COMBINATION THERAPY

Abstract

The invention herein discloses a pharmaceutical combination for use in the treatment of cancer comprising a DNA ligase IV inhibitor and a PARP inhibitor in synergistically effective amounts for simultaneous, separate or sequential administration to a subject in need thereof.

Claims

1. A pharmaceutical combination for use in the treatment of cancer comprising a DNA ligase IV inhibitor and a PARP inhibitor in synergistically effective amounts for simultaneous, separate or sequential administration to a subject in need thereof.

2. The pharmaceutical combination of claim 1, wherein the DNA ligase IV inhibitor comprises a human DNA ligase IV inhibitor.

3. The pharmaceutical combination of claim 1 or 2, wherein the DNA ligase IV inhibitor comprises L189, SCR7, SCR7 pyrazine, S3766-1-X, Rabeprazole or U73122, and optionally L189, SCR7 or S3766-1-X.

4. The pharmaceutical combination of any preceding claim, wherein the PARP inhibitor comprises a PARP-1 inhibitor.

5. The pharmaceutical combination of any preceding claim, wherein the PARP inhibitor comprises Talazoparib, Veliparib, Olaparib, Niraparib or Rucaparib; and optionally Talazoparib or Olaparib.

6. The pharmaceutical combination of any preceding claim, wherein the DNA ligase IV inhibitor comprises L189, SCR7 or S3766-1-X and the PARP inhibitor comprises Talazoparib or Olaparib.

7. The pharmaceutical combination of any preceding claim, wherein the DNA ligase IV inhibitor comprises L189 and the PARP inhibitor comprises Olaparib.

8. The pharmaceutical combination of any preceding claim, wherein the DNA ligase IV inhibitor comprises L189 and the PARP inhibitor comprises Talazoparib.

9. The pharmaceutical combination of any preceding claim, wherein the DNA ligase IV inhibitor comprises SCR7 and the PARP inhibitor comprises Olaparib.

10. The pharmaceutical combination of any preceding claim, wherein the DNA ligase IV inhibitor comprises SCR7 and the PARP inhibitor comprises Talazoparib.

11. The pharmaceutical combination of any preceding claim, wherein the DNA ligase IV inhibitor comprises S3766-1-X and the PARP inhibitor comprises Olaparib.

12. The pharmaceutical combination of any preceding claim, wherein the DNA ligase IV inhibitor comprises S3766-1-X and the PARP inhibitor comprises Talazoparib.

13. The pharmaceutical combination of any preceding claim, wherein the ratio of the DNA ligase IV inhibitor to the PARP inhibitor is selected from any one of 1000:1 to 1:1000, 500:1 to 1:500, 100:1 to 1:100, 50:1 to 1:50, 20:1 to 1:20, 10:1 to 1:10, 5:1 to 1:5, 2:1 to 1:2, 1:1.5 to 1.5:1 and 1:1.

14. The pharmaceutical combination of any preceding claim, wherein the DNA ligase IV inhibitor is present in a greater molar quantity than the PARP inhibitor.

15. The pharmaceutical combination for use in the treatment of cancer of any preceding claim, wherein the cancer is selected from lung cancer, breast cancer, pancreatic cancer, womb (uterus) cancer, ovarian cancer, skin cancer (e.g. melanoma cancer) or colorectal cancer.

16. The pharmaceutical combination for use in the treatment of lung cancer according to claim 15, wherein the lung cancer is selected from small cell lung carcinoma, combined small cell carcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, undifferentiated non-small cell lung cancer, mesothelioma or neuroendocrine tumours.

17. The pharmaceutical combination for use in the treatment of breast cancer according to claim 15, wherein the breast cancer is selected from metastatic breast cancer, ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), invasive ductal breast cancer, invasive lobular breast cancer, inflammatory breast cancer, triple negative breast cancer, medullary carcinoma, tubular carcinoma, mucinous carcinoma, malignant Phyllodes tumour or Paget's disease.

18. The pharmaceutical combination for use in the treatment of pancreatic cancer according to claim 15, wherein the pancreatic cancer is selected from pancreatic ductal adenocarcinoma (PDAC), squamous cell carcinoma, adenosquamous carcinoma, acinar cell carcinoma, solid pseudopapillary neoplasm, pancreatoblastoma, benign precancerous lesions or pancreatic neuroendocrine tumours.

19. The pharmaceutical combination for use in the treatment of skin cancer according to claim 15, wherein the skin cancer is selected from basal cell carcinoma, squamous cell carcinoma or melanoma.

20. The pharmaceutical combination for use in the treatment of ovarian cancer according to claim 15, wherein the ovarian cancer is selected epithelial ovarian cancer, stromal ovarian cancer and germ cell ovarian cancer.

21. A pharmaceutical combination for use in the treatment of cancer of any preceding claim, wherein the cancer is a drug resistant cancer.

22. The pharmaceutical combination for use in the treatment of cancer according to claim 21, wherein the drug resistant cancer is selected from breast, lung or pancreatic cancer.

23. The pharmaceutical combination for use in the treatment of cancer according to any of claims 1 to 6, wherein the cancer is lung cancer and the pharmaceutical combination comprises the DNA ligase inhibitor L189 and the PARP inhibitor Olaparib, and wherein the lung cancer is drug resistant.

24. The pharmaceutical combination for use in the treatment of cancer according to any of claims 1 to 6, wherein the cancer is lung cancer and the pharmaceutical combination comprises the DNA ligase inhibitor L189 and the PARP inhibitor Talazoparib, and wherein the lung cancer is drug resistant.

25. The pharmaceutical combination for use in the treatment of cancer according to any of claims 1 to 6, wherein the cancer is breast cancer and the pharmaceutical combination comprises the DNA ligase inhibitor SCR7 and the PARP inhibitor Olaparib, and wherein the lung cancer is drug resistant.

26. The pharmaceutical combination for use in the treatment of cancer according to any of claims 1 to 6, wherein the cancer is melanoma and the pharmaceutical combination comprises the DNA ligase inhibitor S3766-1-X and the PARP inhibitor Olaparib.

27. The pharmaceutical combination for use in the treatment of cancer according to any of claims 1 to 6, wherein the cancer is ovarian cancer and the pharmaceutical combination comprises the DNA ligase inhibitor S3766-1-X and the PARP inhibitor Olaparib.

28. The pharmaceutical combination for use in the treatment of cancer according to any of claims 1 to 6, wherein the cancer is lung cancer and the pharmaceutical combination comprises the PARR inhibitor Olaparib.

29. The pharmaceutical combination for use in the treatment of cancer according to any of claims 1 to 6, wherein the cancer is pancreatic cancer and the pharmaceutical combination comprises the PARP inhibitor Olaparib.

30. The pharmaceutical combination for use in the treatment of cancer according to any of claims 1 to 6, wherein the cancer is lung cancer and the pharmaceutical combination comprises the PARP inhibitor Talazoparib.

31. The pharmaceutical combination for use in the treatment of cancer according to any of claims 1 to 6, wherein the cancer is pancreatic cancer and the pharmaceutical combination comprises the PARP inhibitor Talazoparib.

32. The pharmaceutical combination of any of claims 23 to 31, wherein the ratio of the DNA ligase IV inhibitor to the PARP inhibitor is selected from any one of 1000:1 to 1:1000, 500:1 to 1:500, 100:1 to 1:100, 50:1 to 1:50, 20:1 to 1:20, 10:1 to 1:10, 5:1 to 1:5, 2:1 to 1:2, 1:1.5 to 1.5:1 and 1:1.

33. The pharmaceutical combination of any of claims 23 to 32, wherein the DNA ligase IV inhibitor is present in a greater molar quantity than the PARP inhibitor.

34. The pharmaceutical combination for use in the treatment of cancer of any preceding claim, wherein the combination is formulated for oral administration or parenteral (e.g. intravenous) administration.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0058] The invention will now be further and more particularly described, by way of example only, and with reference to the accompanying drawings, in which:

[0059] FIG. 1 shows the final dosages of drugs both as monotherapies and in combination incubated with A549 cells for 120 hours for synergy determination.

[0060] FIG. 2 shows raw data returned after performing the cell viability assay.

[0061] FIG. 3 shows the blank corrected cell viability assay raw data.

[0062] FIG. 4 shows cell viability calculated using blank corrected cell viability assay data.

[0063] FIG. 5 shows drug synergism between Olaparib and SCR7 in BT474 cells.

[0064] FIG. 6 shows drug synergism between Talazoparib and L189 in NCIH1975 cells.

[0065] FIG. 7 shows drug synergism between Olaparib and L189 in NCIH1975 cells.

[0066] FIG. 8 shows the effect of RNAi mediated knockdown of ligase IV on ligase IV protein levels in various cancer cell lines.

[0067] FIG. 9 shows the effect of RNAi mediated knockdown of ligase IV on cell viability in various cancer cell lines.

[0068] FIG. 10 shows synergism between RNAi mediated knockdown of ligase IV and Talazoparib in NCIH1975 cells.

[0069] FIG. 11 shows synergism between RNAi mediated knockdown of ligase IV and Olaparib in NCIH1975 cells.

[0070] FIG. 12 shows synergism between RNAi mediated knockdown of ligase IV and Olaparib in

[0071] A549 cells.

[0072] FIG. 13 shows synergism between RNAi mediated knockdown of ligase IV and Olaparib in Capan-1 cells.

[0073] FIG. 14 shows synergism between RNAi mediated knockdown of ligase IV and Talazoparib in Capan-1 cells.

[0074] FIG. 15 shows a 96-well plate map detailing the final dosages of Olaparib and Talazoparib incubated with siRNA transfected cells of a given cell line for HSA synergy determination.

[0075] FIG. 16 shows 5×5 combination plots of Olaparib and S3766-1-X dosages in UACC-62 cancer cells, showing cancer cell kill-rate and Bliss synergy scores.

[0076] FIG. 17 shows 5×5 combination plots of Olaparib and S3766-1-X dosages in OVXF 899L cancer cells, showing cancer cell kill-rate and Bliss synergy scores.

[0077] FIG. 18 shows the LigIV protein binding activity of S37766-1-X.

DETAILED DESCRIPTION OF THE INVENTION

[0078] FIG. 1 shows a 96-well plate map (PLATE 1) where Olaparib (OLA) ranges from 20 μM to 0.08 μM from rows A to F, SCR7 ranges from 60 μM to 0.25 μM from columns 1 to 6, and L189 ranges from 60 μM to 0.25 μM from columns 7 to 12. OLA as a monotherapy ranges from 20 μM to 0.08 μM from wells G1 to G6, whilst Talazoparib (TALA) as a monotherapy ranges from 20 μM to 0.08 μM from wells G7 to G12. As shown in FIG. 1, there is shown a second 96-well plate map (PLATE 2) where TALA ranges from 20 μM to 0.08 μM from rows A to F, SCR7 ranges from 60 μM to 0.25 μM from columns 1 to 6, and L189 ranges from 60 μM to 0.25 μM from columns 7 to 12. SCR7 as a monotherapy ranges from 60 μM to 0.25 μM from wells G1 to G6, whilst L189 as a monotherapy ranges from 60 μM to 0.25 μM from wells G7 to G12. 3-fold serial dilutions were performed to achieve the dosage ranges in both plates. Wells H1 to H4 are DMSO controls, wells H5 to H8 are untreated cell controls, and wells H9 to H12 are blanks; this is the case for both plates.

[0079] FIG. 2 shows a 96-well plate map which presents raw data returned after performing the CellTiter-Glo® luminescent cell viability assay uniformly across all wells of a 96-well plate comprising A549 cells in culture and the drug dosages outlined in PLATE 1 in FIG. 1. The drug dosages outlined in PLATE 1 of FIG. 1 were incubated with A549 cells for 120 hours before performing the cell viability assay. The data obtained from FIG. 1, PLATE 2 were measured, but this data are not shown in FIG. 2.

[0080] FIG. 3 shows a 96-well plate map which presents the raw data returned after performing the CeIlTiter-Glo® luminescent cell viability assay uniformly across all wells of a 96-well plate comprising A549 cells in culture and the drug dosages outlined in PLATE 1 of FIG. 1 that has been blank corrected. Wells H9 to H12 in PLATE 1 of FIG. 1 are blanks containing only cell culture medium without cells, FIG. 3 shows the raw values subtracted by the mean of all blank readings. Blank corrected repeat measurements were also recorded for A549 cells incubated with the drug dosages outlined in PLATE 2 of FIG. 1, but this data is not shown in FIG. 3.

[0081] FIG. 4 shows a 96-well plate map which presents cell viability data for A549 cells after incubation with the dosages outlined in PLATE 1 of FIG. 1 where cell viability is calculated as described in example 1. Cell viability was also calculated, as described in example 1, for A549 cells incubated with the drug dosages outlined in PLATE 2 of FIG. 1, but this data is not shown in FIG. 4.

[0082] FIG. 5 shows a graph with kill-rate on the Y-axis and Olaparib dose on the X-axis showing the kill-rate of Olaparib as a monotherapy (grey), the theoretical additive kill-rate of Olaparib +60 μM SCR7 (dashed line), and the kill-rate of Olaparib in combination with 60 μM SCR7 (black). To an individual skilled in the art, it can be seen that where Olaparib is at 6.67 μM in combination with 60 μM SCR7, there is synergy in BT474 cells.

[0083] FIG. 6 shows a graph with kill-rate on the Y-axis and Talazoparib dose on the X-axis showing the kill-rate of Talazoparib as a monotherapy (grey), the theoretical additive kill-rate of Talazoparib+60 μM L189 (dashed line), and the kill-rate of Talazoparib in combination with 60 μM L189 (black). To an individual skilled in the art, it can be seen that where

[0084] Talazoparib is at 0.25 μM in combination with 60 μM L189, there is synergy in NCIH1975 cells.

[0085] FIG. 7 shows a graph with kill-rate on the Y-axis and Olaparib dose on the X-axis showing the kill-rate of Olaparib as a monotherapy (grey), the theoretical additive kill-rate of Olaparib+60 μM L189 (dashed line), and the kill-rate of Olaparib in combination with 60 μM L189 (black). To an individual skilled in the art, it can be seen that where Olaparib is at 6.67 μM in combination with 60 μM L189, there is synergy in NCIH1975 cells.

[0086] FIG. 8 shows a bar chart with ligase IV protein level plotted against the following cancer cell lines: A549, BT-474, Capan-1 and H1975. For each cancer cell line, the lighter bar represents the level of ligase IV protein in cells treated with small interfering non-targeted RNA (siNT) for 8 days, whilst the darker bar represents cells treated with small interfering RNA targeted to ligase IV mRNA for 8 days causing ligase IV protein knockdown. Small interfering non-targeted RNA is used as a control. Ligase IV protein is knocked down in each cell line after treatment with siRNA targeted to ligase IV mRNA compared to controls.

[0087] FIG. 9 shows a bar chart with cell viability plotted against the following cancer cell lines: A549, BT-474, Capan-1 and H1975. For each cancer cell line, the lighter bar represents cell viability for cells treated with small interfering non-targeted RNA (siNT), whilst the darker bar represents cell viability for cells treated with small interfering RNA targeted to ligase IV mRNA causing ligase IV protein knockdown. Small interfering non-targeted RNA is used as a control. Ligase IV knockdown does not significantly affect cell viability in any cancer cell line compared to controls.

[0088] FIG. 10 shows a graph with H1975 cell kill-rate on the Y-axis and Talazoparib dose on the X-axis showing the kill-rate of Talazoparib as a monotherapy (circles), the kill-rate of LigIV-siRNA as a monotherapy (x marks), the theoretical HSA kill-rate of Talazoparib+LigIV-siRNA (crosses), and the kill-rate of Talazoparib in combination with LigIV-siRNA (squares). Where LigIV-siRNA is used as a monotherapy, a DMSO control is used instead of Talazoparib. Where Talazoparib is used as a monotherapy, a small interfering non-targeted RNA control is used instead of LigIV-siRNA. To an individual skilled in the art, it can be seen that Ligase IV protein knockdown in combination with Talazoparib is synergistic in H1975 cells across the entire concentration range of Talazoparib (μM) tested.

[0089] FIG. 11 shows a graph with H1975 cell kill-rate on the Y-axis and Olaparib dose on the X-axis showing the kill-rate of Olaparib as a monotherapy (circles), the kill-rate of LigIV-siRNA as a monotherapy (x marks), the theoretical HSA kill-rate of Olaparib+LigIV-siRNA (crosses), and the kill-rate of Olaparib in combination with LigIV-siRNA (squares). Where LigIV-siRNA is used as a monotherapy, a DMSO control is used instead of Olaparib. Where Olaparib is used as a monotherapy, a small interfering non-targeted RNA control is used instead of LigIV-siRNA. To an individual skilled in the art, it can be seen that Ligase IV protein knockdown in combination with Olaparib is synergistic in H1975 cells within the range of 0.247—>60 pmol Olaparib.

[0090] FIG. 12 shows a graph with A549 cell kill-rate on the Y-axis and Olaparib dose on the X-axis showing the kill-rate of Olaparib as a monotherapy (circles), the kill-rate of LigIV-siRNA as a monotherapy (x marks), the theoretical HSA kill-rate of Olaparib+LigIV-siRNA (crosses), and the kill-rate of Olaparib in combination with LigIV-siRNA (squares). Where LigIV-siRNA is used as a monotherapy, a DMSO control is used instead of Olaparib. Where Olaparib is used as a monotherapy, a small interfering non-targeted RNA control is used instead of LigIV-siRNA. To an individual skilled in the art, it can be seen that Ligase IV protein knockdown in combination with Olaparib is synergistic in A549 cells within the range of 0.741—20 pmol Olaparib.

[0091] FIG. 13 shows a graph with Capan-1 cell kill-rate on the Y-axis and Olaparib dose on the X-axis showing the kill-rate of Olaparib as a monotherapy (circles), the kill-rate of LigIV-siRNA as a monotherapy (x marks), the theoretical HSA kill-rate of Olaparib+LigIV-siRNA (crosses), and the kill-rate of Olaparib in combination with LigIV-siRNA (squares). Where LigIV-siRNA is used as a monotherapy, a DMSO control is used instead of Olaparib. Where Olaparib is used as a monotherapy, a small interfering non-targeted RNA control is used instead of LigIV-siRNA. To an individual skilled in the art, it can be seen that Ligase IV protein knockdown in combination with Olaparib is synergistic in Capan-1 cells within the range of 0.027—>60 pmol Olaparib.

[0092] FIG. 14 shows a graph with Capan-1 cell kill-rate on the Y-axis and Talazoparib dose on the X-axis showing the kill-rate of Talazoparib as a monotherapy (circles), the kill-rate of LigIV-siRNA as a monotherapy (x marks), the theoretical HSA kill-rate of Talazoparib+LigIV-siRNA (crosses), and the kill-rate of Talazoparib in combination with LigIV-siRNA (squares). Where LigIV-siRNA is used as a monotherapy, a DMSO control is used instead of Talazoparib. Where Talazoparib is used as a monotherapy, a small interfering non-targeted RNA control is used instead of LigIV-siRNA. To an individual skilled in the art, it can be seen that Ligase IV protein knockdown in combination with Talazoparib is synergistic in Capan-1 cells across the entire concentration range of Talazoparib (μM) tested.

[0093] FIG. 15 shows a 96 well plate map where Olaparib ranges from 60 μM to 0.003 μM from A2 to A11, B2 to B11, E2 to El 1 and F2 to F11. Talazoparib ranges from 60 μM to 0.003 μM from C2 to C11, D2 to D11, G2 to G11 and H2 to H11. Rows A to D are seeded with cells treated with LigIV-siRNA whilst rows E to H are seeded with cells treated with small interfering non-targeted RNA. 3-fold serial dilutions were performed to achieve the dosage ranges. Columns 1 and 12 are DMSO controls.

[0094] FIG. 16 (Left) shows a 5×5 combination plot with Olaparib dose ascending from 0 to 10 μM from the bottom row upwards, and S3766-1-X dose ascending from 0 to 100 μM from columns left to right. The leftmost column represents Olaparib as a monotherapy and bottom row represents S3766-1-X as a monotherapy. Shading for each dose pair represents UACC-62 cancer cell kill rate as calculated in example 1. (Right) shows a 5×5 combination plot with Olaparib dose ascending from 0 to 10 μM from the bottom row upwards, and 53766-1-X dose ascending from 0 to 100 μM from columns left to right. The leftmost column represents Olaparib as a monotherapy and bottom row represents 53766-1-X as a monotherapy. Shading in this plot indicates synergy (shading with diagonal lines which ascend from left to right) according to the bliss independence model of synergy determination. The inhibitor(s) where incubated with UACC-62 cells for seven days. The person skilled in the art would understand from this that the combination of Olaparib and 53766-1-X is synergistic as an anticancer treatment in the UACC-62 cancer cell line after 7 days of treatment, particularly at lower concentrations of Olaparib.

[0095] FIG. 17 (Left) shows a 5×5 combination plot with Olaparib dose ascending from 0 to 10 μM from the bottom row upwards, and 53766-1-X dose ascending from 0 to 100 μM from columns left to right. The leftmost column represents Olaparib as a monotherapy and bottom row represents 53766-1-X as a monotherapy. Shading for each dose pair represents OVXF 899L cancer cell kill rate as calculated in example 1. (Right) shows a 5×5 combination plot with Olaparib dose ascending from 0 to 10 μM from the bottom row upwards, and S3766-1-X dose ascending from 0 to 100 μM from columns left to right. The leftmost column represents Olaparib as a monotherapy and bottom row represents S3766-1-X as a monotherapy. Shading in this plot indicates synergy (shading with diagonal lines which ascend from left to right) according to the bliss independence model of synergy determination. The inhibitor(s) where incubated with OVXF 899L cells for seven days. The person skilled in the art would understand from this that the combination of Olaparib and 53766-1-X is synergistic as an anticancer treatment in the OVXF 899L cancer cell line after 7 days of treatment, particularly at lower concentrations of Olaparib.

[0096] FIG. 18 shows LigIV binding activity of 53766-1-X. TRIC traces for 12 53766-1-X concentration points are transformed into a dose-response curve (left) which is modelled to yield the steady-state affinity of the molecular interaction between 53766-1-X and LigIV protein. 53766-1-X binds LigIV protein with a KD of 7.2 μM.

METHODS AND EXAMPLES

Example 1

Investigating the Combination of DNA Ligase IV and PARP Inhibitors on A549 Lung Cancer Cells

[0097] Cell Culture

[0098] A549 cells (Hungarian Academy of Sciences—Semmelweis University—Pathobiochemistry Research Group) were cultured in a 25 cm.sup.3 flask with 5 mL cell culture medium (Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and MycoZap™ Plus-CL) in a humidified CO.sub.2 incubator at 37° C. To seed the cells, cell culture medium was removed from the flask and the cells were washed with 2 ml PBS, then 1 ml trypsine-EDTA solution was added and the cells were incubated in the CO.sub.2 incubator. When the cells detached they were re-suspended in 3 ml cell culture medium to inhibit trypsin and centrifuged with 150×g for 4 minutes at room temperature. The supernatant was discarded and the cells were re-suspended in 5 ml cell culture medium and the exact cell number was determined using hemocytometer after Trypan blue staining. Cells were plated into 2 sterile white 96-well plates at a density of 1000 cells/well in 50 μl cell culture medium and were incubated for 24 hours at 37° C. in CO.sub.2 incubator. After 24 hours the cells were treated with the indicated compounds, giving the final concentrations as shown in FIG. 1, and a final volume of 100 μl. The plates were incubated in a CO.sub.2 incubator at 37° C. for 120 hours.

[0099] Cell Viability Assay

[0100] After the incubation, cell viability was measured with the CeIlTiter-Glo® luminescent cell viability assay (Promega, Madison, Wis., USA). CellTiter GIo™ reagent was added to each well according to the manufacturer's description. The luminescence signal was recorded using a microplate reader (BioTek Synergy 2 Multi-Mode Reader, Winooski, Vt. USA). Raw data was blank corrected and viability was calculated as follows:

Treated/control% (T/C%)

Cell kill-rate=1−(cell viability)

[0101] Dose response curves (using non-linear regression model) were generated and IC.sub.50 values were determined by Graph Pad Prism 5.02 software.

[0102] Determination of Synergy

[0103] Synergy was calculated for each dose pair in all combinations using the Bliss independence model. For each dose-pair in the grid the measured cell viability was converted to effect:

σ.sub.ij, measured=1−ϵ.sub.ij

[0104] Here σ is the effect, ϵ is the viability, and i and j are the doses of compounds.

[0105] Next, theoretical additive effects were determined:

σ.sub.ij, theor=σ.sub.0j+σ.sub.i0−σ.sub.0j*σ.sub.i0

[0106] For each dose-pair the Bliss synergy score, also known as ‘Excess over Bliss’ (EOB) was calculated in the following way:

EOB.sub.i,j=σ.sub.ij, measured−σ.sub.ij, theor

[0107] Here a positive EOB indicates synergism, and a negative EOB indicates antagonism at a given dose pair; the EOB value ranges between (−1 to 1).

[0108] Results

[0109] The invention is now demonstrated with reference to the following experimental data. Cell viability, kill-rate, and synergy were all calculated using the averages across the multiple measurements collected.

TABLE-US-00001 TABLE 1 BT474 cell line-synergistic dose combination of Olaparib and SCR7 BT474 cell line Combination Theoretical Excess Viability/ Kill Rate/σ.sub.ij, Kill Rate/σ.sub.ij, Over Drug Therapy ε.sub.ij measured theor Bliss/EOBij Olaparib 6.67 μM 0.825 0.46 SCR7 60 μM 0.653 Olaparib 6.67 μM 0.443 0.557 0.097 and SCR7 60 μM

TABLE-US-00002 TABLE 2 NCIH1975 cell line-synergistic dose combination of Talazoparib and L189 NCIH1975 cell line Combination Theoretical Excess Viability/ Kill Rate/σ.sub.ij, Kill Rate/σ.sub.ij, Over Drug Therapy ε.sub.ij measured theor Bliss/EOBij Talazoparib 0.25 μM 0.597 0.46 L189 60 μM 0.898 Talazoparib 0.25 μM 0.357 0.643 0.183 and L189 60 μM

TABLE-US-00003 TABLE 3 NCIH1975 cell line-synergistic dose combination of Olaparib and L189 NCIH1975 cell line Combination Theoretical Excess Viability/ Kill Rate/σ.sub.ij, Kill Rate/σ.sub.ij, Over Drug Therapy ε.sub.ij measured theor Bliss/EOBij Olaparib 6.67 μM 0.678 0.39 L189 60 μM 0.897 Olaparib 6.67 μM 0.446 0.554 0.164 and L189 60 μM

Example 2

RNAi Mediated Knockdown of Ligase IV

[0110] The invention has been further demonstrated using RNA interference methods to inhibit ligase IV instead of small molecule compounds. Small interfering RNAs (siRNAs) operate within the RNA interference pathway; siRNAs have been used in this disclosure as RNA interference method of ligase IV inhibition. LigIV-siRNA selectively silences the ligase IV gene and knocks down ligase IV protein levels with no off-target activity (FIG. 8). Inhibitors such as Olaparib and Talazoparib are potent and selective PARP inhibitors and have been used in combination with LigIV-siRNA in various cancer cell lines; synergy has been determined with high confidence which validates the concept of the invention. LigIV-siRNA is synergistic in combination with Talazoparib in H1975 cells (FIG. 10) and Capan-1 cells (FIG. 14). LigIV-siRNA is also synergistic in combination with Olaparib in H1975 cells (FIG. 11), A549 cells (FIG. 12) and Capan-1 cells (FIG. 13). In an embodiment, another means of ligase IV inhibition known in the art may be used for the purpose of the invention.

[0111] Transfection

[0112] The following cancer cell lines A549, BT474, Capan-1 and H1975 were cultured using standard protocols known in the art—for reference see example 1. The cells were seeded onto a multi-well plate and incubated with transfection solution for 24 hours. For each cell line, cells were transfected with LigIV-siRNA whilst a separate sample was transfected with small interfering non-targeted RNA as a control.

[0113] Gel Electrophoresis

[0114] The transfected cells were lysed and the supernatant was recovered for each condition

[0115] (LigIV-siRNA treated and siNT treated) and cell line. The Bradford method was then used to measure total protein content for subsamples of each sample such that protein levels could be normalised before western blot analysis. The samples for each condition and cell line were then run on a polyacrylamide gel (SDS-PAGE) for 15 minutes at 80 V and 1.5-2 hours at 130 V to separate proteins according to their molecular mass. Proteins on the gels were transferred into polyvinylidene difluoride (PVDF) membranes.

[0116] Western Blotting

[0117] The PVDF membranes were blocked and then incubated in primary antibody solution (e.g. DNA Ligase IV (D5N5N) Rabbit mAb (Cell Signalling 14649S)) overnight. The membranes were then incubated in secondary antibody solution for 1 hour before being developed and imaged. Ligase IV protein levels were then normalised to β-Actin and quantified for each cell line and each condition. Ligase IV protein levels were significantly knocked down after transfection with LigIV-siRNA in each cell line compared to controls (FIG. 8). Moreover, ligase IV protein knockdown as a monotherapy had no significant effect on cell viability in each cell line compared to controls (FIG. 9).

[0118] RNAi Cell Viability Assay

[0119] After gene silencing, cells for each cell line—both LigIV-siRNA and siNT treated—were plated into sterile white 96-well plates (one 96-well plate per cell line) and were incubated for 24 hours at 37° C. in CO.sub.2 incubator. After 24 hours the cells were treated with Olaparib or Talazoparib at the indicated concentrations for 5 days treatment time (FIG. 15) in duplicate—3-fold serial dilutions were made. Untreated control wells were supplemented with DMSO containing medium to 0.12% final concentration. After incubation, CellTiter GIo™ reagent was added to each well according to the manufacturer's description. The luminescent signal was recorded and cell viability was calculated—for reference see example 1. Synergy was then determined according to the HSA synergy model.

[0120] According to the HSA model, synergy is observed where the measured kill-rate for a given PARP inhibitor dosage and LigIV-siRNA pair is greater than the theoretical HSA kill-rate for that same PARP inhibitor dosage and LigIV-siRNA pair in a given cell line. HSA (theoretical) is the maximal single agent effect at a given dose pair.

Example 3

S3766-1-X in Combination with Olaparib

[0121] The inventors of the present disclosure have demonstrated that the pharmaceutical combination of a PARP inhibitor and a LigIV inhibitor is synergistic as an anticancer treatment for various lineages of cancer including varieties of breast cancer, lung cancer and pancreatic cancer. The inventors have also demonstrated that the pharmaceutical combination of a PARP inhibitor and a LigIV inhibitor is synergistic as an anticancer treatment for other varieties of cancer including melanoma and ovarian cancer. Compound “53766-1-X”, a derivative of the LigIV inhibitor “SCR7” was tested in combination with Olaparib at different dosage pairs for cancer cell killing activity in both a melanoma cancer cell line (UACC-62) (FIG. 16) and an ovarian cancer cell line (OVXF 899L) (FIG. 17). The cancer cell line “OVXF 899L” is a PDX (Patient Derived Xenograft) cancer cell line. For synergy determination, the same methodology was used as is set forth in example 1. The person skilled in the art would understand from this that the pharmaceutical combination of Olaparib and 53766-1-X is synergistic as an anticancer treatment for both UACC-62 cancer cells and OVXF 899L cancer cells, particularly at lower dosages of Olaparib. These findings highlight the broad utility of such a pharmaceutical combination as an effective anticancer therapy for a wide variety of clinical indications.

Example 4

LigIV Binding Assay

[0122] LigIV binding activity of S3766-1-X was measured on a NanoTemper Dianthus NT.23PicoDuo device. The Dianthus technology is based on the TRIC effect (temperature-related intensity-change of fluorescent molecules). These fluorescence changes are very sensitively dependent on the electronic microenvironment of the fluorescent molecule. Thus, if a fluorescent dye is coupled to a target molecule (protein); its TRIC effect will react to changes by ligand binding or by ligand binding-induced conformational changes. Mixes of the fluorescently-labeled target molecule and increasing concentrations of the ligand molecule (1000 μ-5.6 nM for the S3766-1-X titrant) are placed in 12 adjacent wells. An infrared laser is used to generate a precise temperature gradient while an LED is used for the excitation of fluorescent molecules in the wells. The laser-induced temperature gradient leads to the TRIC-based changes of the fluorescence of the labeled target molecule, which is recorded by fluorescence optics. Different TRIC-responses are obtained for different states of the target molecule, i.e. ligand-unbound and ligand-bound states.

[0123] The comparison of the TRIC traces for all 12 ligand-concentration points reveals that there is a change in the TRIC response that correlates with the ligand concentration. The TRIC traces can finally be transformed to a dose-response curve, which can be fitted to various models to yield the steady-state affinity of the analysed molecular interaction—a K.sub.D of 7.2 μM in the case of S3766-1-X (FIG. 18). The person skilled in the art would understand from this that S3766-1-X binds with high affinity to its intended target—LigIV protein. For assay conditions see table 4.

TABLE-US-00004 TABLE 4 TRIC assay conditions: RED-tris-NTA labeled LIG4 vs. compound +/− dsDNA Fluor. Molecule 50 nM LigIV (DVU1-DVV1, PC13347-1) Fluorophore 25 nM RED-tris-NTA 1.sup.st generation Labeling Labeling buffer: Assay Buffer 100 nM protein/50 nM dye; conditions Incubation time: 30 min Instrument Dianthus NT.23PicoDuo Measuring LED Power: 15%; MST settings: 1-5-1 (s) (initial parameter fluorescence-MST on time-back-diffusion), Duplicates Assay buffer 20 nM Hepes pH 7.5, 30 mM NaCl, 10 mM MgCl2, 5 mM Glutathione, 0.05% Tween