SELECTIVE PARP1 INHIBITORS TO TREAT CANCER

Abstract

The disclosure provides a selective inhibitor of DNA-binding to poly (ADP-ribose) polymerase 1 (PARP1), or a pharmaceutically acceptable salt or solvate thereof, for use in treating, ameliorating or preventing cancer. The treatment may be given to a subject suffering from or at risk of osteoporosis or a subject requiring a long-term therapy.

Claims

1. A method of treating, preventing or ameliorating cancer in a subject, the method comprising administering to a subject in need of such treatment, a therapeutically effective amount of a selective inhibitor of DNA-binding to poly (ADP-ribose) polymerase 1 (PARP1), or a pharmaceutically acceptable salt or solvate thereof, wherein the subject is suffering from or at risk of osteoporosis or requires a long-term therapy.

2. The method of claim 1, wherein the inhibitor does not inhibit the other functions of PARP1 besides DNA-binding.

3. The method of claim 2, wherein the other functions of PARP1 comprise PARP1's role in a cellular response to oxidative stress independent of DNA damage and/or PARP1's role in cell metabolic regulation and metabolic activity, calcium signalling and calcification, and apoptosis.

4. The method of claim 1, wherein the inhibitor does not inhibit or block the NAD+ binding site of PARP1.

5. The method of claim 1, wherein the inhibitor is an inhibitor of the zinc finger of PARP1.

6. The method of claim 1, wherein the subject is a post-menopausal woman, a woman who has had a hysterectomy before the age of 45, a woman who has suffered from absent periods for more than 6 months as a result of over exercising or too much dieting or a man suffering from hypogonadism.

7. The method of claim 1, wherein the subject is suffering from rheumatoid arthritis.

8. The method of claim 1, wherein the cancer is a solid tumour or solid cancer.

9. The method of claim 1, wherein the cancer is blood cancer, bowel cancer, brain cancer, breast cancer, cervical cancer, endometrial cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer or skin cancer.

10. The method of claim 9, wherein the cancer is breast cancer, prostate cancer, myeloma or cervical cancer.

11. The method of claim 1, wherein the long-term therapy is maintenance therapy.

12. The method of claim 1, wherein the inhibitor is not an inhibitor of PARP2 and/or PARP3.

13. The method of claim 1, wherein the inhibitor is a gold complex, optionally a gold (I) complex.

14. (canceled)

15. The method of claim 1, wherein the inhibitor is a polymeric water-soluble complex.

16. The method of claim 1, wherein the inhibitor is a compound of Formula I, Formula II, Formula III, Formula IV or Formula V: ##STR00004## or a pharmaceutically acceptable salt and/or solvate thereof.

17. The method of claim 16, wherein the compound is a compound of Formula I or Formula II.

18. The method of claim 17, wherein the compound is a compound of Formula IIa: ##STR00005## or a pharmaceutically acceptable salt and/or solvate thereof.

19. The method of claim 17, wherein the inhibitor is sodium aurothiomalate, potassium aurothiomalate or calcium aurothiomalate, optionally wherein the inhibitor is a compound of Formula Ia: ##STR00006##

20. (canceled)

21. The method of claim 1, wherein the inhibitor is used in combination with a drug that damages DNA.

22. The method of claim 21, wherein the inhibitor is used in combination with an ataxia-telangiectasia mutated and rad3-related protein kinase (ATR) inhibitor, a checkpoint inhibitor, a vascular endothelial growth factor (VEGF) inhibitor or a wee1 inhibitor, optionally wherein the checkpoint inhibitor is a programmed cell death protein 1 (PD-1) inhibitor, a programmed death-ligand 1 (PD-L1) inhibitor or a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitor.

23-25. (canceled)

Description

[0059] FIG. 1 is a graph showing how PARP1 and PARP2 activity is split between DNA-dependent and DNA-independent reactions;

[0060] FIG. 2 is a graph showing the percentage inhibition of PARP1 for different concentrations of auranofin and aurothiomalate;

[0061] FIG. 3 is a graph showing the percentage inhibition of PARP1 and PARP2 for different concentrations of aurothiomalate;

[0062] FIG. 4 is a graph showing the percentage inhibition of PARP1 and PARP2 for different concentrations of aurothioglucose;

[0063] FIG. 5 is a PARP amino acid sequence alignment;

[0064] FIG. 6 is a graph showing the percentage inhibition of PARP1 and PARP2 for different concentrations of minocycline;

[0065] FIG. 7 shows scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of cross-sections of the long limb bone from rats where the rats were (a) untreated; (b) fed a high adenine/low protein diet which caused chronic kidney disease (CKD); or (c) fed a high adenine/low protein diet which caused CKD and administered minocycline; and

[0066] FIG. 8 shows analysis of the bone density of the long limb bone in the rats.

EXAMPLE 1ASSAYING OF DNA-DEPENDENT AND DNA-INDEPENDENT PARP1 ACTIVITY AND INHIBITOR DOSE-RESPONSES

[0067] The PARP inhibitor assay is a direct fluorescence-based concentration measurement of reaction product formation. The assay reagents are sold as a commercial kit (see http://www.merckmillipore.com/GB/en/product/PARP1-Enzyme-Activity-Assay,MM_NF-17-10149). To measure PARP inhibition, the NAD+ substrate concentration should be set at Km (the Michaelis constant) to enable identifications of all types of inhibitors (competitive, uncompetitive and non-competitive (allosteric) (the latter represents a mode of action of Zn-finger inhibitors)), direct calculation of inhibitor potency (Ki) and in vivo modelling. (See in and literature sited therein: Michael G. Acker, Douglas S. Auld. Considerations for the design and reporting of enzyme assays in high-throughput screening applications. Perspectives in Science (2014) 1, 56-73). All other PARP inhibitor assays reported in the literature (and including those available commercially) either alter NAD+ significantly to label it for measurement, or only include very small concentrations of NAD+(if at all) such that the competitive kinetics are not representative.

[0068] PARP activity and inhibition was measured for human full length active PARP1 (CS207770, Merck), PARP2 (ab198766, Abeam) and PARP3 (ab79638, Abeam) proteins. Inhibitor compounds (Sodium Aurothiomalate and Aurothioglucose, Sigma-Aldrich and Auranofin, Bio-Techne) at different concentrations (1, 10 and 100 nM, 1, 10 and 100 M final) were added to the reaction buffer, concocted as a 1:1 mixture of Merck kit buffer with 50 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl.sub.2, 0.05% Tween-20, pH 8.0, Sigma), and incubated with PARP1 (2.5 ng/L final), PARP2 (2.2 ng/L final) or PARP3 (55 ng/L final) at room temperature for 30 min.

[0069] Further, activated DNA (2 ng/L final), 13-NAD (60 and 400 M final for PARP1/2 and PARP3, respectively) and Nicotinamidase (200 ng/L final) were added and incubated at 37 C. for 45 min. Total reaction volume was 25 L.

Controls were executed as follows: [0070] 1. Control of 0% inhibition contained reaction sample without inhibitor; [0071] 2. Control of 100% inhibition of PARP1/2/3 activity contained reaction sample without -NAD; and [0072] 3. Control of 100% inhibition of DNA-dependent activity contained reaction sample without DNA.

[0073] After the plates were cooled down to room temperature, 25 L of Merck proprietary reagent was added to the reaction mixture and incubated at mild shaking for 45 min. Fluorescence measurement was carried out at excitation wavelength of 410 nm and emission of 460 nm in Fluostar Omega microplate reader (BMG Labtech).

[0074] Calculation of PARP1/2/3 Activity

[0075] Total PARP1/2/3 activity was calculated as a difference between control (1) and control (2). DNA-independent activity was calculated as a difference between control (1) and control (3). DNA-dependent activity was calculated as the difference between the total PARP1/2/3 activity and DNA-independent activity. As shown in FIG. 1, about 80% of PARP1 activity is DNA-dependent. However, potentially up to 30% of PARP1 activity can be DNA-independent.

[0076] Calculation of PARP inhibition

[0077] Inhibitory values were converted into percentages according to controls. Controls (1) and (3) were used in the case of PARP1, because only inhibition of DNA-dependent activity was observed, and the results are shown in FIG. 2. Controls (1) and (2) were used in the case of PARP2/3, because inhibition of total PARP2/3 activity (both DNA-dependent and DNA-independent reactions) was observed. FIGS. 3 and 4 show the percentage inhibition of PARP1 and PARP2 for different concentrations of aurothiomalate and aurothioglucose, respectively.

[0078] IC50 values were determined as inhibitor concentration at 50% inhibition, and are given in table 1.

TABLE-US-00001 TABLE 1 IC.sub.50 values for Auranofin, Aurothiomalate and Aurothioglucose IC.sub.50/nm Sodium Auranofin Aurothiomalate Aurothioglucose PARP1 1400 98 48 7 120 7 PARP2 1160 81 1190 190 4452 623 PARP3 1160 93 1140 20

[0079] As shown in FIG. 2 and Table 1, auranofin, as a mixed group aurothio- and phosphine compound only inhibits PARP1 and PARP2 at very high concentrations. Accordingly, auranofin is not suitable as a drug candidate, as doses this high are not known to be safe.

[0080] However, sodium aurothiomalate and aurothioglucose, i.e. pure aurothio compounds, have an IC.sub.50 for PARP1 which is 30-10 more potent than auranofin, so both are within acceptable safety dosage. Furthermore, as shown in FIGS. 3 and 4 and Table 1, neither aurothiomalate nor aurothioglucose inhibit PARP2 or PARP3, and so can be viewed as selective PARP1 inhibitors.

EXAMPLE 2EFFECT OF PARP2 INHIBITION ON BONE DENSITY

[0081] In order to prove that inhibiting PARP2 is a significant risk factor for osteoporosis, we first identified a PARP2-specific inhibitor, using the PARP inhibitor assay described in Example 1. Using this assay, the inventors found that minocycline is a specific PARP2 inhibitor and inhibits PARP2 with an IC.sub.50 of 2.8 M, and inhibits PARP1 with an IC.sub.50 of 204.5 M, see FIG. 6. It will be noted that the PARP2 vs PARP1 selectivity factor for minocycline is greater than 70.

[0082] The effects of minocycline on bone calcification processes were evaluated in an in vivo rat model. The rats were fed a high adenine/low protein diet in order to develop chronic kidney disease (CKD) and associated hyperphosphatemia and medial vascular calcification. It is also expected to cause increased rates of bone turnover, allowing the inventors to examine whether inhibition of PARP2 enzymatic activity during bone remodelling affected mineralization.

[0083] 14 of the rats on the high adenine/low protein diet were treated with 50 mg/kg/day of minocycline for 6 weeks. At the end of the study period cross sections of the long limb bone were analysed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), see FIG. 7, and the area fraction of solid bone in the cortical area of the bone cross section was quantified from these images, see FIG. 8. Statistical significance was determined by Mann-Whitney test.

[0084] As shown in FIG. 8b, a 25% reduction in the area fraction of solid bone was observed in the rats treated with the minocycline when compared to both the control and the rats which had been fed the high adenine/low protein diet but not treated with minocycline.

CONCLUSIONS

[0085] The inventors believe that the reason sodium aurothiomalate and aurothioglucose inhibit PARP1 and not PARP2/3 is because they inhibit the PARP1 Zn finger domain/domains from binding to DNA, a pre-requisite step in the activation of PARP1 in DNA repair. It is thought that the Zn.sup.2+ ion is released and replaced by a Au.sup.+ ion and there is a conformational change. The resultant gold finger domain does not bind to DNA and therefore SSBs are not repaired. Accordingly, the synthetic lethality mechanism aimed at killing cancer cells is preserved.

[0086] The inventors have shown that PARP1 has DNA-independent activity. This activity is maintained in the presence of sodium aurothiomalate and aurothioglucose. Thus, PARP1 is available to undertake its other essential cellular DNA-independent roles in non-cancerous cells in the rest of the body.

[0087] The inventors have shown that PARP2 inhibition affects osteoblast function. Such inhibition would be particularly problematic in a patient suffering from or at increased risk of osteoporosis, e.g. a patient suffering from breast cancer or prostate cancer. Inhibition of osteoblast function would also be problematic, and greatly increase the risk of osteoporosis, in patients requiring long-term treatments, such as patients receiving maintenance therapy.

[0088] Furthermore, PARP2/3 activity is not inhibited by aurothiomalate and aurothioglucose, thus both enzymes are preserved to undertake their essential cellular roles, and osteoblast function will not be affected. Accordingly, the inventors have shown that aurothio compounds, such as aurothiomalate and aurothioglucose, could be used as highly selective oncology drugs for cancer therapy and/or as a second line of treatment to reduce drug resistance to other PARP inhibitors that target the catalytic site of PARP enzymes. This will be particularly beneficial for patients suffering from or at risk of osteoporosis. It will be noted that these compounds offer a significant advantage over approved drugs such as olaparib (LYNPARZA) which inhibit both PARP1 and PARP2.