WO2016051213A1 - Par inhibition - Google Patents

Abstract

The present invention relates to a compound capable of inhibiting PAR by forming a supramolecular structure with PAR, for use in inhibiting PAR or for use in treating or preventing a disease susceptible to PAR inhibition. The present invention further relates to a method of inhibiting PAR and a method of treating or preventing a disease susceptible to PAR inhibition, using a compound capable of inhibiting PAR by forming a supramolecular structure with PAR. Preferably the compound is dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof. The present invention also relates to a PAR inhibitor for use as a cardioprotective agent or for use in treating or preventing the cardiotoxic side effects of anthracyclines. The present invention further relates to a method of treating or preventing the cardiotoxic side effects of anthracyclines, using a PAR inhibitor. Furthermore, the present invention relates to a PAR inhibitor for use in treating or preventing extravasation. The present invention further relates to a method of treating or preventing extravasation, using a PAR inhibitor. Moreover, the present invention relates to a polymer of (i) polyadenylated RNA or DNA, and (ii) dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, which may be used to treat or prevent a disease susceptible to PAR inhibition. Moreover, the present invention relates to a combination of a PAR inhibitor and a PARP inhibitor, or a combination of a PAR inhibitor and a second PAR inhibitor, which may be used to treat or prevent a disease susceptible to PAR inhibition. Lastly, the present invention relates to the use of dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, for pre-treatment of mesenchymal stem cells.

Description

PAR Inhibition

Field of the invention The present invention relates to a compound capable of inhibiting PAR by forming a supramolecular structure with PAR, for use in inhibiting PAR or for use in treating or preventing a disease susceptible to PAR inhibition. The present invention further relates to a method of inhibiting PAR and a method of treating or preventing a disease susceptible to PAR inhibition, using a compound capable of inhibiting PAR by forming a supramolecular structure with PAR. Preferably the compound is dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof.

The present invention also relates to a PAR inhibitor for use as a cardioprotective agent or for use in treating or preventing the cardiotoxic side effects of anthracyclines. The present invention further relates to a method of treating or preventing the cardiotoxic side effects of anthracyclines, using a PAR inhibitor.

Furthermore, the present invention relates to a PAR inhibitor for use in treating or preventing extravasation. The present invention further relates to a method of treating or preventing extravasation, using a PAR inhibitor.

Moreover, the present invention relates to a polymer of (i) polyadenylated RNA or DNA, and (ii) dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, which may be used to treat or prevent a disease susceptible to PAR inhibition.

Moreover, the present invention relates to a combination of a PAR inhibitor and a PARP inhibitor, or a combination of a PAR inhibitor and a second PAR inhibitor, which may be used to treat or prevent a disease susceptible to PAR inhibition.

Lastly, the present invention relates to the use of dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, for pre-treatment of mesenchymal stem cells.

Background of the invention Dexrazoxane has been in clinical use since 1981. Initially, it was considered as an antineoplastic agent, but the antineoplastic potential was insufficient for further development. Subsequently, dexrazoxane was found to protect against the cardiotoxicity of anthracyclines and was licensed for use in Europe in 1992 for the prevention of cardiomyopathy associated with doxorubicin administration. Today, dexrazoxane is clinically used as a cardioprotective agent in patients receiving anthracycline-based chemotherapy and to prevent anthracycline extravasation injury. Dexrazoxane hydrochloride (marketed as Cardioxane® or Zinecard®) has been approved for use as a cardioprotective agent. It has been used to protect the heart against the cardiotoxic side effects of anthracyclines, such as doxorubicin, epirubicin, daunorubicin and idarubicin. Dexrazoxane hydrochloride is administered intravenously as a sterile, pyrogen-free lyophilizate. The intravenous administration of dexrazoxane is in acidic condition with HC1 adjusting the pH, because dexrazoxane is rapidly degraded at pH above 7.

The exact mechanism by which dexrazoxane exerts its cardioprotective effect has not been fully elucidated, however based on the available evidence the following mechanism has been suggested (Summary of Product Characteristics for Cardioxane®, 2014). The dose-dependent cardiotoxicity observed during anthracycline administration may be due to anthracycline-induced iron-dependent free radical oxidative stress on the relatively unprotected cardiac muscle. Dexrazoxane, an analogue of EDTA (ethylene diamine tetra-acetic acid), is hydrolysed in cardiac cells to the ring-opened form. Both dexrazoxane and its ring-opened form are capable of chelating metal ions. It is generally thought that they can provide cardioprotection by scavenging metal ions, thus preventing the Fe³⁺-anthracycline complex from redox cycling and forming reactive radicals. When used as cardioprotective agent, dexrazoxane hydrochloride is administered by a short intravenous infusion over 15 minutes, approximately 30 minutes prior to anthracycline administration at a dose equal to 10 times the anthracycline dose. Since typically 50 mg/m² doxorubicin or 60 mg/m² epirubicin is used per administration, typically 500 mg/m² or 600 mg/m² dexrazoxane as its hydrochloride is administered. Dosages are calculated per body surface area in m². Prior to administration dexrazoxane hydrochloride lyophilizate is reconstituted and diluted with an aqueous diluent such that 500mg dexrazoxane as its hydrochloride (589mg) are dissolved in 50125ml of aqueous diluent. Following reconstitution and dilution, the solution ready for intravenous administration has a concentration of dexrazoxane of 4-10 mg/ml and a pH of 2.2-4.2.

Dexrazoxane hydrochloride (marketed as Savene® or Totect®) has also been approved for use as a treatment of extravasation resulting from intravenous anthracycline chemotherapy. Extravasation is an adverse event in which chemotherapies containing anthracyclines leak out of the blood vessel and necrotize the surrounding tissue.

Again, the exact mechanism by which dexrazoxane treats or prevents extravasation has not been fully elucidated. Dexrazoxane is believed to have two major mechanisms of action: chelation of iron and inhibition of topoisomerase 2. It is currently believed that one or both of these mechanisms are responsible for dexrazoxane's ability to treat or prevent extravasation, although it is not known to what extent each of these mechanisms contributes (Summary of Product Characteristics for Savene®, 2011).

When used for the treatment of extravasation, dexrazoxane hydrochloride is administered by intravenous infusion over 1-2 hours, within the first six hours after extravasation and for two consecutive days thereafter. The recommended dose is 1000 mg/m² on day 1 and 2, and 500 mg/m² on day 3. Dosages are calculated per body surface area in m². For patients with a body surface area of more than 2m², a single dose should not exceed 2000mg. For a body surface area of 1.2-3.5 rn², the daily dose to be administered varies between 600-2000 mg.

Prior to administration dexrazoxane hydrochloride lyophilizate is reconstituted and diluted with an aqueous diluent. Following reconstitution and dilution, the solution ready for intravenous administration has a concentration of dexrazoxane of 1.2-4 mg/ml.

Summary of the invention

Poly(ADP-ribose) (PAR) is the polymer product of poly(ADP-ribose) polymerase (PARP). In a first aspect, the present invention provides a compound for use in inhibiting PAR, wherein the compound or a metabolite thereof is capable of inhibiting PAR by forming a supramolecular structure with PAR. The first aspect of the present invention further provides a compound for use in treating or preventing a disease susceptible to PAR inhibition, wherein the compound or a metabolite thereof is capable of inhibiting PAR by forming a supramolecular structure with PAR. The first aspect of the present invention further provides a method of inhibiting PAR using a compound or a metabolite thereof which is capable of inhibiting PAR by forming a supramolecular structure with PAR.

The first aspect of the present invention further provides a method of treating or preventing a disease susceptible to PAR inhibition, comprising administering to a patient in need thereof a compound or a metabolite thereof which is capable of inhibiting PAR by forming a supramolecular structure with PAR.

The compound or the metabolite thereof, capable of inhibiting PAR by forming a supramolecular structure with PAR, preferably forms the supramolecular structure with PAR through non-covalent interactions, including ionic attractions and hydrogen bonding. Preferably the compound is dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, or a pharmaceutically acceptable salt thereof, wherein the compound may be in racemic, enantiomerically enriched or enantiomerically pure form.

In a preferred embodiment of the first aspect of the present invention, the compound is dexrazoxane, levrazoxane, razoxane, desmethyl dexrazoxane (ICRF-154), methyl dexrazoxane (ICRF-193), ICRF-202, ICRF-192, ICRF-158, bimolane, methyl bimolane, sobuzoxane, merbarone or mitindomide, or a pharmaceutically acceptable salt thereof. In one embodiment, the compound is dexrazoxane or a pharmaceutically acceptable salt thereof. In an alternative embodiment, the compound is not dexrazoxane or a pharmaceutically acceptable salt thereof.

In another preferred embodiment of the first aspect of the present invention, the disease is cancer, a disease of the cardiovascular system, central nervous system, gastrointestinal tract, immune system or skin, a disease associated with the ear or eye, a hepatic disease, an infection (including a viral infection), a metabolic disease, a musculoskeletal disease, an oral disease, a renal disease, a respiratory disease, or ischaemia/reperfusion injury to an organ. In one embodiment, the disease is not anthracycline-induced cardiotoxicity or extravasation. In another embodiment, the disease is not a brain tumour. In another embodiment, the disease is not a tumour of the central nervous system. In another embodiment, the disease is not breast cancer. In another embodiment, the disease is not cancer. In another preferred embodiment of the first aspect of the present invention, the compound is used in combination with a PARP inhibitor. Examples of PARP inhibitors include, but are not limited to, 3-aminobenzamide, Iniparib (BSI 201), BMN-673, Olaparib (AZD-2281), Rucaparib (AG014699, PF-01367338), Veliparib (ABT-888), CEP 9722, Niraparib (MK4827), BGB-290, PJ34 and INO-1001, and pharmaceutically acceptable salts thereof.

In a second aspect, the present invention provides a PAR inhibitor for use as a cardioprotective agent. The second aspect of the present invention further provides a PAR inhibitor for use in treating or preventing the cardiotoxic side effects of anthracyclines (such as doxorubicin, epirubicin, daunorubicin and idarubicin).

The second aspect of the present invention further provides a method of treating or preventing the cardiotoxic side effects of anthracyclines (such as doxorubicin, epirubicin, daunorubicin and idarubicin), comprising administering a PAR inhibitor to a patient in need thereof.

In a preferred embodiment of the second aspect of the present invention, the PAR inhibitor is dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, or a pharmaceutically acceptable salt thereof, which may be in racemic, enantiomerically enriched or enantiomerically pure form. In another preferred embodiment, the PAR inhibitor is dexrazoxane, levrazoxane, razoxane, desmethyl dexrazoxane (ICRF-154), methyl dexrazoxane (ICRF-193), ICRF- 202, ICRF-192, ICRF-158, bimolane, methyl bimolane, sobuzoxane, merbarone or mitindomide, or a pharmaceutically acceptable salt thereof. In one embodiment, the PAR inhibitor is not dexrazoxane or a pharmaceutically acceptable salt thereof.

In another preferred embodiment of the second aspect of the present invention, the PAR inhibitor is used in combination with a PARP inhibitor. Examples of PARP inhibitors include, but are not limited to, 3-aminobenzamide, Iniparib (BSI 201), BMN- 673, Olaparib (AZD-2281), Rucaparib (AG014699, PF-01367338), Veliparib (ABT- 888), CEP 9722, Niraparib (MK4827), BGB-290, PJ34 and INO-1001, and pharmaceutically acceptable salts thereof.

In another preferred embodiment of the second aspect of the present invention, the patient to be treated is a human below the age of 18 years. Preferably the PAR inhibitor is administered to the human below the age of 18 years in an amount of 200-2000 mg/m², preferably 400-1000 mg/m², preferably 500-600 mg/m², wherein the amount is calculated per body surface area in m². Preferably the PAR inhibitor is administered to the human below the age of 18 years by intravenous infusion, preferably over 10-30 minutes, preferably 15-60 minutes before anthracycline administration.

In a third aspect, the present invention provides a PAR inhibitor for use in treating or preventing extravasation.

The third aspect of the present invention further provides a method of treating or preventing extravasation, comprising administering a PAR inhibitor to a patient in need thereof.

In a preferred embodiment of the third aspect of the present invention, the extravasation is caused by intravenous anthracycline chemotherapy (such as doxorubicin, epirubicin, daunorubicin and idarubicin). In another preferred embodiment of the third aspect of the present invention, the PAR inhibitor is dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, or a pharmaceutically acceptable salt thereof, which may be in racemic, enantiomerically enriched or enantiomerically pure form. In another preferred embodiment, the PAR inhibitor is dexrazoxane, levrazoxane, razoxane, desmethyl dexrazoxane (ICRF-154), methyl dexrazoxane (ICRF-193), ICRF- 202, ICRF-192, ICRF-158, bimolane, methyl bimolane, sobuzoxane, merbarone or mitindomide, or a pharmaceutically acceptable salt thereof. In one embodiment, the PAR inhibitor is not dexrazoxane or a pharmaceutically acceptable salt thereof.

In another preferred embodiment of the third aspect of the present invention, the PAR inhibitor is used in combination with a PARP inhibitor. Examples of PARP inhibitors include, but are not limited to, 3-aminobenzamide, Iniparib (BSI 201), BMN-673, Olaparib (AZD-2281), Rucaparib (AG014699, PF-01367338), Veliparib (ABT-888), CEP 9722, Niraparib (MK4827), BGB-290, PJ34 and INO-1001, and pharmaceutically acceptable salts thereof.

In another preferred embodiment of the third aspect of the present invention, the patient to be treated is a human below the age of 18 years. Preferably the PAR inhibitor is administered to the human below the age of 18 years in an amount of 200-2000 mg/m² per day for 1, 2, 3, 4 or 5 consecutive days, preferably 500-1000 mg/m² per day for 1, 2, 3 or 4 consecutive days, wherein the amount is calculated per body surface area in m². Preferably the PAR inhibitor is administered to the human below the age of 18 years by intravenous infusion, preferably over 0.5-4 hours.

In a fourth aspect, the present invention provides a polymer of:

(i) polyadenylated RNA or DNA, and

(ii) dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, or a pharmaceutically acceptable salt thereof, which may be in racemic, enantiomerically enriched or enantiomerically pure form.

Analogues, derivatives and metabolites of dexrazoxane and levrazoxane include, but are not limited to, desmethyl dexrazoxane (ICRF-154), methyl dexrazoxane (ICRF-193), ICRF-202, ICRF-192, ICRF-158, bimolane, methyl bimolane and sobuzoxane, and pharmaceutically acceptable salts thereof. Preferably the polymer is a polymer of (i) polyadenylated RNA or DNA, and (ii) dexrazoxane or a pharmaceutically acceptable salt thereof.

Preferably the polymer of the fourth aspect of the present invention can be administered orally or intravenously. The polymer is more stable than dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, and therefore adapted to be administered orally. The polymer is stable at pH 7-8 and therefore adapted to be administered intravenously.

Preferably the polymer of the fourth aspect of the present invention has a longer shelf- life than dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof.

The polymer of the fourth aspect of the present invention can be used to treat or prevent a disease susceptible to PAR inhibition. Preferably the disease is cancer, a disease of the cardiovascular system, central nervous system, gastrointestinal tract, immune system or skin, a disease associated with the ear or eye, a hepatic disease, an infection (including a viral infection), a metabolic disease, a musculoskeletal disease, an oral disease, a renal disease, a respiratory disease, or ischaemia/reperfusion injury to an organ.

In a fifth aspect, the present invention provides a combination of a PAR inhibitor and a PARP inhibitor, or a combination of a PAR inhibitor and a second PAR inhibitor.

The combination of the fifth aspect of the present invention can be used to treat or prevent a disease susceptible to PAR inhibition. Preferably the combination has a synergistic effect when used to treat or prevent a disease susceptible to PAR inhibition. Preferably the disease is cancer, a disease of the cardiovascular system, central nervous system, gastrointestinal tract, immune system or skin, a disease associated with the ear or eye, a hepatic disease, an infection (including a viral infection), a metabolic disease, a musculoskeletal disease, an oral disease, a renal disease, a respiratory disease, or ischaemia/reperfusion injury to an organ.

In a preferred embodiment of the fifth aspect of the present invention, the PAR inhibitor is dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, or a pharmaceutically acceptable salt thereof, which may be in racemic, enantiomerically enriched or enantiomerically pure form. In another preferred embodiment, the PAR inhibitor is dexrazoxane, levrazoxane, razoxane, desmethyl dexrazoxane (ICRF-154), methyl dexrazoxane (ICRF-193), ICRF- 202, ICRF-192, ICRF-158, bimolane, methyl bimolane, sobuzoxane, merbarone or mitindomide, or a pharmaceutically acceptable salt thereof. In one embodiment, the PAR inhibitor is dexrazoxane or a pharmaceutically acceptable salt thereof. In another preferred embodiment of the fifth aspect of the present invention, the PARP inhibitor is 3-aminobenzamide, Iniparib (BSI 201), BMN-673, Olaparib (AZD-2281), Rucaparib (AG014699, PF-01367338), Veliparib (ABT-888), CEP 9722, Niraparib (MK4827), BGB-290, PJ34 or INO-1001, or a pharmaceutically acceptable salt thereof.

In another preferred embodiment of the fifth aspect of the present invention, the second PAR inhibitor is Iduna, ME0328, AG- 14361, UPF 1069, AZD 2461 or A-966492, or a pharmaceutically acceptable salt thereof.

In a sixth aspect, the present invention provides use of a compound for pre-treatment of mesenchymal stem cells, wherein the compound is dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, or a pharmaceutically acceptable salt thereof, wherein the compound may be in racemic, enantiomerically enriched or enantiomerically pure form. Analogues, derivatives and metabolites of dexrazoxane and levrazoxane include, but are not limited to, desmethyl dexrazoxane (ICRF-154), methyl dexrazoxane (ICRF-193), ICRF-202, ICRF-192, ICRF- 158, bimolane, methyl bimolane and sobuzoxane, and pharmaceutically acceptable salts thereof. Preferably the compound is dexrazoxane or a pharmaceutically acceptable salt thereof.

Unless specified otherwise, the following paragraphs refer to all aspects of the present invention. The compounds of the present invention inhibit or sequester PAR and therefore are PAR inhibitors, PAR sequestrators, PAR antagonists or PAR blockers, and these terms can be used interchangeably.

Non-limiting examples of compounds of the present invention are depicted in Figure 1. In a preferred embodiment of the present invention, the compound is dexrazoxane, levrazoxane, razoxane, desmethyl dexrazoxane (ICRF-154), methyl dexrazoxane (ICRF- 193), ICRF-202, ICRF-192, ICRF-158, bimolane, methyl bimolane, sobuzoxane, merbarone or mitindomide, or a pharmaceutically acceptable salt thereof. Analogues, derivatives and metabolites of dexrazoxane and levrazoxane include, but are not limited to, desmethyl dexrazoxane (ICRF-154), methyl dexrazoxane (ICRF-193), ICRF-202, ICRF-192, ICRF-158, bimolane, methyl bimolane and sobuzoxane, and pharmaceutically acceptable salts thereof.

The compounds of the present invention can be used both, in their free base form and their acid addition salt form. For the purposes of this invention, a salt of a compound of the present invention is an acid addition salt. Acid addition salts are preferably pharmaceutically acceptable, non-toxic addition salts with suitable acids, including but not limited to inorganic acids such as hydrohalogenic acids (for example, hydrofluoric, hydrochloric, hydrobromic or hydroiodic acid) or other inorganic acids (for example, nitric, perchloric, sulphuric or phosphoric acid); or organic acids such as organic carboxylic acids (for example, propionic, butyric, glycolic, lactic, mandelic, citric, acetic, benzoic, salicylic, succinic, malic or hydroxysuccinic, tartaric, fumaric, maleic, hydroxymaleic, mucic or galactaric, gluconic, pantothenic or pamoic acid), organic sulphonic acids (for example, methanesulphonic, trifluoromethanesulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, benzenesulphonic, toluene-p-sulphonic, naphthalene-2-sulphonic or camphorsulphonic acid) or amino acids (for example, ornithinic, glutamic or aspartic acid). The acid addition salt may be a mono- or di-acid addition salt. A preferred salt is a hydrofluoric, hydrochloric, hydrobromic, hydroiodic, sulphuric, phosphoric or organic acid addition salt. A more preferred salt is a hydrochloric acid addition salt.

The compound of the present invention may be in racemic form, which means that the compound comprises about equal amounts of enantiomers, for example ±10%. The compound may be in enantiomerically enriched form, which means that the compound comprises more than 60% of one enantiomer, preferably more than 70%, preferably more than 80%. The compound may be in enantiomerically pure form, which means that the compound comprises more than 90% of one enantiomer, preferably more than 95%, preferably more than 99%. In a preferred embodiment of the first, fourth and fifth aspects of the present invention, a disease susceptible to PAR inhibition includes cancer, a disease of the cardiovascular system, central nervous system, gastrointestinal tract, immune system or skin, a disease associated with the ear or eye, a hepatic disease, an infection (including a viral infection), a metabolic disease, a musculoskeletal disease, an oral disease, a renal disease, a respiratory disease, or ischaemia/reperfusion injury to an organ. In one embodiment, the disease susceptible to PAR inhibition is not anthracycline-induced cardiotoxicity or extravasation. In another embodiment, the disease susceptible to PAR inhibition is not a brain tumour. In another embodiment, the disease susceptible to PAR inhibition is not a tumour of the central nervous system. In another embodiment, the disease susceptible to PAR inhibition is not breast cancer. In another embodiment, the disease susceptible to PAR inhibition is not cancer.

In a preferred embodiment of any aspect of the present invention, the patient to be treated is a human. Now that the present inventor has found that dexrazoxane, levrazoxane, merbarone, mitindomide and analogues, derivatives and metabolites thereof are PAR inhibitors, it is clear that they are useful in the treatment or prevention of a large number of diseases, including but not limited to the diseases listed in the following paragraphs, which have all been indicated in the literature as being mediated substantially or in some significant part by PARP/PAR.

PAR inhibitors are useful in the treatment or prevention of cancer, in particular tumours that have inherent DNA repair defects (including tumours with germline BRCA 1/2 mutations, tumours that are deficient in BRCA 1/2, and tumours with defective homologous repair [HR] mechanisms that result from epigenetic modifications of BRCA 1/2 and/or mutations and defects in various proteins critical to HR pathways including RAD51, RAD54, DSSi, RPAl, ATM, CHK2 and PTEN (phosphatase and tensin homolog), Fanconi's anaemia protein, TNKS (tankyrase) and EMSY); tumours that are characterized by an upregulation of PARP activity; and tumours that are resistant to treatment with PARP inhibitors (in particular when resistance is not due to the development of alternative means of HR repair that may, for example, include the restoration of the wild-type BRCA1/2 open reading frame).

PAR inhibitors are useful in the treatment or prevention of cancer, in particular for the following:

• As monotherapy or in combination with DNA-damaging agents for the treatment of patients with tumours with defects in DNA repair mechanisms, in particular the homologous recombination pathway, for example due to BRCA mutations

• In combination with an inhibitor of phosphoinositide 3-kinase (PI3K) for the treatment of patients with breast cancer tumours with elevated activity of the PI3K pathway • To sensitize resistant cells to the effects of alkylating agents in patients with cancer or any condition for which therapy with an alkylating agent is indicated or deemed appropriate

• As single agent therapy for the treatment of patients with breast and ovarian cancer with BRCA mutations

• For the treatment of patients with BRCA mutation-associated solid tumours that include prostate, pancreatic, lung and endometrial tumours, other than BRCA mutation-associated breast and ovarian cancers

• As single agent therapy or in combination with chemotherapy for the treatment of patients with various cancer types that include breast, ovarian, colorectal and glioma

• In combination with chemotherapy or radiation for the treatment of patients with various cancer types that include breast, colorectal, glioblastoma multiforme and melanoma

• In combination with gemcitabine for the treatment of patients with breast and lung cancers

• To enhance the effects of temozolomide in patients with cancer

• In combination with temozolomide for the treatment of patients with advanced solid tumours

• In combination with temozolomide for the treatment of patients with melanoma · In combination with temozolomide for the treatment of patients with advanced solid malignancies that include melanoma, desmoid tumour, prostatic and pancreatic cancer, and leiomyosarcoma

• In combination with temozolomide for the treatment of patients with chemotherapy-na^'ive metastatic melanoma

· In combination with temozolomide for the treatment of patients with metastatic breast cancer with unknown BRCA mutation status

• In combination with temozolomide for the treatment of patients with advanced malignant melanoma

• In combination with temozolomide for the treatment of patients with metastatic colorectal cancer

• In combination with temozolomide and radiation therapy for the treatment of patients with newly-diagnosed glioblastoma multiforme

• In combination with dacarbazine for the treatment of patients with advanced melanoma

· As single agent therapy for the treatment of patients with advanced breast and ovarian cancer, that includes BRCAl- and BRCA2-deficient tumours As single agent therapy for the treatment of patients with metastatic colorectal cancer

In combination with paclitaxel for the treatment of patients with metastatic triple negative breast cancer

As single agent therapy for the treatment of patients with recurrent advanced breast, epithelial ovarian, peritoneal or fallopian tube carcinoma, that includes tumours with BRCA mutations

As single agent therapy for the treatment of patients with advanced high-grade serous and undifferentiated ovarian cancer and triple negative breast cancer, that includes tumours with BRCA mutations

As single agent therapy for the treatment of patients with previously-treated serous ovarian cancer

In combination with low dose metronomic cyclophosphamide for the treatment of patients with chemotherapy-resistant ER/PR positive, HER2/neu-negative metastatic breast cancer in patients who received prior regimens that included taxane and capecitabine

In combination with an mFOLFOX-6 regimen for the treatment of patients with metastatic unresectable pancreatic cancer

In combination with gemcitabine and carboplatin for the treatment of treatment- naive and pre-treated patients with triple negative breast cancer

In combination with gemcitabine and carboplatin for the treatment of patients with recurrent platinum-sensitive ovarian, fallopian tube and peritoneal carcinoma, in tumours of unknown BRCA status

In combination with gemcitabine and carboplatin for the treatment of patients with metastatic triple negative breast cancer

In combination with topotecan for the treatment of patients with advanced breast cancer in tumours of unknown BRCA status

In combination with bevacizumab for the treatment of patients with advanced solid malignancies

In combination with metronomic cyclophosphamide for the treatment of patients with advanced solid tumours that includes ovarian, carcinoid, breast, colon, pancreatic, urothelial, melanoma, sarcoma, endometrial tumours, tumours of unknown primary origin, and including tumours with BRCA mutations

As single agent therapy for the treatment of patients with unspecified advanced refractory solid tumours • In combination with carboplatin and paclitaxel for the treatment of patients with advanced (IIIB-IV) non-small cell ling cancer

• For the management of intestinal adenomas in patients with familial adenomatous polyposis in the prevention of colorectal carcinogenesis

· In combination with either irinotecan or oxaliplatin for the treatment of patients with colon cancer

• In combination with an inhibitor of histone deacetylase (HDAC) for the treatment of patients with hepatocellular cancer in which tumour cells have been shown to be sensitive to inhibition by both an inhibitor of PARP and HDAC

· As adjunctive therapy to sensitize tumour cells to the individual or combined cytotoxic effects of N-methyl-N-nitrosourea (NMU), i,3-bis-(2-chloroethyl)-i- nitrosourea (carmustine, BCNU), bleomycin, camptothecin and ionizing radiation

• As an anti-angiogenic agent for the treatment of patients with cancer

• As prophylactic treatment for non-neoplastic BRCA2-deficient cells in patients who are heterozygous for BRCA2

• As primary follow-up treatment after surgery in patients in whom BRCA2 deficiency has led to tumourigenesis

• In combination with ionizing radiation for the treatment of patients with head and neck cancer

· In combination with ionizing radiation for the treatment of patients with lymphoma

• In combination with whole brain radiation for the treatment of patients with brain metastases

• In combination with chest wall and nodal irradiation for the treatment of patients with inflammatory or loco-regionally recurrent breast cancer

· In combination with DNA-damaging agents for the treatment of patients with sporadic tumours that have no intrinsic DNA repair defects

• In combination with platinum-based agents for the treatment of patients with pancreatic cancer that has a BRCA2-related DNA repair defect

• In combination with gemcitabine plus radiotherapy for the treatment of patients with pancreatic cancer

• To potentiate the effects of monofunctional alkylating agents such as dacarbazine (DTIC)

• To potentiate the cytotoxic effects of topoisomerase 1 poisons that include camptothecin derivatives such as topotecan

· As a chemosensitizing agent for the treatment of patients with diverse types of cancer who are receiving therapy with melphalan • As a preventative agent in cancers highly associated with inflammatory phenotypes that are characterized by oxidative clustered DNA lesions

• As a resistance-modifying agent in patients with ovarian cancer who are receiving treatment with cisplatin

• As a resistance-modifying agent in patients with cancer who are receiving a regimen that includes cisplatin, etoposide or mitoxantrone, either alone or in combination

• To attenuate the development and progress of intestinal mucositis in patients receiving cancer chemotherapy that includes 5-fluorouracil

• In combination with radiopharmaceuticals that include radium 223 for the treatment of patients with castration-resistant prostate cancer that is metastatic to bone

PAR inhibitors are useful in the treatment or prevention of diseases of the cardiovascular system, such as cardiomyopathy, heart failure, ischaemia, reperfusion injury, toxic myocardial injury, myocardial infarction, endothelial dysfunction, vascular disease, atherosclerosis, circulatory shock, and conditions that are characterized by oxidative and nitrosative stress (including myocardial reperfusion injury and heart transplantation), and useful in the peri-operative management of patients undergoing cardiopulmonary bypass surgery. In particular, PAR inhibitors are useful in the treatment or prevention of the following diseases of the cardiovascular system:

• For the treatment of anthracycline-induced cardiomyopathy that includes heart failure

• For the treatment of acute and chronic heart failure

• For the treatment of acute cardiomyopathy

• For the treatment of cardiomyopathy including iatrogenic cardiomyopathies

• For the treatment of diabetic cardiomyopathy

• For the treatment of ischaemia/ reperfusion injury to the heart

• For the treatment of ischaemia/ reperfusion injury associated with aortic clamping

• For the treatment of myocardial ischaemia/ reperfusion injury

• For the treatment of toxic myocardial injury

• For the attenuation of age-related endothelial dysfunction

• For the attenuation of hypertension-associated endothelial dysfunction and the treatment of vascular disease that is associated with hypertension

• To prevent remodelling, preserve systolic function and delay transition of hypertensive cardiomyopathy to heart failure • For the treatment of cardiovascular disease that is characterized by endothelial dysfunction

• For the management of atherosclerotic disease and vascular disease that is associated with atherosclerosis

· For the treatment of patients with various conditions that are characterized by oxidative and nitrosative stress and which include myocardial reperfusion injury and heart transplantation

• For the treatment of patients who have had a myocardial infarction

• For the peri-operative management of patients undergoing cardiopulmonary bypass surgery

• For the prevention and reversal of the atherosclerotic process and associated cardiovascular clinical sequelae

• For the treatment of circulatory shock PAR inhibitors are useful in the treatment or prevention of diseases of the central nervous system, such as brain injury, injuries to the central nervous system characterized by reperfusion (including stroke, cerebrovascular accidents, and transient ischaemic attack), stroke, ischaemia, traumatic spinal cord injury, injury to hippocampal neurons, cortical cells, glioma, microglia, motor neurons, astrocytes or corpus striatum, amyotrophic lateral sclerosis (motor neurone disease, Lou Gehrig's disease), cortical trauma, MPTP-induced parkinsonism, ataxia telangiectasia (Louis- Bar syndrome), multiple sclerosis, neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease and Huntingdon's disease), meningitis-associated CNS complications, bacterial meningitis, transverse myelitis, neurotrauma, and encephalomyelitis, useful to ameliorate neurotoxicity and catecholamine depletion associated with or induced by intravenous administration of MPTP, and useful to attenuate loss of NAD+ in patients with ataxia telangiectasia and to attenuate cerebral vasospasm following subarachnoid haemorrhage. In particular, PAR inhibitors are useful in the treatment or prevention of the following diseases of the central nervous system:

• For the treatment of traumatic and post-traumatic brain injury

• For the treatment of injuries to the central nervous system that are characterized by reperfusion including stroke, cerebrovascular accidents, and transient ischaemic attack (TLA; ministroke)

· For the treatment of stroke

• For the treatment of focal ischaemia of the brain • For the treatment of brain ischaemia attributable to cardiac arrest

• For the treatment of traumatic spinal cord injury

• For the treatment of injuries to hippocampal neurons

• For the treatment of injuries to cortical cells

· For the treatment of injuries to glioma and microglia

• For the treatment of injuries to motor neurons and astrocytes

• For the treatment of patients with amyotrophic lateral sclerosis (motor neurone disease, Lou Gehrig's disease)

• For the treatment of injuries to the corpus striatum

· For the treatment of cortical trauma

• For the treatment of ischaemia and inflammation associated with injuries to the neonatal brain

• For the amelioration of neurotoxicity associated with intravenous administration of the heroin-like drug MPTP (i-methyl-4-phenyl-i,2,3,6-tetrahydropyridine)

· For the amelioration of striatal and cortical catecholamine depletion induced by intravenous administration of MPTP

• For the treatment of patients with MPTP-induced parkinsonism

• For the treatment of ataxia telangiectasia (Louis-Bar syndrome)

• To attenuate loss of nicotinamide adenine dinucleotide (NAD+) in patients with ataxia telangiectasia

• To attenuate cerebral vasospasm following subarachnoid haemorrhage

• For the treatment of multiple sclerosis

• For the treatment of patients with Parkinson's disease

• For the treatment of patients with Alzheimer's disease

· For the treatment of patients with Huntingdon's disease

• For the treatment of meningitis-associated CNS complications

• For the treatment of acute bacterial meningitis

• For the treatment of transverse myelitis

• For the treatment of neurotrauma

· For the treatment of allergic encephalomyelitis

PAR inhibitors are useful in the treatment or prevention of diseases associated with the ear, such as ischaemia/reperfusion injury to the cochlea. PAR inhibitors are useful in the treatment or prevention of diseases associated with the eye, such as ischaemia/reperfusion injury to a part of the eye such as the retina, degenerative eye disease (including retinitis pigmentosa), and uveitis, and useful for increasing survival of retinal ganglion cells following injury. In particular, PAR inhibitors are useful in the treatment or prevention of the following diseases associated with the eye:

· For the treatment of ischaemia/ reperfusion injury to the retina

• To increase survival of retinal ganglion cells following injury, including axotomy to the optic nerve

• For the treatment and management of patients with degenerative eye disease that includes those with retinitis pigmentosa

· For the treatment of patients with uveitis

PAR inhibitors are useful in the treatment or prevention of diseases of the gastrointestinal tract, such as gastric disease, ischaemia/reperfusion injury to the gastrointestinal tract or the mesentery, inflammatory bowel disease, colitis and pancreatitis. In particular, PAR inhibitors are useful in the treatment or prevention of the following diseases of the gastrointestinal tract:

• For the treatment of H Pylori-associated gastric disease

• For the treatment of ischaemia/ reperfusion injury to the gastrointestinal tract

• For the treatment of inflammatory bowel disease

· For the treatment of colitis that includes ulcerative colitis

• For the treatment of ischaemia/ reperfusion injury to the mesentery

• For the treatment of acute pancreatitis and the management of the associated systemic inflammatory response that includes pancreatitis-associated lung injury PAR inhibitors are useful in the treatment or prevention of hepatic diseases, such as hepatocyte injury, ischaemia/reperfusion injury to the liver, and liver failure. In particular, PAR inhibitors are useful in the treatment or prevention of the following hepatic diseases:

• For the treatment of hepatocyte injury that is characterized by inflammation

· For the treatment of ischaemia/ reperfusion injury to the liver

• For the treatment of liver failure

• For the treatment of acetaminophen-induced and other forms of toxic liver failure

PAR inhibitors are useful in the treatment or prevention of diseases of the immune system, such as compromised immune function, contact hypersensitivity, an autoimmune disorder (including autoimmune nephritis), an inflammatory disorder, immune-mediated nephritides, and rejection in patients who have undergone organ transplantation. In particular, PAR inhibitors are useful in the treatment or prevention of the following diseases of the immune system:

• For the prevention and treatment of compromised immune function associated with stress-related diseases that include stress of physical, somatic or psychological origin

• For the prevention and treatment of contact hypersensitivity

• For the prevention of rejection in patients who have undergone allograft heart transplantation

• For the prevention of rejection in patients who have undergone allograft kidney transplantation

• For the treatment of chronic autoimmune disorders that includes autoimmune nephritis

• For the treatment of patients with inflammatory disorders that are associated with an overactivity of PARP

· For the treatment of immune-mediated nephritides that includes nephritis in patients with systemic lupus erythematosus (SLE)

PAR inhibitors are useful in the treatment or prevention of an infection, such as a viral infection (including HlV-i infection and cytomegalovirus infection).

PAR inhibitors are useful in the treatment or prevention of metabolic diseases, such as type I/II diabetes, secondary disease in patients with type I/II diabetes (including cardiovascular dysfunction, endothelial dysfunction, macrovascular disease, metabolic deficits, peripheral neuropathy, microvascular injury (including retinopathy and nephropathy), and myocardial dysfunction), diabetic cardiomyopathy, chronic inflammatory injury, organ failure, haemorrhagic shock, vascular hyporeactivity, and conditions that are characterized by oxidative and nitrosative stress (including autoimmune β-cell destruction associated with diabetes mellitus), and useful to attenuate systemic inflammation and multi-organ damage in patients with microbial sepsis. In particular, PAR inhibitors are useful in the treatment or prevention of the following metabolic diseases:

• For the prevention or attenuation of the onset of type I diabetes (insulin-dependent) in susceptible and at-risk individuals

• To prevent further destruction of islet cells in newly-diagnosed patients with type I diabetes

• For the treatment of cardiovascular dysfunction in patients with type I/II diabetes • For the treatment of endothelial dysfunction in patients with type I/II diabetes

• For the treatment of macrovascular disease of the cardiac and cerebrovascular circulation in patients with type I/II diabetes

• For the treatment and prevention of metabolic deficits associated with type I/II diabetes

• For the prevention and treatment of functional and structural manifestations of peripheral neuropathy in patients with type I/II diabetes

• For the treatment and prevention of microvascular injury that includes retinopathy and nephropathy in patients with type I/II diabetes

· For the treatment and prevention of myocardial dysfunction that is mediated independently of coronary artery disease in patients with type I/II diabetes

• For the treatment and attenuation of the development of diabetic cardiomyopathy that is characterized by early diastolic function

• For the treatment of patients with chronic inflammatory injury including that caused by microorganisms

• For the prevention or treatment of patients with multiple organ failure of various aetiologies

• To attenuate systemic inflammation and multi-organ damage in patients with microbial sepsis

· For the treatment of patients with haemorrhagic shock that includes patients with septic shock

• For the treatment of vascular hyporeactivity in patients with haemorrhagic shock that in includes patients with septic shock

• For the treatment of patients with various conditions that are characterized by oxidative and nitrosative stress and which include autoimmune β-cell destruction associated with diabetes mellitus

PAR inhibitors are useful in the treatment or prevention of musculoskeletal diseases, such as ischaemia/reperfusion injury to skeletal muscle, arthritis, rheumatoid arthritis, and tempero mandibular joint dysfunction (TMJ), and useful for managing sports injury to skeletal muscle. In particular, PAR inhibitors are useful in the treatment or prevention of the following musculoskeletal diseases:

• For the treatment of ischaemia/ reperfusion injury to skeletal muscle

• For the treatment of rheumatoid arthritis

· For the treatment of temperomandibular joint dysfunction (TMJ) • For the management of sports injury to skeletal muscle in which damage is associated with eccentric loading

PAR inhibitors are useful in the treatment or prevention of oral diseases, such as periodontal disease.

PAR inhibitors are useful in the treatment or prevention of renal diseases, such as ischaemia/reperfusion injury to the kidney, and cisplatin-induced nephropathy. PAR inhibitors are useful in the treatment or prevention of respiratory diseases, such as acute respiratory distress syndrome (ARDS), an acute episode of an allergen-induced asthma-like reaction, interstitial pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), pleurisy, obliterative bronchiolitis, and lung injury, useful for managing airway allergic inflammation and secondary disease in asthma (including lung inflammation, bronchial hyperactivity, airway damage, remodelling, and airway allergic inflammation), and useful for preserving normal permeability and surfactant synthesis in oxidatively-damaged pulmonary epithelial cells. In particular, PAR inhibitors are useful in the treatment or prevention of the following respiratory diseases:

· For the treatment of acute respiratory distress syndrome (ARDS) of various aetiologies

• For the treatment and prevention of an acute episode of an allergen-induced, asthma-like reaction

• For the management of chronic lung inflammation, bronchial hyperactivity, airway damage and remodelling in patients with severe unresponsive asthma

• For the management of airway allergic inflammation, including asthma

• For the preservation of normal permeability and surfactant synthesis in oxidatively- damaged pulmonary epithelial cells

• For the treatment of patients with interstitial pulmonary fibrosis

· For the treatment of patients with chronic obstructive pulmonary disease (COPD)

• For the treatment of pleurisy

• For the treatment of obliterative bronchiolitis in lung transplant recipients

• For the prevention and treatment of ventilator-induced lung injury in patients receiving mechanical ventilation PAR inhibitors are useful in the treatment or prevention of diseases of the skin, such as vesicant-induced skin damage, and dermal inflammation and injury. In particular, PAR inhibitors are useful in the treatment or prevention of the following diseases of the skin:

• For the prevention and treatment of vesicant-induced damage such as that associated with sulphur mustard (mustard gas; bis-(2-chloroethyl) sulphide)

• For the treatment of sunburn-related dermal inflammation and injury

PAR inhibitors are useful in the treatment or prevention of ischaemia/reperfusion injury to an organ, in particular treatment of ischaemia/reperfusion injury to an organ during a surgical procedure that temporarily ablates or compromises perfusion.

PAR inhibitors may protect stem cells. Therefore PAR inhibitors are useful in the pre- treatment of stem cells, such as mesenchymal stem cells.

Brief description of the accompanying figures

Figure l shows non-limiting examples of compounds of the present invention.

Figure 2 shows the conversion of dexrazoxane into two single-ring-opened intermediates B and C, and then into the double-ring-opened metabolite ADR-925.

Figure 3 shows the conversion of levrazoxane into two single-ring-opened intermediates B and C, and then into the double-ring-opened metabolite.

Figure 4 depicts the self-assembly of dexrazoxane with two strands of polyadenylated RNA.

Figures 5A and 5B depict the self-assembly of dexrazoxane intermediate B with one and two strands of polyadenylated RNA respectively.

Figure 6 depicts the self-assembly of dexrazoxane intermediate B with one strand of polyadenylated and polyguanidylated RNA.

Figure 7 depicts the self-assembly of dexrazoxane intermediate C with two strands of polyadenylated RNA. Figure 8 depicts the self-assembly of dexrazoxane with two strands of PAR.

Figures 9A-9E depict the self-assembly of dexrazoxane intermediate B with two strands of PAR. Different conformers of dexrazoxane intermediate B are depicted in the five illustrations of Figures 9A-9E.

Figure 10 depicts the self-assembly of dexrazoxane intermediate B with two strands of PAR. In this illustration, this supramolecular structure shows the incorporation of water molecules as H₃0⁺ (only a few molecules are shown). Consequently, this formation is a hydrogel.

Figure 11 depicts the self-assembly of dexrazoxane intermediate C with two strands of PAR.

Figure 12 depicts the self-assembly of levrazoxane with two strands of polyadenylated RNA.

Figures 13A and 13B depict the self-assembly of levrazoxane intermediate B with one and two strands of polyadenylated RNA respectively.

Figure 14 depicts the self-assembly of levrazoxane intermediate B with one strand of polyadenylated and polyguanidylated RNA. Figure 15 depicts the self-assembly of levrazoxane intermediate C with two strands of polyadenylated RNA.

Figure 16 depicts the self-assembly of levrazoxane with two strands of PAR. Figures 17A-17E depict the self-assembly of levrazoxane intermediate B with two strands of PAR. Different conformers of levrazoxane intermediate B are depicted in the five illustrations of Figures 17A-17E.

Figure 18 depicts the self-assembly of levrazoxane intermediate B with two strands of PAR. In this illustration, this supramolecular structure shows the incorporation of water molecules as H₃0⁺ (only a few molecules are shown). Consequently, this formation is a hydrogel.

Figure 19 depicts the self-assembly of levrazoxane intermediate C with two strands of PAR.

In all of Figures 4-19, the dashed lines represent hydrogen bonding.

Figures 20A-20C depict the enrichment of mitochondria from liver homogenate. Figure 20A shows the enrichment of mitochondria via Western blot from 5 g whole liver lysate and isolated mitochondrial fraction with rat heart mitochondria isolate positive control (Abeam) using anti-AIF (ab325i6) and anti-porin (abi5895). Figure 20B shows the relative signal intensity of AIF and porin bands normalised to whole liver lysate (A, Lane 1) using ImageJ area under the curve analysis. Figure 20C shows Oxphos (abii04i3) Western blot detection of mitochondrial complex subunits.

Figure 21 depicts the measurement of mitochondria integrity following isolation. Figure 21 shows JC-i assay relative fluorescence signal following 1 g/mL 30 minute valinomycin treatment, relative to untreated control mitochondria. Error bars show standard deviation of 3 independent isolations.

Figure 22 depicts dexrazoxane toxicity on mitochondria as measured by JC-i staining following 30 minutes incubation at room temperature. Results are shown as percentage signal of untreated controls.

Figures 23A-23B show that dexrazoxane prevents AIF release from isolated mitochondria. Figure 23A shows AIF release in isolated mitochondria following incubation with PAR and dexrazoxane. PAR and dexrazoxane were incubated together for 10 minutes before being immediately added to the mitochondrial suspension. Top band is AIF, lower band is porin (loading control). "+" is mitochondrial lysate (positive control). Figure 23B shows the optical densitometry of the Western blot shown in Figure 23A.

Detailed description of the invention

I. Dexrazoxane Dexrazoxane is a bisdioxopiperazine with the following structure:

Dexrazoxane undergoes an initial metabolism to its two single-ring-opened intermediates A^-(2-amino-2-oxoethyl)-A^-[(iS')-2-(3,5-dioxo-i-piperazinyl)-i-methyl- ethyl]glycine [B] and A^-(2-amino-2-oxoethyl)-A^-[(2S)-2-(3,5-dioxo-i-piperazinyl)- propyl]glycine [C] and is then further metabolized to its presumed active metal- chelating metabolite A^,A^'-[(iS')-i-methyl-i,2-ethanediyl]bis[(A^-(2-amino-2-oxoethyl)]- glycine [ADR-925] (see Figure 2). The first ring-opening reaction is catalyzed by dihydropyrimidinase (DHPase) and the second by dihydroorotase (DHOase), but not vice versa. DHOase is present in a variety of tissues including the heart, liver and kidney and in erythrocytes and leukocytes. However, although DHPase is present in the liver and the kidney, it is not present in the heart. It is likely that the enantiomer of dexrazoxane, levrazoxane, is metabolised in a similar two-step way, see Figure 3; while several reports describe a DHPase catalyzed ring-opening reaction of levrazoxane, reports of a subsequent DHOase catalyzed hydrolysis are not evident within the available literature. Although dexrazoxane is a bisdioxopiperazine that readily enters cells, it was until now unclear how the ionic metabolites B, C and ADR-925 transfer across cell membranes.

Studies on the metabolism of dexrazoxane in humans and in the rat showed that B, C and ADR-925 quickly appeared in the plasma after bolus intravenous administration. By contrast, under physiological conditions in vitro, hydrolysis of dexrazoxane to ADR- 925 is slow, with dexrazoxane hydrolyzing to B and C with a ti/₂ of 9.3 h at 37°C and pH 7.4, and to the final hydrolysis product ADR-925 with a ti/₂ of 23 h. The fact that the monoanionic B and C and dianionic ADR-925 are present in human and rat plasma at relatively high concentrations also suggested that these three metabolites were being released from the cells in which they were formed, since blood itself does not promote the hydrolysis of dexrazoxane. The level of intermediate B exceeds that of C.

The present inventor has now found that the chemistry of dexrazoxane is consistent with that of a self-assembling species that has the capacity to form anti-parallel supramolecular structures with other molecules through non-covalent interactions that include ionic attractions and hydrogen bonding. Modelling studies (using ChemDraw Ultra Version 10 (PerkinElmer) and MarvinSketch (ChemAxon)) suggest that in vivo, both systemically and within the intracellular environment, dexrazoxane and its single- ring-opened intermediates B and C can self-assemble in the presence of other molecules that include polyadenylated RNA, polyadenylated DNA, polyguanidylated RNA, polyguanidylated DNA and the polymer PAR. Dexrazoxane and its intermediates B and C sequester strands of polyadenylated RNA, polyadenylated DNA, polyguanidylated RNA, polyguanidylated DNA or PAR by a mechanism that is analogous to that of classical Watson-Crick base pairing.

In the case of RNA, modelling studies suggest that dexrazoxane and its intermediates B and C, through their interactions with RNA, especially polyadenylated or polyguanidylated regions, may affect transcription. Moreover, systemically, short strands of polyadenylated or polyguanidylated RNA may facilitate transfer of dexrazoxane and its intermediates B and C. Figures 4-7 depict the self-assembly of dexrazoxane and its intermediates B and C with polyadenylated or polyguanidylated RNA. In the case of PAR, modelling of the interactions of dexrazoxane and its intermediates B and C with PAR, indicates that an anti-parallel supramolecular structure results, which can incorporate water forming a hydrogel. Figures 8-11 depict the self-assembly of dexrazoxane and its intermediates B and C with PAR. In the presence of overactive/hyperactive PARP, this newly-described phenomenon, whereby dexrazoxane and its intermediates B and C can sequester PAR, has considerable therapeutic significance.

Levrazoxane and its intermediates B and C similarly self-assemble in the presence of other molecules such as polyadenylated RNA, polyadenylated DNA, polyguanidylated RNA, polyguanidylated DNA and the polymer PAR, forming anti-parallel supramolecular structures. Figures 12-15 depict the self-assembly of levrazoxane and its intermediates B and C with polyadenylated or polyguanidylated RNA, and Figures 16-19 depict the self-assembly of levrazoxane and its intermediates B and C with PAR.

Self-organization through self-assembly is a distinguishing feature of intact and viable cells. Moreover, self-assembly is a thermodynamically driven process whereby the end- product is energetically favoured. Thus, while some of the different conformations shown in Figures 4-19 may not be preferred in vitro, self-assembly in vivo may still occur given that the assembled product will occupy a lower energy level. Many xenobiotics have the capacity to self-assemble in vivo. Because of this, such molecules and/or their metabolites are able to exploit opportunities for transport and membrane transfer, compete with endogenous substrates for self-assembly, or catalyze the self-assembly of other molecules. The capacity of dexrazoxane and levrazoxane and their intermediates B and C to self- assemble in the presence of polyadenylated RNA offers some insight into the transport and transfer of these compounds, which hitherto has represented a challenge to the precepts of classical theory that is characterized by the pH-partition hypothesis. It is currently believed that dexrazoxane and levrazoxane may, upon conversion to their intermediates B and C within the liver, self-assemble onto cardiac-specific microRNAs that are elaborated within the liver and then transported to the heart. Indeed, several hundred different microRNAs have been shown to be upregulated during heart failure, thus reinforcing the hypothesis that in some part, intermediates B and C hitch a ride upon microRNAs with many of them targeting the heart. Clearly, early anthracycline- induced cardiomyocyte damage would be a precipitating signal for the upregulation of such a transport system.

Dexrazoxane and levrazoxane catalyze the self-assembly of PAR according to classical rules of Watson-Crick base pairing and, as with their intermediates B and C, the strands display an anti-parallel orientation. Importantly, as a lipid-soluble moiety, the parent compound can transfer passively into the cardiomyocyte. However, within the anthracycline-compromised cardiomyocyte there is a high intracellular concentration of PAR. Thus, as part of a self-assembled supramolecular structure, dexrazoxane/ levrazoxane is not free to leave the interior and it becomes trapped. And, as stated above, self-assembly is a thermodynamically driven process whereby the end-product is energetically favoured. Consequently, there is a net transfer of free dexrazoxane/ levrazoxane down a concentration gradient from out to in, which means that the anthracycline-compromised cardiomyocyte represents a deep compartment for the accumulation of dexrazoxane/levrazoxane. This is an extremely important PK/PD property that for the first time offers the only plausible explanation for the remarkable and long-lasting cardioprotective effects of dexrazoxane.

II. PARPs and their polymer product PAR Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that use NAD⁺ as a substrate to synthesize polymers of ADP-ribose (PAR) as post-translational modifications of proteins. PARPs have important cellular roles that include preserving genomic integrity, telomere maintenance, transcriptional regulation, and cell fate determination. The diverse biological roles of PARPs have made them attractive therapeutic targets, which have fuelled the pursuit of small molecule PARP inhibitors.

Poly(ADP-ribose) polymerase-i (PARP-i) has emerged as a prominent target in chemotherapy due to its important role in maintenance of genomic integrity. Its functional roles in the DNA damage response and cell fate determination have fuelled development of PARP-i inhibitors. Some of these compounds have entered clinical trials with promising therapeutic applications towards treatment of cancer. In combination with DNA damaging agents (e.g. temozolomide, cisplatin) or irradiation, PARP-i inhibitors are effective chemosensitizers. As monotherapy, PARP-i inhibitors selectively kill tumours harbouring DNA repair deficiencies such as genetic deletion of genes involved in the BRCAi and BRCA2 homologous recombination DNA repair pathway.

PARP-i accounts for about 90% of total cellular PARP activity. In the nucleus, activated PARP-i catalyzes the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD⁺) onto nuclear acceptor proteins. This process, known as poly(ADP- ribosyl)ation, causes chromatin relaxation and functions as a scaffold that facilitates the recruitment and assembly of the DNA repair proteins. As polymer chains can reach more than 200 units on the acceptor, poly(ADP-ribosyl)ation may result in remarkable conformational change of the acceptor protein, thereby functioning importantly in diverse biological processes, including transcriptional regulation, chromatin remodelling, DNA repair, cell proliferation, and apoptosis. Although the role of PARP in the DNA-damage response has long been recognized, recent work indicates that the polymer product PAR acts at the mitochondria to directly induce cell death through stimulation of apoptosis-inducing factor (AIF) release (Curr Opin Chem Biol 2007 11 644-653).

Overactivation of PARP contributes to the development of cell dysfunction and tissue injury in various pathophysiological conditions associated with oxidative and nitrosative stress, including myocardial reperfusion injury, heart transplantation, diabetic cardiomyopathy and chronic heart failure. Within the nervous system, hypoxia or hypoxia-reoxygenation injury results in neuronal cell death, including apoptosis and necrosis. Oxidative stress results in mitochondrial dysfunction and the release of cytochrome c and AIF, which are associated with apoptosis through caspase-dependent and caspase-independent pathways, respectively. In the case of mild DNA damage, the cell activates PARP to facilitate DNA repair. Severe DNA damage may cause PARP overactivation, leading to depletion of the cellular NAD⁺/ATP stores and occurrence of cell necrosis. On the other hand, PARP activation induces PAR polymer formation primarily in the nucleus. As discussed above, PAR polymers can translocate to the mitochondria and mediate the release of AIF from the mitochondria. AIF then translocates to the nucleus and induces cell death. The PAR-mediated release of AIF that results in caspase-independent cell death is a form of cell death that is distinct from apoptosis, necrosis and autophagy and is termed parthanatos.

III. Current theories regarding dexrazoxane's mode of action

There are currently two theories on how dexrazoxane works, in particular how it exerts its cardioprotective effect and how it treats or prevents extravasation, namely chelation of iron and inhibition of topoisomerase 2. Both of these theories are incorrect for at least the reasons set out in the following paragraphs. The present inventor has now found that the way dexrazoxane works is by inhibiting or sequestering PAR by forming a self-assembled supramolecular structure with PAR as set out above. fi) Chelation of iron Zatloukalova et al (Can J Physiol 2012 90 473-484) investigated the role of iron chelation using dexrazoxane in a rat model of myocardial infarction. While dexrazoxane significantly reduced mortality, reduced myocardial calcium overload, histological impairment, and peripheral haemodynamic disturbances, additional in vitro studies revealed that dexrazoxane had no effect upon iron-catalyzed processes that characterize this model of cardiac impairment. From their observations, Zatloukalova et al concluded that the general hypothesis that iron chelation is the main mechanism of cardioprotection, was not confirmed.

Popelova et al (J Pharmacol Exp Ther 2008 326 259-269) investigated whether the orally-active iron-chelator deferiprone is an effective cardioprotective agent in a rabbit model of chronic anthracycline toxicity. From the results of this study these workers strongly suggested that oral treatment with deferiprone has no beneficial effect on chronic anthracycline cardiotoxicity. Popelova et al also investigated the iron-chelator deferasifox using the same model and similarly showed no cardioprotective effects. They added that this result is surprising given that other workers have shown that deferasifox rapidly, effectively and efficiently removed iron as Fe³⁺ from its complex with doxorubicin, rapidly entered myocytes, and displaced iron from an intracellular iron-calcein complex (Hasinoff et al Free Radic Biol Med 2003 35 1469-1479).

In discussing their results, Popelova et al added that the well-established iron chelator desferrioxamine (as a mesylate salt) (also known as deferioxamine) has also been shown to be ineffective against chronic anthracycline cardiotoxicity in spontaneously hypertensive rats, whereas in that study dexrazoxane was able to attenuate the cardiotoxicity and mortality induced by doxorubicin (Cancer Chemother Pharmacol 1994 35 93-100). Similarly, earlier studies by the same group showed that the cardioprotective effects of dexrazoxane were superior to that of all members of a series of aroylhydrazone iron chelators (Pharmacol Res 2005 51 223-231; J Pharmacol Exp Ther 2006 319 1336-1347; Toxicology 2007 235 150-166). While these agents did provide partial protection, surprisingly dose-escalation of all aroylhydrazones resulted in a loss of cardioprotective effects.

In summarizing their experience with the orally-active iron-chelator, deferiprone, Popelova et al proposed that the observed lack of a cardioprotective agent in an animal model of chronic anthracycline cardiotoxicity could mean that iron-catalyzed formation of reactive oxygen species is not the pivotal and ultimate executor responsible for chronic anthracycline cardiotoxicity. Most importantly, they proposed that their study clearly emphasizes the limitations of in vitro studies and that there is a real need to review the mechanism of action of dexrazoxane that consistently outperforms other agents.

(2) Inhibition of topoisomerase 2

Anthracyclines

Since their first discovery nearly fifty years ago (Nature 1964 201), anthracyclines such as doxorubicin, daunorubicin, epirubicin and idarubicin, have been successfully developed as potent anticancer therapeutics with significant efficacy in lymphomas and many solid tumours. Particularly in patients with breast cancer, they are the primary choices of therapy.

However, cardiotoxicity represents a limiting complication in the use of anthracyclines. Cardiotoxicity is reported to be dose-related, with sharp rises in left ventricular dysfunction with cumulative doses >400 to 450 mg/m² for doxorubicin. Using cardiac imaging, the incidence of heart failure was reported as 5%, 26% and 48% in patients receiving 400, 550 and 700 mg/m² doxorubicin (Cancer 2003 97 (11) 2869-2879). In the Product Characteristics for Doxorubicin 2mg/ml Concentrate for Solution for Infusion, it is stated that the probability of developing chronic heart failure (CHF), estimated around 1% to 2% at a cumulative dose of 300 mg/m², increases progressively up to the total cumulative dose of 450-550 mg/m². Thereafter, the risk of developing CHF increases markedly and it is recommended that practitioners do not exceed a maximum cumulative dose of 550 mg/m². Using these and other reports as a basis for decision making, most oncologists typically limit the dose of doxorubicin to 450-500 mg/ m².

Children are especially vulnerable, with rates of significant left ventricular dysfunction of 5% at 15 years of follow-up, increasing to 10% for cumulative doses of≥550 mg/m² (New Engl J Med 1991 324 (12) 808-815). In clinical practice, the observations and recommendations on cumulative dosing of doxorubicin limits the total dose an individual patient could receive. For particularly problematic cancers, many oncologists would prefer to use higher doses (Circulation 2013 128 (2) 98-100).

Topoisomerases and anthracyclines DNA topoisomerases are essential enzymes for cells that modulate DNA topology by regulating the over- or underwinding of DNA strands during cellular processes, such as DNA transcription, replication, or recombination (Proc Natl Acad Sci 1987 84 (20) 7024-7027). Mammalian cells contain two distinct topoisomerase 2 (Top2) isoforms. The alpha isoform (Top2a) is present in high amounts in proliferating and undifferentiated cells, and its expression is cell cycle-dependent, reaching a maximum in G₂/M phase (Cell Growth Differ 1991 2 (4) 209-214). Conversely, the beta isoform (Τορ2β) is expressed at relatively steady levels throughout the cell cycle and is the major Top2 form in quiescent cells. Whereas Top2a is implicated predominantly in DNA replication, Τορ2β has important roles in the regulation of gene transcription (Int J Biochem Cell Biol 2012 44 (6) 834-837; Nat Rev Cancer 2009 9 (5) 327-337; Nat Rev Mol Cell Biol 2002 3 (6) 430-440; Bioessays 1998 20 (3) 215-226). Notably, following the seminal observation by Capranico et al in 1992 that only Τορ2β is detected in adult murine heart (Biochim Biophys Acta 1992 1132 (1) 43-48), it is the consensus today that Τορ2β is selectively expressed in the human heart and that Top2a is absent (Circulation 2013 128 (2) 98-100). Support for this consensus is derived from the outcomes of gene deletion and knockdown studies (Clin Pharmacol Ther 2014 95 (1) 45-52; PLOS One 2013 8 (10) 676676; J Biol Chem 2006 281 35997-36003).

Three decades earlier, it was established that the Top2 isoenzymes are a molecular target of anthracyclines (Science 1984 226 (4673) 466-468). With the subsequent identification of two isoforms of mammalian Top2, it has been postulated that it is the alpha isoform, Top2a that is the primary target of anthracycline's anti-cancer effect. This is based on two important observations. First, as already discussed above, Top2a expression level is markedly elevated in rapidly proliferating cells and tumour cells. Second, only Top2a is essential in cell proliferation, because it is required in the chromosomal segregation process (Mol Biol Cell 2004 15 (12) 5700-5711). By contrast, in more recent times, it has been proposed that the selective expression of Τορ2β in cardiac cells may represent a critical determinant of anthracycline-induced cardiotoxicity. However, it is not understood why doxorubicin, a commonly used anthracycline, appears to preferentially target cardiomyocytes, and why doxorubicin- induced damage progresses to cardiomyopathy with an often difficult-to-predict development and progression. How topoisomerases deal with tangled DNA

A feature common to all topoisomerases is the DNA strand passage event. It is this event that enables relaxation of DNA supercoils (tangles) that are an inevitable consequence when DNA unwinds. However, the ability to pass a single- or double- stranded segment of DNA freely through another comes with a heavy price. In order to "untangle" supercoiled DNA, it is necessary to cut DNA strands. Topoisomerases have evolved as enzymes that catalyze the formation of breaks in the genetic material. In an effort to maintain genomic integrity during this cleavage reaction, topoisomerases covalently attach to the newly-generated DNA termini, i.e. cut part of each DNA strand, via phosphotyrosyl bonds. Under normal circumstances, these covalent enzyme- DNA "cleavage complexes" are fleeting catalytic intermediates and are present in low steady-state concentrations. Consequently, they are tolerated by the cell. However, conditions that significantly increase the physiological concentration or lifetime of these breaks unleash a myriad of deleterious side effects, including mutations, insertions, deletions, and chromosomal aberrations. Thus, all topoisomerases are fundamentally dualistic in nature. Although they catalyze essential reactions in the cell, they possess an inherent dark side capable of inflicting great harm to the genome of an organism.

In resolving DNA tangles, the normal catalytic cycle of Τορ2β, for example, consists of a transient enzyme-bridged DNA double-strand break, where attachment of Τορ2β to the DNA is via a 5' tyrosyl phosphodiester covalent linkage. Normally, these transiently- cut ends enable relaxation of tangled DNA by a progressive restoration to a less chaotic and lower energy topology. Relaxation is achieved as a tangled strand is able to pass through the space created by the cut ends, following which the cut ends are religated. The process is either repeated at the same site or the Τορ2β protein migrates to another supercoil. Doxorubicin is a catalytic poison that prevents religation of cut DNA

Drugs such as doxorubicin have the capacity to stabilize the cut-strands intermediate and prevent religation of the cut ends. Because prevention of religation results in deadend complexes that can lead to cell death, drugs in this category are known as "Top2 poisons". That is, doxorubicin has converted Τορ2β into a poison - use of the word "poison" is somewhat ambiguous and it is used interchangeably to describe the agent and the topoisomerase. An increasing formation of cleavable complexes represents an accumulation of DNA damage that invariably results in cell death.

Dexrazoxane is a catalytic inhibitor that is not associated with cutDNA

By contrast, dexrazoxane belongs to a class of molecules that are able to function as "Top2 catalytic inhibitors". These compounds are known to antagonize the formation of the Top2-DNA covalent complex described above by stabilizing the ATP-bound closed clamp conformation of Top2 before scission of DNA. That is, dexrazoxane inhibits the catalytic activity of Τορ2β by binding the enzyme in proximity of the ATP binding site and inhibiting the ATPase activity of Τορ2β (Proc Natl Acad Sci USA 2003 100 (19) 10629-10634). This action results in the Top2 enzyme being trapped as a closed clamp on DNA, but without any breaks in the DNA strands. Consequently, catalytic inhibitors, notably the bisdioxopiperazines that include dexrazoxane, do not induce a DNA damage response, at least in the short term (Nature Cell Biology 2009 11 (2) 204-210; Embo Reports 2005 6 (8) 729-735; Chem Biol 2003 10 (12) 1267-79; Biochemical Pharmacology 1997 54 (7) 755-759). Cell death, however, can be induced by some catalytic inhibitors by eliminating the essential enzymatic processes of Top2 (Nat Rev Cancer 2009 9 (5) 338-350).

Does dexrazoxane protect the cardiomyocyte by eliminating Τορ2β as a target for doxorubicin?

Outcomes from an in vitro study by Lyu et al (Cancer Res 2007 67 (18) 8839-8846) are the basis for a new hypothesis on the cardioprotective mechanism of action of dexrazoxane. Lyu et al observed preferential proteasome-dependent degradation of Τορ2β in H9C2 cardiomyoblast-derived cells after exposure to dexrazoxane, coupled with a lack of significant changes to Top2a. From their observations, these workers argued that selective degradation of Τορ2β could prevent anthracycline-induced damage to the cardiomyocyte, while sparing the Top2a isoform in cancer cells. Following this seminal study by Lyu et al, Zhang et al (Nat Med 2012 18 (110) 1639- 1642) reported that the cardiomyocyte-specific deletion of the Τορ2β gene protected mice from developing doxorubicin-associated cardiotoxicity. Subsequently, these observations have led to the proposal by Vejpongsa and Yeh (Clin Pharmacol Ther 2014 95 (1) 45-52) that pretreatment with dexrazoxane degrades Τορ2β with disruption of Τορ2β-ϋΝΑ complex assemblies (as discussed later, degradation of Τορ2β protein occurs when Τορ2β is part of the Τορ2β-ϋΝΑ complex). In turn this diminishes the target for doxorubicin leading to abrogated doxorubicin-induced cardiotoxicity.

Importantly, the preferential degradation of Τορ2β targets the protein as part of the Τορ2β-ϋΝΑ complex and does not affect Τορ2β mRNA. That is, in principal, degraded Τορ2β protein can be replaced through the synthesis of new protein.

Lyu et al (Cancer Res 2007 67 (18) 8839-8846) proposed two mechanisms for the antagonism by dexrazoxane of doxorubicin-induced DNA damage in the cardiomyocyte. Both mechanisms are dependent upon the presence of Τορ2β, whereby dexrazoxane protects the cardiomyocyte by precluding access of doxorubicin to Τορ2β. In the first mechanism they proposed that binding of dexrazoxane to free Τορ2β prevents binding of Τορ2β to DNA. That is, if Τορ2β cannot bind to DNA, then there is no opportunity for Τορ2β to catalyze the formation of cut strands and no target for doxorubicin.

It is a general consensus that in the cardiomyocyte, Τορ2β is a principal target of anthracyclines in inducing toxicity and subsequent cardiomyopathy, and that unequivocally dexrazoxane is protective in this setting. Moreover, the outcomes of Lyu et al showed that in addition to binding to Τορ2β, exposure to dexrazoxane in vitro results in the preferential proteasome-mediated degradation of Τορ2β protein. However, when taken together it does not follow that the cardioprotection afforded by dexrazoxane is the result of dexrazoxane either binding to and/or inducing proteasome-dependent degradation of the anthracycline target, Τορ2β.

The first mechanism proposed by Lyu et al is flawed for the following reasons. Dexrazoxane does not bind covalently to Top2 isoenzymes (BMC Pharmacol 2004 4 31 Open access 10.1186/1471-2210-4-31). Indeed, washing processes involved in assays will release bound dexrazoxane from the enzyme (Brian Hasinoff, personal communication to Roti Roti and Salih, Biol Reprod 2012 86 (3) Article 96 1-11). Consequently, the binding of dexrazoxane to Top2 isoforms is a dynamic equilibrium process, whereby dissociated dexrazoxane becomes available for metabolic conversion and unchanged Top2 isoform is released, that upon binding to DNA becomes a target for doxorubicin. Accordingly, because the binding of dexrazoxane with Top2 isoforms is reversible, then the effects of direct binding of dexrazoxane upon Top2-mediated processes must be considered as transient and equilibria will be critically context- dependent.

That is, given that Τορ2β binds covalently to DNA, but only non-covalently to dexrazoxane, then the movement of Τορ2β is dynamically shifted away from dexrazoxane and toward DNA.

Importantly, regardless of any effects of dexrazoxane binding directly to free Τορ2β, as discussed later in this report, dexrazoxane is efficiently metabolized in humans with the result that available dexrazoxane for binding to Τορ2β is soon depleted. The liver contains both dihydropyrimidinase (DHPase) and dihydroorotase (DHOase) that completely catalyze the hydrolysis of dexrazoxane to the double-ring-opened metabolite, ADR-925. Thus, in the liver the two liver enzymes act in concert, and sequentially, on dexrazoxane, first to produce the two single-ring-opened metabolites, and then to produce ADR-925 (Drug Metab Dispo 2005 33 (6) 719-725).

In cancer patients, dexrazoxane is rapidly converted to two single-ring-opened metabolites with the subsequent appearance of the double-ring-opened metabolite, ADR-925. Schroeder et al (Cancer Chemother Pharmacol 2003 52 (2) 167-174) studied the metabolism of dexrazoxane in cancer patients with brain metastases treated with high-dose etoposide. They reported that the two single-ring-opened hydrolysis intermediates of dexrazoxane appeared in the plasma at low levels upon completion of the dexrazoxane infusion and then rapidly decreased with half-lives of 0.6 and 2.5 hours. Since dexrazoxane circulates as >98% in the free form in plasma and it is lipid- soluble, then it will passively transfer across cell membranes. Consequently, intracellular conversion of dexrazoxane will result in a concentration gradient down which dexrazoxane will move from plasma to cell interior. Restated, the rapid disappearance of dexrazoxane means that within a very short time frame the ratio of free doxorubicin (with a half-life in some patients of up to 48 hours (Drugs.com FDA 2014)) to free dexrazoxane will rapidly and non-linearly increase by orders of magnitude. This means that the amount of intracellular binding of dexrazoxane to Τορ2β will rapidly become negligible and of no pharmacodynamic importance, and competition for binding to Τορ2β-ϋΝΑ complexes will be dominated by the increasingly greater amounts of free doxorubicin relative to the rapidly-disappearing amounts of free dexrazoxane. The second mechanism proposed by Lyu et al (Cancer Res 2007 67 (18) 8839-8846) does not involve free Τορ2β, but is focused upon Τορ2β that is attached to DNA and is at a stage in the catalytic cycle at which scission of DNA has not yet been induced. Dexrazoxane binds to and stabilizes the ATP-bound closed-clamp conformation of Τορ2β before scission of DNA. That is, dexrazoxane inhibits the further catalytic activity of Τορ2β, but without any breaks in the DNA strands.

The second mechanism proposed by Lyu et al is flawed for the following reasons. A primary function of Τορ2β is to resolve the deranged topology (tangles) that is a consequence of DNA unwinding during transcriptional activity in the synthesis of cellular protein. However, both Τορ2β poisons and catalytic inhibitors alike block this process by stabilizing Τορ2β attached to DNA, with cut ends in the case of poisons or without cut ends in the case of catalytic inhibitors. Thus, both categories of inhibitor induce "transcriptional arrest" (Proc Natl Acad Sci USA 2003 100 (6) 3239-3244) by interfering with the progress along the unwinding DNA strands of the RNA polymerase elongation complex that catalyzes the transcription of DNA in order to synthesize precursors of mRNA.

The enzyme, 26S proteasome, resolves Top2β-mediated transcription block by catalyzing the degradation of Τορ2β and thereby removing the "roadblock". Degradation of Τορ2β induced by both poisons and catalytic inhibitors is generally selective for Τορ2β and with no significant degradation of Top2a and this much- observed phenomenon has been reported in studies using VM-26 (teniposide), VM-16 (etoposide), ICRF-193, BNS-22, lycobetaine, and dexrazoxane, and in an array of cell types (J Biol Chem 2013 288 (10) 7182-7192; Biology Open 2012 doi: 10.1242/010.20121834 Biology Open 1 Sept 15 2012 863-873; Chem Biol 2011 18 (6) 743-751; J Nucleic Acid 2010 pii:7i0589 doi:io.4o6i/20io/7i0589; Mol Cancer Ther 2009 8 (5) 1075-1085; PLOS One 2009 4 (12) e8i04; Cancer Res 2007 67 (18) 8839- 8846; Proc Natl Acad Sci USA 2007 104 (26) 11014-11019; J Biol Chem 2006 281 35997-36003; Proc Natl Acad Sci USA 2003 100 (6) 3239-3244; FEBS Lett 2003 546 (2-3) 374-378; Br J Cancer 2001 85 (10) 1585-1591; J Biol Chem 2001 276 (44) 40652- 40658; J Biol Chem 1997 272 (39) 24159-24164).

Preferential degradation of Τορ2β with no effect upon Top2a cannot be explained by differences between binding affinity of a drug for each isoenzyme, but may be attributable to the different functions of each isoenzyme and their different intracellular spatial locations (J Biol Chem 2001 276 (44) 40652-40658; Proc Natl Acad Sci USA 2003 100 (6) 3239-3244). Thus, for example, Τορ2β but not Top2a is committed to transcription, and its location is directly upon unwinding and tangled DNA strands. Consequently, when dexrazoxane inhibits the catalytic activity of Τορ2β in resolving tangled DNA, as already discussed, the subsequent transcriptional arrest will activate the 26S proteasome-mediated degradation of Τορ2β; any antagonism of Top2a (at a different location) by dexrazoxane will not result in transcriptional arrest and degradation of Top2a protein will not be initiated. Moreover, immunohistochemical studies have shown that Top2a is predominately present in proliferating tissues including tumours, while Τορ2β is present in numerous cells including terminally-differentiated cells such as the adult cardiomyocyte which contains οη^ Τορ2β.

At least for the cleavage complexes induced by Top2 poisons, in 2001 it was reasoned by Mao et al (J Biol Chem 2001 276 (44) 40652-40658) that proteasome-mediated degradation of Top2 exposes the Top2-concealed DNA strand breaks that then results in activation of cell death mechanisms. Exposure of the DNA breaks will be followed by PARP binding to the cut ends, hyperactivation of PARP and the subsequent elaboration of the death signal, PAR.

In summary, preferential degradation of Τορ2β is a ubiquitous phenomenon that is observed using both catalytic inhibitors and Top2 poisons, and is by no means unique to dexrazoxane. If antagonism of Τορ2β activity by preferential degradation of Τορ2β protein represented the mechanism for the cardioprotective effect of dexrazoxane, then given that this is a property shared by many other compounds (and many for which inhibition of Top2 isoenzymes remains to be assayed), then it would be realistic to expect that dexrazoxane is a member of a large category of "me too" cardioprotective drugs occupied by numerous competing molecules that share a common mechanism of action (e.g. compare NSAIDS, ACE inhibitors, etc). But this is not the case.

Dexrazoxane is rapidly metabolized in humans. In patients with various cancers, the terminal elimination half-life of dexrazoxane and doxorubicin is reported as approximately 2 hours, and up to 48 hours, respectively. It is a consensus that dexrazoxane, but not its ring-opened metabolites, is a catalytic inhibitor of topoisomerase 2 isoforms (Biochem Pharmacol 1995 50 (7) 953-958; Mol Pharmacol 1997 52 (5) 839-845; J Chromatogr B 2001 760 (2) 263-269; Cancer Res 2007 67 (18) 1780-1758; Drag Metab Dispos 2008 36 (9) 1780-1785). As already discussed, in cancer patients, dexrazoxane is rapidly metabolized to two single-ring- opened metabolites with the subsequent appearance of the double-ring-opened metabolite ADR-925. Schroeder et al (Cancer Chemother Pharmacol 2003 52 (2) 167- 174) studied the metabolism of dexrazoxane in cancer patients with brain metastases treated with high-dose etoposide. They reported that the single-ring-opened hydrolysis intermediates of dexrazoxane appeared in the plasma at low levels upon completion of dexrazoxane infusion and then rapidly decreased with half-lives of 0.6 and 2.5 hours. A plasma concentration of 10 μΜ ADR-925 was also detected at the completion of the dexrazoxane infusion period, indicating that dexrazoxane was rapidly metabolized in humans. A plateau level of 30 μΜ ADR-925 was maintained for 4 hours and then slowly decreased.

Dexrazoxane is slowly metabolized in vitro. Under physiological conditions (37°C and pH 7.4), dexrazoxane is slowly hydrolyzed to the single-ring-opened metabolites (ti/₂ of 9.3 hours), and to the final hydrolysis product ADR-925 (ti/₂ of 23 hours) (Drug Metab Dispos 1999 27 (2) 265-268; Int J Pharma 1994 107 67-76; J Pharma Sci 1994 83 (1) 64; Drug Metab Dispos 1990 18 (3) 344-349). By contrast, after intravenous administration to patients with cancer the plasma concentrations of dexrazoxane were observed to decline with an average half-life of 2.2 +/- 1.2 hours (Summary of Product Characteristics for Cardioxane^®, 2014). Given the slow rate at which dexrazoxane hydrolysis-activation occurs in vitro, outcomes from the laboratory setting using cell preparations within the short time frames of the study period, represent the effects of constant exposure of cells to high concentrations of dexrazoxane. Consequently, considerable caution must be exercised in extrapolating laboratory findings using cell preparations exposed continuously to high concentrations of dexrazoxane to the clinical setting with rapidly-declining concentrations of dexrazoxane.

Significant amounts of Τορ2β are not degraded despite continuous exposure to high levels of dexrazoxane. Lyu et al (Cancer Res 2007 67 (18) 8839-8846) treated H9C2 rat cardiomyocytes with 100 and 200 μπιοΙ/L dexrazoxane, which are clinically-relevant concentrations, for o, 1, 2, 4 and 6 hours. Following incubation, cells were lysed and protein levels of Top2a and Τορ2β were determined by Western blotting using anti- Τορ2α/Τορ2β followed by detection using enhanced chemiluminescence with visualization using X-ray films. These visualizations show a time-dependent disappearance of Τορ2β, but with no significant effect upon the levels Top2a, with approximately 50% of Τορ2β protein detected at 2 hours, and lower, but still detectable levels observable at 4 and 6 hours.

Yan et al (Mol Cancer Ther 2009 8 (5) 1075-1085 and Supplement) in a later study investigated the effects of dexrazoxane upon expression of Top2a and Τορ2β using HTETOP cells which is a human tumour cell line derived from the human fibrosarcoma cell line, HT1080. Unlike the earlier data of Lyu et al (Cancer Res 2007 67 (18) 8839- 8846), in which degradation of Τορ2β was assessed semi-quantitatively by inspection of X-ray visualization of chemiluminescence signals, Yan et al subjected each protein band to densiometric analysis using an NIH J freeware imaging processing system. Consequently, the outcomes of Yan et al are provided as numeric quantities (Supplement to Cancer Ther 2009 (5) 1075-1085). Using dexrazoxane at a concentration of 100 μπιοΙ/L and 200 μπιοΙ/L, they showed that after incubation for 24 hours, approximately 30% of Τορ2β protein was detected at both concentrations. And approximately 60%, 50% and 40% of Τορ2β protein was detected at 3, 6 and 12 hours respectively, using 100 μπιοΙ/L dexrazoxane. That is, these amounts represent undegraded and intact Τορ2β protein.

The in vitro data of Lyu et al is used to propose a mechanism to explain the cardioprotective effect of dexrazoxane. In recent times, on the basis of the outcomes from the study by Lyu et al (Cancer Res 2007 67 (18) 8839-8846), several workers have proposed that pretreatment with dexrazoxane degrades Τορ2β, and it is this mechanism, by removing Τορ2β, that precludes doxorubicin from binding to the Τορ2β-ϋΝΑ complex, thereby preventing cardiotoxicity in patients receiving doxorubicin (Clin Pharmacol Ther 2014 95 (1) 45-52; PLOS One 2013 8 (10) 676676).

Despite continuous exposure of the cells to high concentrations of dexrazoxane, both in vitro studies presented above show that Τορ2β protein is still detectable at the final assay point (at 6 hours in the semi-quantitative study of Lyu et al, and at 24 hours in the densiometric study by Yan et al). In the study by Yan et al, approximately 40% of Τορ2β protein was detected at 12 hours (Supplement to Cancer Ther 2009 (5) 1075- 1085). Thus, in both cell preparations dexrazoxane did not eliminate Τορ2β activity. Consequently, if these findings do have any clinical relevance, then it is not unreasonable to assume that the amounts of Τορ2β protein detected at later times constitute a sufficient target for the toxic effects of doxorubicin. In humans the terminal half-life of doxorubicin is up to 48 hours (Drugs.com FDA 2014), and that for dexrazoxane is approximately 2 hours. Dexrazoxane is administered as a 15 minute infusion 30 minutes before administration of doxorubicin. At 12 hours after the administration of doxorubicin (equivalent to 12.75 hours after the initial administration of dexrazoxane), approximately 1% and 85% of dexrazoxane and doxorubicin respectively will remain, the amounts being calculated as the proportion of peak plasma levels following administration.

In humans, the metabolism of dexrazoxane to the single-ring-opened intermediates and subsequently to the double-ring-opened metabolite, ADR-925, is extremely rapid. Schroeder et al (Cancer Chemother Pharmacol 2003 52 (2) 167-174) investigated the metabolism of dexrazoxane in cancer patients with brain metastases treated with high- dose etoposide. They showed that the two single-ring-opened hydrolysis intermediates of dexrazoxane appeared in the plasma at low levels upon completion of dexrazoxane infusion and then rapidly decreased with half-lives of 0.6 and 2.5 hours. A plasma concentration of 10 μπιοΙ/L of ADR-925 was also detected at the completion of the dexrazoxane i.v. infusion period. From their observations these workers concluded that in this setting, dexrazoxane is rapidly metabolized. These observations are in complete contrast to investigations using cell preparations, where under physiological conditions (37°C and pH 7.4) dexrazoxane is only slowly hydrolyzed to the single-ring-opened metabolites (ti/₂ of 9.3 hours) and to the final hydrolysis product ADR-925 (ti/₂ of 23 hours) (Drug Metab Dispos 1999 27 (2) 265-268; Int J Pharma 1994 107 67-76; J Pharma Sci 1994 83 (1) 64; Drug Metab Dispos 1990 18 (3) 344-349). In humans, at 12 hours following the administration of doxorubicin (12.75 hours following initiation of the infusion of dexrazoxane), there is virtually no dexrazoxane remaining in plasma. Given that dexrazoxane is <2% bound to plasma proteins, and that the uncharged parent compound will rapidly equilibrate across cell membranes, then intracellular concentrations will likewise be at or near zero. Moreover, since it is only the parent compound that is a catalytic inhibitor of Τορ2β (Cardiovasc Toxicol 2007 7 (2) 140-144; Mol Pharmacol 2003 64 (3) 670-678; Anticancer drugs 2002 13 (3) 255-258; Biochem Pharmacol 1995 50 (7) 953-958), then undegraded Τορ2β protein remaining at 12 hours will represent an exclusive target for doxorubicin, which at 12 hours is present at a very substantial concentration. Both the outcomes of Lyu et al and Yan et al, notably that of the more elaborate and longer study of Yan et al, show that at the end of the observation period undegraded Τορ2β could be detected. In the study by Yan et al, at 12 hours they detected approximately 40% of undegraded Τορ2β protein (Supplement to Cancer Ther 2009 (5) 1075-1085).

That there will be substantial Τορ2β protein remaining at 12 hours is supported by the observations of Hasinoff et al, who showed that in Chinese Hamster Ovary [CHO] cells and K562 cells continuous long term [days] exposure to dexrazoxane is necessary in order to achieve maximal suppression of Top2 activity (Mol Pharmacol 2001 59 (3) 453-461; J Pharmacol Exper Ther 2000 29 (2) 474-483; Biochem Pharmacol 1997 53 (12) 1843-1853; Biochem Pharmacol 1995 50 (7) 953-958).

Consequently, it is highly unlikely that after 12 hours in a clinical setting with rapid loss of circulating dexrazoxane all Τορ2β activity (measured as protein), if indeed any activity, will be suppressed by dexrazoxane. Moreover, since it is Τορ2β protein that is degraded and the Τορ2β synthetic machinery remains intact, then as also demonstrated by Hasinoff et al, removal of dexrazoxane results in restoration of Top2-mediated effects (Mol Pharmacol 2001 59 (3) 453-461). That is, the machinery exists to replace degraded Τορ2β.

Τορ2β is a key player in cell viability and survival. Importantly, in the terminally- differentiated post-mitotic cell that includes the cardiomyocyte, Τορ2β occupies a key role in the regulation of gene transcription and other functions (Int J Biochem Cell Biol 2012 44 (6) 834-837; Nat Rev Cancer 2009 9 (5) 327-337; Nat Rev Cancer 2009 9 (5) 338-350; Nat Rev Mol Cell Biol 2002 3 (6) 430-440; Bioessays 1998 20 (3) 215- 226). Indeed, it has been shown that depletion of Top2Beta results in down-regulation of peroxiredoxin 2 that leads to cell death (peroxiredoxins are ubiquitous and abundant proteins that are important for antioxidant defence and regulate cell signalling pathways). Restated, substantial loss of Τορ2β, unless restored, will result in a loss of viability of the cardiomyocyte that will likely lead to cell death or considerable loss-of- function.

Indeed, it is worth iterating the obvious paradox whereby, if the formation of ternary doxorubicin-Top2β-DNA cleavable complexes is precluded by the dexrazoxane-induced proteasome-mediated degradation of Τορ2β, then this must also represent a potentially toxic effect of dexrazoxane. That is, the ability of doxorubicin to form ternary cleavable complexes is entirely dependent upon the capacity of Τορ2β to bind DNA as part of its normal role in maintaining fidelity in transcription; if Τορ2β is binding with DNA then it is performing an important role.

There is no evidence that prolonged and maximal suppression of Τορ2β by dexrazoxane-induced proteasome-mediated Τορ2β degradation occurs in a clinical setting. Indeed, the pharmacokinetic profile of dexrazoxane in humans precludes such a notion. If such dexrazoxane-induced degradation of Τορ2β protein was the mechanism by which dexrazoxane exerted its cardioprotective effects, then clinical use would be at the expense of potentially very damaging toxicity. A long history of clinical use with ongoing surveillance in many patient cohorts provides no support for such novel dexrazoxane toxicity attributable to either local and/or widespread degradation of Τορ2β protein.

Dexrazoxane is administered several hours after doxorubicin in the management of extravasation injury

Extravasation injury allows doxorubicin an opportunity to have uninterrupted access to Τορ2β in the absence of pre-administration of dexrazoxane. Langer et al (J Clin Oncol 2000 18 (16) 3064) reported in initial preclinical studies using mice that a single, non-toxic, systemic injection of dexrazoxane administered up to 6 hours after a subcutaneous injection of daunorubicin, idarubicin, doxorubicin or epirubicin reduces the frequency, size and duration of the resulting wounds. The same group showed that dexrazoxane was virtually 100% effective in abrogating doxorubicin damage, and that the effect was the same even with a delay of 3 hours (Clin Cancer Res 2000 6 (9) 3680-3686). And this is how dexrazoxane is used in the clinic for the treatment of extravasation injury - after the event. Indeed, Aigner et al (Dermatology 2014 Nov 27 288-292 Epub ahead of print) report complete recovery following the use of dexrazoxane seventy-two hours after extensive epirubicin extravasation. However, even if subsequent administration of dexrazoxane were to induce the degradation of all of the Τορ2β protein within the zone of extravasation injury and as a result were to remove the target for doxorubicin to induce any further DNA damage, the damage has already been done, removing Τορ2β is too late. However, the Τορ2β theory derived from the findings of Lyu et al could only be entertained in the management of extravasation injury, if in the subsequent absence of Τορ2β induced by dexrazoxane the original damage becomes self-remitting given that doxorubicin now has no target. Although this view cannot be immediately rejected, it seems an unlikely event. PAR- mediated cell death (parthanatos) is an irreversible process (Proc Natl Acad Sci USA 2006 103 (48) 18308-18313). Pharmacodynamically, anthracycline-induced Τορ2β- mediated cell damage resulting in permanent and cumulative DNA damage (the mechanism of action of anthracyclines) will result in hyperactivation of PARP and PAR- mediated cell death. Consequently, based on what is known, there are no grounds for believing that removing Τορ2β subsequent to doxorubicin-mediated damage could change the natural course of the extravasation injury. However, the progress of this devastating injury could be halted by blocking PAR, the mechanism proposed by the present inventor.

Therefore the outcomes from the in vitro study of Lyu et al have no relevance to the use of dexrazoxane in a clinical setting. IV. Experimental Studies

Anthracycline-induced DNA damage results in the rapid synthesis of PAR and the subsequent release by PAR of mitochondrial apoptosis-inducing factor (AIF) with AIF- mediated cell death (Shin et al, Sci Rep 2015 5 i5798ff). The present inventor has surprisingly found that dexrazoxane catalyzes the formation of hierarchical hybrid supramolecular structures through classical Watson-Crick non-covalent base-pairing of adjacent PAR polymers with an anti-parallel orientation. The following studies, using isolated rat liver mitochondria, were performed to confirm experimentally that dexrazoxane inhibits PAR-induced AIF release from isolated mitochondria by sequestration of PAR.

Materials and Methods

Mitochondria Isolation

Mitochondria were isolated from 0.5 g of freshly excised rat liver tissue using a Dounce homogeniser kit (Abeam abnoi69). The liver tissue was homogenised in 2 mL of mitochondrial isolation buffer using 25-30 Dounce strokes before centrifugation at i,ooog for 10 minutes at 4°C. The supernatant was then transferred to fresh micro centrifuge tubes before further centrifugation at i2,ooog for 10 minutes at 4°C. Pelleted mitochondria were washed by re-suspension in 1 mL of mitochondrial isolation buffer before again being centrifuged at i2,ooog for 10 minutes at 4°C. 100 μί, of the supernatant was frozen at -20°C for sample purity determination. Mitochondria pellets were again re-suspended in mitochondrial isolation buffer containing protease inhibitor cocktail (Abeam ab20im) before protein concentration determination via BCA (Thermo-Fisher, 23227).

JC-i Mitochondrial Viability Assay

Isolated mitochondria were re-suspended in mitochondrial sucrose buffer (300mM sucrose, o.imM EDTA, lomM HEPES pH 7.4) at 500 g/mL following protein concentration determination. 100 μ\, of 500 g/mL mitochondria was then transferred to micro centrifuge tubes before being treated for 30 minutes with indicated concentrations of dexrazoxane or 1 g/mL valinomycin. JC-i reaction buffer was prepared from 5x stock in ddH20 before addition of 0.2 mg/mL JC-i stain (final concentration in reaction was 0.2 g/mL). 90 μΐ, of JC-i staining buffer was then added before addition of 10 μΐ, of 500 μg/mL mitochondria. Plates were incubated at room temperature for 7 minutes before reading at

AIF Release Assay

Isolated mitochondria were re-suspended at 4 mg/mL in mitochondria buffer (300mM sucrose, o.imM EDTA, lomM HEPES pH 7.4). For dexrazoxane and PAR-combination experiments, dexrazoxane and PAR were incubated together or alone on ice for 10 minutes at the concentrations indicated after which they were then added to 100 μΐ, of 4 mg/mL mitochondria and incubated at room temperature for 30 minutes. Following completion of the AIF release assay, supernatants were collected and subjected to the BCA protein assay to determine protein concentration. The mitochondria used in the assay were pelleted, then lysed using RIPA buffer (Abeam abi50034) and subjected to the BCA assay to determine protein content. Resulting lysates were then used in the appropriate Western blot to ensure equal protein loading.

Western Blot Samples that were subjected to Western blot procedure were resolved by SDS-PAGE on 12% pre-cast gels for 25 minutes at 250 volts, before being transferred to PVDF membrane. Membranes were then blocked for 2 hours in 4% milk (0.5% PBS- Tween2o), then probed overnight at 4°C with the appropriate primary antibody (anti- AIF antibody (Abeam ab325i6), anti-porin antibody (Abeam abi5895) or Oxphos antibody cocktail (Abeam abii04i3)). Following 3x 0.5% PBS-Tween20 washes, membranes were then probed with either anti-Rabbit-HRP conjugate antibody (Abeam ab672i) or anti-mouse-HRP conjugate antibody (Abeam ab6789) for 1 hour at 4°C. Membranes were then washed 3x in 0.5% PBS-Tween20 and imaged via ECL reagent (GE-Healthcare, RPN2232).

Results

Study 1 - To isolate mitochondria from rat liver and demonstrate that the isolated mitochondria are suitable for further analysis.

As shown in Figure 20, isolated mitochondria showed enrichment in both porin and AIF signals compared to whole liver lysate (Figures 20A and 20B), whereas the supernatant fraction following mitochondria centrifugation showed very little AIF and porin protein, indicating that the mitochondrial isolation preparation was efficient and did not cause excess mitochondrial damage. Isolated mitochondria were also compared to Abcam's rat heart mitochondrial lysate for both AIF/porin (Figures 20A and 20B), and Abcam's Oxphos antibody cocktail mix (abii04i3) (Figure 20C). AIF and porin protein levels were similar in 5 g of Abcam's rat heart mitochondrial lysate, compared to 5 g of the prepared mitochondrial isolation, further indicating the efficiency of the preparation.

As shown in Figure 21, isolated mitochondria maintained their membrane potential, as assayed by JC-i staining. Treatment with ^g/mL of the electron transport chain un- coupling drug valinomycin resulted in a significant drop in JC-i fluorescence, indicating that the mitochondria remained intact and maintained their membrane potential following isolation.

Thus the mitochondrial isolation preparation was efficient and did not cause excess mitochondrial damage. The isolated mitochondria maintained their membrane potential following isolation, indicating they were suitable for in vitro experimentation. Study 2 - To determine a non-toxic concentration of dexrazoxane that is suitable for incubation with isolated mitochondria. The toxicity of dexrazoxane upon isolated mitochondria was assessed by JC-i staining. As shown in Figure 22, dexrazoxane only reduced mitochondrial viability at concentrations far greater than would be expected to be achieved in vivo (Hasinoff and Aoyama, Drug Metab Dispos 1999 27(2) 265-268) and thus is considered to be minimally toxic to isolated mitochondria.

Study 3 - To investigate the ability of dexrazoxane to inhibit PAR-induced AIF release from isolated mitochondria.

To investigate the ability of dexrazoxane to inhibit PAR-induced AIF release from isolated mitochondria, ιοοηΜ PAR was used to remain consistent with previously published work (Yu et al, Proc Natl Acad Sci 2006 103(48) 18314-18319) (Wang et al, Science Signaling 2011 4(167) ra2o). Figure 23A shows that dexrazoxane reduced PAR- induced AIF release in isolated mitochondria, and that inhibition is inversely-related to dose with the greatest effect observed at a concentration of ιοηΜ of dexrazoxane. Equal protein loading on the gel was demonstrated by the porin loading control (lower band). In order to aid analysis and allow quantification of the Western blot shown in Figure 23A, optical densitometry was also carried out (Figure 23B). This further analysis verified the Western blot and demonstrated the inverse relationship between inhibition and dose with the greatest effect observed at a concentration of dexrazoxane of ιοηΜ. Taken together these two analyses thus provide evidence that dexrazoxane and PAR combine to reduce AIF release in isolated mitochondria.

Discussion This study represents the first time that AIF release has been attempted from isolated mitochondria derived from rat liver; previous studies have used either mouse brain or cultured cells (Yu et al, Proc Natl Acad Sci 2006 103(48) 18314-18319; Baek et al, Molecules and Cells 2013 36(3) 258-266; Wang et al, Science Signaling 2011 4(167) ra2o). The present inventor succeeded in the isolation of viable and live mitochondria from rat liver that were suitable for this study. To ensure that the prepared mitochondrial isolation both contained and was enriched in mitochondria relative to whole liver lysate, the present inventor probed for porin, a protein found on the outer mitochondrial membrane, and also utilised a combination of specific antibodies raised against mitochondrial specific proteins (Oxphos cocktail). The results of these two analyses (Figure 20) showed that the mitochondrial preparation methodology produced mitochondrial fractions enriched in mitochondria. To ensure that the mitochondria produced by the isolation protocol were undamaged and suitable for in vitro analysis, the present inventor utilised the JC-i assay, which relies upon the uptake of the cationic carbocyanine dye JC-i into the mitochondrial matrix in a membrane- potential dependent manner. In healthy cells JC-i is converted into red fluorescent aggregates. However, in unhealthy or dying cells, with low membrane potential, no accumulation of the JC-i dye is possible (Baek et al 2013). This is shown in Figure 21, whereby use of the antibiotic valinomycin, which causes mitochondrial membrane depolarisation, is associated with a dramatic drop in JC-i dye uptake and associated fluorescence. The JC-i assay was also utilised to investigate the toxicity of dexrazoxane on isolated mitochondria. Dexrazoxane was found to be remarkably non-toxic to isolated mitochondria with concentrations as high as 10 μΜ revealing no decrease in JC-i staining relative to control (untreated) (Figure 22). Such high concentrations are however not reached in vivo (Hasinoff and Aoyama, 1999) and so a maximum concentration of ιοοηΜ dexrazoxane was deemed more appropriate for use in study 3.

The novel application of dexrazoxane in sequestering PAR and preventing AIF release was the main focus of this study and required the previously mentioned foundations (studies 1 and 2) to be in place before proceeding to the final investigation of dexrazoxane's ability to bind PAR and prevent AIF release (study 3, Figure 23). Consistent with the extensive modelling studies also presented within this application, the present inventor observed that dexrazoxane has the unique capacity to inhibit AIF release from mitochondria. Interestingly study 3 indicated that dexrazoxane dose- dependently shows an inverse relationship in its ability to sequester PAR, whereby smaller concentrations of dexrazoxane (ιθμΜ) inhibit AIF release to a more significant degree relative to higher concentrations of dexrazoxane (5θμΜ and ιθθμΜ). This observation is consistent with the complex stoichiometry of non-covalent interactions between a polymer and a small molecule upon mixing. It is currently believed that maximal inhibition of PAR-induced AIF release by dexrazoxane is realised at a thermodynamic equilibrium at which there exists a maximum number of interactions between PAR polymer molecules and dexrazoxane molecules, and that in this self- assembling system as presented herein, the time taken to reach equilibrium is inversely-related to the concentration of dexrazoxane. Thus, at very early times with relatively high concentrations of dexrazoxane, little if any inhibition of AIF release is observed. V. Treatment of diseases

The way dexrazoxane, levrazoxane, merbarone, mitindomide and analogues, derivatives and metabolites thereof work is by inhibiting or sequestering PAR by forming a self-assembled supramolecular structure with PAR as set out above.

The self-assembly with PAR explains why dexrazoxane provides sustainable cardioprotection. The proposition that dexrazoxane and its single-ring-opened metabolites exert a cytoprotective/cardioprotective effect through catalyzing hybrid self-assembly with PAR, is entirely consistent with the sustained protection afforded by an agent that is otherwise rapidly metabolized in humans, especially when the damaging influence, i.e. doxorubicin, has a remarkably long half-life. The thermodynamically-favoured self-assembly will have a much longer half-life than the free parent compound. The sequestration of dexrazoxane by PAR offers an additional compartment for the rapid distribution of dexrazoxane that previously will not have received consideration. This compartment will not exist in normal volunteers, but will be present in patients receiving a Τορ2β poison such as an anthracycline. That is, and importantly, distribution of dexrazoxane into this compartment will occur simultaneously, but not necessarily coinci dentally with anthracycline-induced hyperactivation of PARP.

Given the robust symmetry of the self-assembly of dexrazoxane and both single-ring- opened intermediates with PAR, in the terminally-differentiated cardiomyocyte such self-assemblies may well be a permanent feature that in some part explains the long- term sustained benefits of dexrazoxane, for which there is currently no explanation (Lancet Oncol 2010 11 (10) 950-961; J Korean Med Sci 2010 25 (9) 1336-1342).

That dexrazoxane provides such long-term benefits is consistent with the well- documented observation that in the absence of dexrazoxane, doxorubicin has a greater probability of being associated with the development of a chronic cardiomyopathy in which genetic and other intracellular organelle-associated lesions accumulate in the long-term (Cardiovasc Pathol 2010 19 (5) 6167-174). Moreover, the onset of symptoms may take place many years after exposure to doxorubicin. When viewed in this perspective, there is a need to accept that dexrazoxane could have long-term intracellular residence.

In the absence of self-assembly with PAR, it is difficult to explain how protection by dexrazoxane could extend beyond a few hours or even a few minutes. Certainly, in the absence of self-assembly with PAR, there is no prospect for even beginning to understand long term benefits. In the immediate term, self-assembly with PAR qualifies dexrazoxane as a "smart drug" given that cells with hyperactive PARP render these cells as deep compartments for the accumulation of dexrazoxane given the inward concentration gradient that exists.

Now that the present inventor has found that dexrazoxane, levrazoxane, merbarone, mitindomide and analogues, derivatives and metabolites thereof are PAR inhibitors, it is clear that they are useful in the treatment or prevention of a large number of diseases, including but not limited to the diseases listed above, which have all been indicated in the literature as being mediated substantially or in some significant part by PARP/PAR.

Moreover, dexrazoxane, levrazoxane, merbarone, mitindomide and analogues, derivatives and metabolites thereof can now be used in a new way to protect the heart against the cardiotoxic side effects of anthracyclines and to treat or prevent anthracycline-induced extravasation injury, namely by using them to inhibit PAR. This represents a new clinical situation for the use of dexrazoxane, levrazoxane, merbarone, mitindomide and analogues, derivatives and metabolites thereof.

Claims

Claims

1. A compound for use in inhibiting PAR, wherein the compound or a metabolite thereof is capable of inhibiting PAR by forming a supramolecular structure with PAR.

2. A compound for use in inhibiting PAR, wherein the compound is dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, or a pharmaceutically acceptable salt thereof, wherein the compound may be in racemic, enantiomerically enriched or enantiomerically pure form.

3. A compound for use in treating or preventing a disease susceptible to PAR inhibition, wherein the compound or a metabolite thereof is capable of inhibiting PAR by forming a supramolecular structure with PAR.

4. A compound for use in treating or preventing a disease susceptible to PAR inhibition, wherein the compound is dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, or a pharmaceutically acceptable salt thereof, wherein the compound may be in racemic, enantiomerically enriched or enantiomerically pure form.

5. A method of inhibiting PAR using a compound or a metabolite thereof which is capable of inhibiting PAR by forming a supramolecular structure with PAR.

6. A method of inhibiting PAR using dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, or a pharmaceutically acceptable salt thereof, wherein the compound may be in racemic, enantiomerically enriched or enantiomerically pure form.

7. A method of treating or preventing a disease susceptible to PAR inhibition, comprising administering to a patient in need thereof a compound or a metabolite thereof which is capable of inhibiting PAR by forming a supramolecular structure with PAR.

8. A method of treating or preventing a disease susceptible to PAR inhibition, comprising administering to a patient in need thereof dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, or a pharmaceutically acceptable salt thereof, wherein the compound may be in racemic, enantiomerically enriched or enantiomerically pure form.

A compound as claimed in claim 3 or 4, or a method as claimed in claim 7 or 8, wherein the disease is cancer, a disease of the cardiovascular system, central nervous system, gastrointestinal tract, immune system or skin, a disease associated with the ear or eye, a hepatic disease, an infection (including a viral infection), a metabolic disease, a musculoskeletal disease, an oral disease, a renal disease, a respiratory disease, or ischaemia/reperfusion injury to an organ.

A compound or a method as claimed in any one of claims 1 to 9, wherein the compound is dexrazoxane, levrazoxane, razoxane, desmethyl dexrazoxane (ICRF- 154), methyl dexrazoxane (ICRF-193), ICRF-202, ICRF-192, ICRF-158, bimolane, methyl bimolane, sobuzoxane, merbarone or mitindomide, or a pharmaceutically acceptable salt thereof.

A compound or a method as claimed in claim 10, wherein the compound is dexrazoxane or a pharmaceutically acceptable salt thereof.

A compound or a method as claimed in claim 10, wherein the compound is not dexrazoxane or a pharmaceutically acceptable salt thereof.

A compound or a method as claimed in any one of claims 1 to 12, wherein the salt is a hydrofluoric, hydrochloric, hydrobromic, hydroiodic, sulphuric, phosphoric or organic acid addition salt.

A PAR inhibitor for use as a cardioprotective agent.

15. A PAR inhibitor for use in treating or preventing the cardiotoxic side effects of anthracyclines.

16. A method of treating or preventing the cardiotoxic side effects of anthracyclines, comprising administering a PAR inhibitor to a patient in need thereof.

17. A PAR inhibitor as claimed in claim 14 or 15, or a method as claimed in claim 16, wherein the PAR inhibitor is dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, or a pharmaceutically acceptable salt thereof, which may be in racemic, enantiomerically enriched or enantiomerically pure form.

18. A PAR inhibitor or a method as claimed in claim 17, wherein the PAR inhibitor is not dexrazoxane or a pharmaceutically acceptable salt thereof.

19. A PAR inhibitor or a method as claimed in any one of claims 14 to 18, wherein the PAR inhibitor is used in combination with a PARP inhibitor.

20. A PAR inhibitor or a method as claimed in claim 19, wherein the PARP inhibitor is 3-aminobenzamide, Iniparib (BSI 201), BMN-673, Olaparib (AZD-2281), Rucaparib (AG014699, PF-01367338), Veliparib (ABT-888), CEP 9722,

Niraparib (MK4827), BGB-290, PJ34 or INO-1001, or a pharmaceutically acceptable salt thereof.

21. A PAR inhibitor for use in treating or preventing extravasation.

22. A method of treating or preventing extravasation, comprising administering a PAR inhibitor to a patient in need thereof.

23. A PAR inhibitor as claimed in claim 21, or a method as claimed in claim 22, wherein the extravasation is caused by intravenous anthracycline chemotherapy.

24. A PAR inhibitor or a method as claimed in any one of claims 21 to 23, wherein the PAR inhibitor is dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, or a pharmaceutically acceptable salt thereof, which may be in racemic, enantiomerically enriched or enantiomerically pure form.

25. A PAR inhibitor or a method as claimed in claim 24, wherein the PAR inhibitor is not dexrazoxane or a pharmaceutically acceptable salt thereof.

26. A PAR inhibitor or a method as claimed in any one of claims 21 to 25, wherein the PAR inhibitor is used in combination with a PARP inhibitor.

27. A PAR inhibitor or a method as claimed in claim 26, wherein the PARP inhibitor is 3-aminobenzamide, Iniparib (BSI 201), BMN-673, Olaparib (AZD-2281), Rucaparib (AG014699, PF-01367338), Veliparib (ABT-888), CEP 9722, Niraparib (MK4827), BGB-290, PJ34 or INO-1001, or a pharmaceutically acceptable salt thereof.

28. A polymer of:

(i) polyadenylated RNA or DNA, and

(ii) dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, or a pharmaceutically acceptable salt thereof, which may be in racemic, enantiomerically enriched or enantiomerically pure form.

29. A polymer as claimed in claim 28, wherein the polymer is a polymer of (i) polyadenylated RNA or DNA, and (ii) dexrazoxane or a pharmaceutically acceptable salt thereof.

30. A polymer as claimed in claim 28 or 29, for treating or preventing a disease susceptible to PAR inhibition.

31. A polymer as claimed in claim 30, wherein the disease is cancer, a disease of the cardiovascular system, central nervous system, gastrointestinal tract, immune system or skin, a disease associated with the ear or eye, a hepatic disease, an infection (including a viral infection), a metabolic disease, a musculoskeletal disease, an oral disease, a renal disease, a respiratory disease, or ischaemia/reperfusion injury to an organ.

32. A combination of a PAR inhibitor and a PARP inhibitor, or a combination of a PAR inhibitor and a second PAR inhibitor.

33. A combination as claimed in claim 32, wherein the PAR inhibitor is dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, or a pharmaceutically acceptable salt thereof, which may be in racemic, enantiomerically enriched or enantiomerically pure form.

34· A combination as claimed in claim 33, wherein the PAR inhibitor is dexrazoxane or a pharmaceutically acceptable salt thereof.

35. A combination as claimed in any one of claims 32 to 34, wherein the PARP inhibitor is 3-aminobenzamide, Iniparib (BSI 201), BMN-673, Olaparib (AZD-

2281), Rucaparib (AG014699, PF-01367338), Veliparib (ABT-888), CEP 9722, Niraparib (MK4827), BGB-290, PJ34 or INO-1001, or a pharmaceutically acceptable salt thereof.

36. A combination as claimed in any one of claims 32 to 34, wherein the second PAR inhibitor is Iduna, ME0328, AG- 14361, UPF 1069, AZD 2461 or A-966492, or a pharmaceutically acceptable salt thereof.

37. A combination as claimed in any one of claims 32 to 36, for treating or preventing a disease susceptible to PAR inhibition.

38. A combination as claimed in claim 37, wherein the disease is cancer, a disease of the cardiovascular system, central nervous system, gastrointestinal tract, immune system or skin, a disease associated with the ear or eye, a hepatic disease, an infection (including a viral infection), a metabolic disease, a musculoskeletal disease, an oral disease, a renal disease, a respiratory disease, or ischaemia/reperfusion injury to an organ.

39. Use of a compound for pre-treatment of mesenchymal stem cells, wherein the compound is dexrazoxane, levrazoxane, merbarone or mitindomide, or an analogue, derivative or metabolite thereof, or a pharmaceutically acceptable salt thereof, wherein the compound may be in racemic, enantiomerically enriched or enantiomerically pure form.