PREPARATIONS OF RADIUM-224 AND PROGENIES FOR USE IN RADIONUCLIDE THERAPY IN COMBINATION WITH DNA REPAIR INHIBITORS

Abstract

The present invention related to a combination of radium-224 (.sup.224Ra) and/or progeny of .sup.224Ra, and a DNA repair inhibitor for use in the treatment of cancer. The DNA repair inhibitor can for example be a poly (ADP-ribose) polymerase inhibitor (PARPi), a MGMT inhibitor, a DNA-dependent protein kinase inhibitor (DNA-PK inhibitor), an ataxia telangiectasia and Rad3-related (ATR) kinase inhibitor, an ataxia telangiectasia mutated (ATM) kinase inhibitor, a Wee1 kinase inhibitor, or a checkpoint kinase 1 and 2 (CHK 1/2) inhibitor. The radium-224 (.sup.224Ra) and/or progeny of .sup.224Ra can be comprised in nano- and/or micro sized particles.

Claims

1. A method of inhibiting a cancer comprising administering a product combination comprising: a) Radium-224 (224Ra) and/or a progeny of .sup.224Ra, and b) a DNA repair inhibitor,

2-19. (canceled)

20. The method according to claim 1, wherein the DNA repair inhibitor is selected from the group consisting of a poly (ADP-ribose) polymerase inhibitor (PARPi), a MGMT inhibitor, a DNA-dependent protein kinase inhibitor (DNA-PK inhibitor), an ataxia telangiectasia and Rad3-related (ATR) kinase inhibitor, an ataxia telangiectasia mutated (ATM) kinase inhibitor, a Wee1 kinase inhibitor, and a checkpoint kinase 1 and 2 (CHK1/2) inhibitor.

21. The method according to claim 1, wherein the progeny of .sup.224Ra is .sup.220Rn, .sup.216Po, .sup.212Pb or .sup.212Bi.

22. The method according to claim 1, wherein the PARPi is selected from the group consisting of Olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib, Pamiparib, CEP 9722, E7016, and 3-Aminobenzamide.

23. The method according to claim 1, wherein the PARPi is Olaparib or Niraparib.

24. The method according to claim 1, wherein the PARPi is Rucaparib.

25. The method according to claim 1, wherein the PARPi is Talazoparib.

26. The method according to claim 1, further comprising nano- and/or micro sized particles.

27. The method according to claim 26, wherein the nano- and/or microparticles are made of CaCO.sub.3, or a calcium phosphate.

28. The method according to claim 27, wherein the CaCO.sub.3 is selected from the group consisting of PEG modified CaCO.sub.3, protein modified CaCO.sub.3, carbohydrate modified CaCO.sub.3, lipid modified CaCO.sub.3, vitamin modified CaCO.sub.3, organic compound modified CaCO.sub.3, polymer modified CaCO.sub.3 and inorganic crystal modified CaCO.sub.3.

29. The method according to claim 26, wherein the size of the particle is from 1 nm to 500 μm.

30. The method according to claim 26, wherein the product composition is a particle suspension comprising monodisperse or polydisperse particles.

31. The method of claim 1, wherein the cancer is selected from the group consisting of ovarian cancer, colorectal cancer, stomach cancer, liver cancer, peritoneal cancer, pleural cancer, pleural effusion, malignant mesothelioma, pericardial cancer and bladder cancer.

32. The method of claim 1, wherein the cancer is a metastatic cancer, selected from the group consisting of sarcomas, osteocarcoma, lung cancer, non-small-cell-lung cancer, pancreatic cancer, breast cancer, neoplastic meningitis, glioblastoma and astrocytoma, melanoma and prostate cancer.

33. The method of claim 1, wherein the amount of radionuclide is 1 kBq to 10 GBq per dosing, or with an amount of radionuclide that is 50 MBq to 100 GBq suitable for multidose industrial scale production.

34. The method according to claim 1, wherein the product combination further comprises a diluent, vehicle, carrier, carrier solution, surfactant, or excipient.

35. The method according to claim 34, wherein the carrier is selected from particles, proteins, antibodies, antibody fragments, or peptides.

36. The method according to claim 1, wherein a) and b) are administered together or separately.

37. The method according to claim 1, wherein i) a) and b) are administered within the same day, ii) b) is started one or several days before start of a), or iii) b) is initiated one or several days after start of a).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0118] FIG. 1: SKOV-3 cells treated with the combination of one-point concentration of one drug with escalating concentrations of the combining drug resulted in synergistic interaction of .sup.224Ra and (A) niraparib and (B) olaparib that were time point and dose dependent as illustrated by the CI grayscale and number matrix.

EXAMPLES

Example 1. Production of .SUP.224.Ra

[0119] The .sup.224Ra-generator was prepared by mixing a .sup.228Th source with an actinide resin and loading it on a column. A source of .sup.228Th in 1 M HNO.sub.3 was purchased from Eckert & Ziegler (Braunschweig, Germany) or Oak Ridge National Laboratory (TN, USA), and an actinide resin based on the DIPEX® Extractant was acquired from Eichrom Technologies LLC (Lisle, IL) in the form of a pre-packed cartridge of 2 mL. The material in an actinide resin cartridge was extracted and the resin was preconditioned with 1 M HCl (Sigma-Aldrich). A slurry of approximately 0.25 mL actinide resin, 0.25 mL 1 M HCl and 0.1 mL .sup.228Th in 1 M HNO.sub.3 was prepared in a vial (4 mL vial, E-C sample, Wheaton, Millville, NJ) and incubated with gentle agitation for immobilization of .sup.228Th for 4 h at room temperature and let to rest for a few days. The generator column was prepared in a 1 mL filtration column (Isolute SPE, Biotage AB, Uppsala, Sweden) by first applying 0.2 mL of inactive actinide resin, before the portion containing .sup.228Th was loaded on top. The inactive resin was introduced in the bottom of the column to serve as a catcher layer if .sup.228Th was released during operation of the generator. Later, the capacity of the generator was increased. A slurry consisting of 0.4 mL actinide resin, 0.5 mL .sup.228Th in 1 M HNO.sub.3 and 0.5 mL 1 M HCl was prepared as described above, before it was loaded onto the generator column.

[0120] Radium-224 could be eluted regularly from the generator column in 1-2 mL of 1 M HCl. For further purification, the crude eluate from the generator column was loaded directly onto a second actinide resin column. The second column was washed with 1 M HCl. This eluate was evaporated to dryness in a closed system. The vial was placed in a heater block and flushed with N.sub.2-gas through a Teflon tube inlet and outlet in the rubber/Teflon septum on the vial. The acid vapor was led into a beaker of saturated NaOH by a stream of N.sub.2-gas. The radioactive residue remaining after evaporation was dissolved in 0.2 mL or more of 0.1 M HCl. A radioisotope calibrator (CRC-25R, Capintec Inc., Ramsey, NJ) was used to measure the total extracted activity in the process.

Example 2—Combination of Radiotherapy using Radium-224 with DNA Repair Inhibitor

[0121] Ovarian cancer cell lines: ES-2 (clear cell carcinoma) and SKOV-3 (adenocarcinoma), were used to investigate the pharmacodynamic interactions resulting from the paired combination of Radium-224 (.sup.224Ra) with a DNA repair inhibitor exemplified by the poly (ADP-ribose) polymerase inhibitors (PARPi); niraparib and olaparib.

[0122] Methodology: The cells in supplemented McCoy's 5A-modified growth medium were plated at a volume of 200 μl and cell concentration of 5,000 cells/ml in black 96-well plates treated for cell culture (Thermo Fisher, MA USA). The cells were incubated for 24 hours under controlled culture conditions of 5% CO.sub.2, 37° C. and 95% humidity in a cell incubator for 22-24 hours.

[0123] Thereafter, escalating concentrations of .sup.224Ra (0.2-150 kBq/ml), niraparib (0.05-13.2 μM) and olaparib (0.15-92.2 μM) were added to the cells (in duplicates) for the assessment of single agent cytotoxicity and determination of the IC.sub.50 for each agent. The IC.sub.50 of the single agent is a guide for the appropriate choice for the concentrations to use for the paired combinations. Additionally, the cells were simultaneously exposed to paired combinations of .sup.224Ra with either of the PARPi at escalating concentrations (in duplicates). This was done to assess the pharmacodynamic interactions resulting from the combination of the treatment agents. The cells were further incubated with the treatment agents over a period of 5 days.

[0124] At variant timepoints following the addition of the treatment agents i.e. days 3, 4 and 5, cell proliferation was assessed by determining the DNA content in each well which is proportional to the total cell number per well.

[0125] The growth medium was aspirated, and the cells were incubated with a dye that binds cellular nucleic acids using the CyQuant NF cell proliferation assay kit (Thermo Fisher) following the manufacturers' protocol. The fluorescence was measured using the Fluoroskan Ascent Fluorometer (Thermo Fisher).

[0126] The pharmacodynamic interactions of the output were determined by calculating the combination index (CI) where CI<0.90 is synergistic, CI of 0.90-1.1 is additive and CI>1.1 is antagonistic as described in Table 1.

TABLE-US-00001 TABLE 1 Description of the combination index range defining the pharmacodynamic interactions: synergism, additive and antagonism, as described by Chou and Talalay. CI range Description <0.1 Very strong synergism 0.1-0.3 Strong synergism 0.3-0.7 Synergism  0.7-0.85 Moderate synergism 0.85-0.9  Slight synergism 0.9-1.1 Additive 1.1-1.2 Slight antagonism  1.2-1.45 Moderate antagonism 1.45-3.3  Antagonism 3.3-10  Strong antagonism >10 Very strong antagonism

[0127] Results: Data presented in Table 2 shows the IC.sub.50 concentrations of .sup.224Ra, niraparib and olaparib in ES-2 and SKOV-3 cells. These concentrations elaborate on the sensitivity of each cell line to the different treatments and it is clear to observe that SKOV-3 is less sensitive to PARPi than ES-2 cells.

TABLE-US-00002 TABLE 2 IC.sub.50 of single agent treatment. Single agent IC.sub.50 Day 3 Day 4 Day 5 .sup.224Ra Niraparib Olaparib .sup.224Ra Niraparib Olaparib .sup.224Ra Niraparib Olaparib Cell line (kBq) (μM) (μM) (kBq) (μM) (μM) (kBq) (μM) (μM) ES-2 0.77 0.69 3.51 0.33 0.78 1.23 0.66 0.62 2.21 SKOV-3 4.37 5.16 21.38 1.84 2.83 23.60 0.44 2.2 1.83

[0128] When paired combinations were evaluated, the IC50 of each individual agent in the combination consequently decreased as shown in Table 3

TABLE-US-00003 TABLE 3 IC.sub.50 of paired combination treatment Paired combination IC.sub.50 Day 3 Day 4 Day 5 .sup.224Ra Niraparib Olaparib .sup.224Ra Niraparib Olaparib .sup.224Ra Niraparib Olaparib Cell line (kBq) (μM) (μM) (kBq) (μM) (μM) (kBq) (μM) (μM) ES-2 0.38 0.42 — 0.22 0.25 — 0.32 0.36 — 0.38 — 1.43 0.13 — 0.50 0.25 — 0.93 SKOV-3 5.62 2.45 — 0.98 0.43 — 0.50 0.22 — 3.35 — 10.21 0.88 — 2.67 0.26 — 0.81

[0129] An evaluation of the combination indices of paired combinations at concentrations similar to and less than the single agent IC.sub.50 revealed that the pharmacodynamic interactions of the pairs were mainly synergistic. This is shown in Table 4 for ES-2 cells and Table 5 for SKOV-3 cells.

TABLE-US-00004 TABLE 4 The combination index of the paired combinations of .sup.224Ra with niraparib and olaparib in the ES-2 cell line. [.sup.224Ra] [Niraparib] μM [Olaparib] μM (kBq) 0.18 0.73 0.62 2.47 Day 3 0.16 0.99 0.66 0.89 0.55 0.65 0.58 0.52 0.57 0.61 Day 4 0.16 0.77 0.67 0.99 0.61 0.65 0.75 0.73 0.54 0.53 Day 5 0.16 0.99 0.61 0.73 0.60 0.65 0.46 0.48 0.49 0.47

TABLE-US-00005 TABLE 5 The combination index of the paired combinations of .sup.224Ra with niraparib and olaparib in the SKOV-3 cell line. [.sup.224Ra] [Niraparib] μM [Olaparib] μM (kBq) 0.41 1.65 2.88 11.5 Day 3 0.94 1.43 0.81 0.41 0.55 3.76 0.57 0.74 0.34 0.80 Day 4 0.94 0.40 0.50 0.40 0.40 3.76 0.44 0.67 0.40 0.51 Day 5 0.94 0.80 0.57 0.57 0.63 3.76 0.56 0.68 0.63 0.60

Example 3—Evaluation of One-point Concentration of One Drug with Escalating Concentrations of the Combining Drug in SKOV-3 Cells

[0130] The combination effect of .sup.224Ra with olaparib and niraparib was evaluated in SKOV-3 at one-point concentrations of one drug with escalating concentrations of the combining drug. Skov-3 has in previous examples been shown to be the least sensitive cell line to all drug tested. The assay used and calculations made were done as described in Example 2. The chosen one-point concentrations were a fraction of the IC50 of each drug at 72 hours to ensure that the level of cytotoxicity of each individual drug was below the inhibitory threshold, in order to capture the interaction effect of the combination. The drug IC50 fractions used were 25% for niraparib, 46% for olaparib and 46% for .sup.224Ra.

[0131] The combination of .sup.224Ra and the PARPi resulted in synergistic interactions, depending on the timepoint of assessment. The combination of .sup.224Ra with olaparib was synergistic which emphasises on the benefit of drug combination in tumours with low drug sensitivity.

[0132] Results are shown in FIG. 1.

ITEMS

[0133] 1. A combination of: [0134] a) Radium-224 (224Ra) and/or progeny of .sup.224Ra, and [0135] b) a DNA repair inhibitor, [0136] for use in the treatment of cancer.

[0137] 2. The combination for use according to item 1, wherein the a DNA repair inhibitor is selected from the group consisting of a poly (ADP-ribose) polymerase inhibitor (PARPi), a MGMT inhibitor, a DNA-dependent protein kinase inhibitor (DNA-PK inhibitor), an ataxia telangiectasia and Rad3-related (ATR) kinase inhibitor, an ataxia telangiectasia mutated (ATM) kinase inhibitor, a Wee1 kinase inhibitor, and a checkpoint kinase 1 and 2 (CHK1/2) inhibitor.

[0138] 3. The combination for use according to items 1-2, wherein the progeny of .sup.224Ra is selected from the group consisting of .sup.220Rn, .sup.216Po, .sup.212Pb and .sup.212Bi.

[0139] 4. The combination for use according to any of the previous items, wherein the progeny of .sup.224Ra is .sup.220Rn.

[0140] 5. The combination for use according to any of the previous items, wherein the progeny of .sup.224Ra is .sup.216Po.

[0141] 6. The combination for use according to any of the previous items, wherein the progeny of .sup.224Ra is .sup.212Pb.

[0142] 7. The combination for use according to any of the previous items, wherein the progeny of .sup.224Ra is .sup.212Bi.

[0143] 8. The combination for use according to any of the previous items, wherein the PARPi is selected from the group consisting of Olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib, Pamiparib, CEP 9722, E7016, and 3-Aminobenzamide.

[0144] 9. The combination for use according to any of the previous items, wherein the PARPi is Olaparib.

[0145] 10. The combination for use according to any of the previous items, wherein the PARPi is Rucaparib.

[0146] 11. The combination for use according to any of the previous items, wherein the PARPi is Niraparib.

[0147] 12. The combination for use according to any of the previous items, wherein the PARPi is Talazoparib.

[0148] 13. The combination for use according to any of the previous items, further comprising nano- and/or micro sized particles.

[0149] 14: The combination for use according to any of the previous items, wherein the carriers are selected from the group consisting of particles, proteins, including antibodies, antibody fragment, or a peptide.

[0150] 15. The combination for use according to item 13, wherein the nano- or microparticles are made of CaCO.sub.3, or calcium phosphates including Ca-Hydroxyaptatite, or fluoroapatite.

[0151] 16. The combination for use according to item 13, wherein the nano- or microparticles are made of MgCO.sub.3, SrCO.sub.3 or BaCO.sub.3

[0152] 17. The combination for use according to item 15, wherein the CaCO.sub.3 is selected from the group consisting of PEG modified CaCO.sub.3, protein modified CaCO.sub.3, carbohydrate modified CaCO.sub.3, lipid modified CaCO.sub.3, vitamin modified CaCO.sub.3, organic compound modified CaCO.sub.3, polymer modified CaCO.sub.3 and/or inorganic crystal modified CaCO.sub.3.

[0153] 18. The combination for use according to items 14-17, wherein the size of the particle is from 1 nm to 500 μm.

[0154] 19. The combination for use according to items 14-17, wherein the composition is a particle suspension comprising monodisperse or polydisperse particles.

[0155] 20. The combination for use according to items 1-19, which is used in the treatment of cancer, and selected from the group consisting of ovarian cancer, colorectal cancer, stomach cancer, liver cancer, peritoneal cancer, pleural cancer, pleural effusion, malignant mesothelioma, pericardial cancer and bladder cancer.

[0156] 21. The combination for use according to items 1-19, which is used in the treatment of metastatic cancer, and which treatment is selected from the group consisting of sarcomas, osteocarcoma, lung cancer, non-small-cell-lung cancer, pancreatic cancer, breast cancer, neoplastic meningitis, glioblastoma and astrocytoma, melanoma and prostate cancer.

[0157] 22. The combination for use according to items 1-21, wherein the amount of radionuclide is 1kBq to 10GBq per dosing, or with an amount of radionuclide that is 50 MBq to 100 GBq suitable for multidose industrial scale production.

[0158] 23. The combination for use according to items 1-22, wherein the combination or composition comprises one or more selected from the group consisting of a diluent, vehicle, carrier solution, surfactant, and/or excipient.

[0159] 24. The combination for use according to items 1-23, wherein a) and b) are administered together or separately.

[0160] 25. The combination for use according to items 1-24, wherein a) and b) are administered within the same day.

[0161] 26. The combination for use according to items 1-24, wherein b) is started one or several days before start of a).

[0162] 27. The combination for use according to items 1-24, wherein b) is initiated one or several days after start of a).