows that the IC50 values for normal cells are much lower than for all cholangiocarcinoma cancer cell lines. The therapeutic index is 0.4, 0.4, 0.4 and 0.2, respectively. This is an indication that gemcitabine is not a good treatment for cholangiocarcinoma.

[0683] The drug combination gemcitabine plus cisplatin is also a preferred clinical treatment cholangiocarcinoma. The combination index data for the cell lines CC-SW-1 and TFK-1 shows some improvement in therapeutic index, however, addition of cisplatin to gemcitabine for cell line EGI-1 destroys the effect of gemcitabine. The combination has no effect on the therapeutic effect for cell line HuCCT.

Example 34: Panobinostat Combination Therapies that Show Synergy in at Least One CCA Cell Line

[0684] The Table below shows which panobinostat combination therapies show synergy in at least one CCA cell line.

TABLE-US-00014 Synergy shown in cell line Drug combination CC-SW-1 EGI-1 HuCC-T1 TFK1 Panobinostat and dactolisib x x x Panobinostat and dasatinib x x Panobinostat and trametinib x x Panobinostat and daporinad x Panobinostat and luminespib x Panobinostat and gemcitabine x Panobinostat and doxorubicin x x x x Panobinostat and topotecan x x Panobinostat and SB-743921 x x Panobinostat and elesclomol x Panobinostat and carboplatin x Panobinostat and cisplatin x Panobinostat and methotrexate x Panobinostat and molibresib x

[0685] In summary, the present inventors have undertaken extensive testing of anticancer drugs and combinations thereof. In this respect, there are currently thousands of known compounds with some reported activity against one or more cancer form. To arrive at the results set out herein, the inventors first selected a library of suitable compounds comprising of 384 compounds. Such a library of compounds would generate more than 120,000 different combinations comprising two substances. Through extensive testing of single compounds and combinations of compounds the inventors have identified 20 combinations that show good efficacy against at least one of the cell lines tested herein. This is about 0.02% of the theoretical number of combinations based on initial selection of 384 different compounds.