MODULATION OF OSTEOGENESIS AND OR ANGIOGENESIS BY

MODULATING PEROXIDASE FUNCTIONALITY

PRIORITY CLAIM

[001] This application claims priority to Australian provisional patent application number 2014902437 filed on 25 June 2014, the content of which is hereby incorporated by reference.

FIELD

[002] The present disclosure relates to methods for modulating osteogenesis and/or angiogenesis by modulating peroxidase functionality. The present disclosure also relates to compositions and products for modulating osteogenesis and/or angiogenesis by modulating peroxidase functionality.

BACKGROUND

[003] The process of angiogenesis involves coordinated endothelial cell proliferation, invasion, migration, and tube formation. This process involves a balance between pro- angiogenic factors and anti-angiogenic factors. Angiogenesis is crucial for normal development such as wound healing and bone remodelling and also plays an essential role in tumour development and other pathological disorders.

[004] The notion of targeting angiogenesis for therapeutic purposes has attracted immense interest over the last few decades. The discovery of new pro- and anti- angiogenic factors and the ability to regulate their expression endogenously or administer them exogenously has potential implications to multiple conditions, and presents a unique avenue for the development of therapeutics.

[005] For example, the concept of targeting angiogenesis with novel anti-angiogenic therapeutics has become a real strategy not only for the treatment of cancers, but also their recurrence. A variety of potential angiogenesis inhibitors have been developed which show therapeutic potential.

Substitute Sheet

(Rule 26) RO/AU [006] The concept of promoting angiogenesis in ischemic tissues remains a potential therapeutic approach, for example for the treatment of myocardium and skeletal muscles that lack sufficient blood supply. A number of pro-angiogenic factors are in clinical trial.

[007] Osteogenesis, or bone formation, is a temporally controlled, multistep process involving a variety of factors. Osteogenesis not only occurs during bone formation but also during bone repair, and the ability to control osteogenesis has a variety of therapeutic applications.

[008] It is also not widely recognised that angiogenesis and bone formation are coupled during skeletal development and fracture healing. Angiogenic factors may interact with bone cells to improve bone formation and bone regeneration. Angiogenesis and osteogenesis may share a number of mechanistic commonalities.

[009] There is a continuing need to identify the molecular mechanisms by which osteogenesis and/or angiogenesis are controlled and to identify new possible targets for therapeutic intervention. The present disclosure relates to the determination that peroxidases, aside from their role in defence mechanisms, have a fundamental role in angiogenesis and bone formation.

SUMMARY

[0010] The present disclosure relates to methods, compositions and products for modulating osteogenesis and/or angiogenesis.

[001 1] Certain embodiments of the present disclosure provide a method of modulating osteogenesis and/or angiogenesis in a subject, the method comprising administering to the subject an effective amount of an agent that modulates peroxidase functionality, thereby modulating osteogenesis and/or angiogenesis in the subject.

[0012] Certain embodiments of the present disclosure provide a method of modulating osteogenesis and/or angiogenesis in a biological system, the method comprising exposing the biological system to an effective amount of an agent that modulates peroxidase functionality, thereby modulating osteogenesis and/or angiogenesis in the biological system.

[0013] Certain embodiments of the present disclosure provide a method of preventing and/or treating a fracture in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that modulates peroxidase functionality, thereby preventing and/or treating the fracture.

[0014] Certain embodiments of the present disclosure provide a method of modulating bone grafting in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that modulates peroxidase functionality, thereby modulating bone grafting in the subject.

[0015] Certain embodiments of the present disclosure provide a method of modulating remodelling of bone in a biological system, the method comprising exposing the biological system to an effective amount of an agent that modulates peroxidase functionality, thereby modulating remodelling of bone.

[0016] Certain embodiments of the present disclosure provide a method of modulating bone mineralization in a biological system, the method comprising exposing the biological system to an effective amount of an agent that modulates peroxidase functionality, thereby modulating bone mineralization.

[0017] Certain embodiments of the present disclosure provide a method of modulating collagen production by osteoblasts in a biological system, the method comprising exposing the biological system to an effective amount of an agent that modulates peroxidase functionality, thereby modulating collagen production.

[0018] Certain embodiments of the present disclosure provide a method of modulating angiogenesis in a biological system, the method comprising exposing the biological system to an effective amount of an agent that modulates peroxidase functionality, thereby modulating angiogenesis in the biological system.

[0019] Certain embodiments of the present disclosure provide a method of preventing and/or treating a disease condition or state associated with undesired and/or dysfunctional angiogenesis in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that modulates peroxidase functionality, thereby preventing and/or treating the disease, condition or state in the subject.

[0020] Certain embodiments of the present disclosure provide a method of inhibiting growth and/or metastasis of a cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits peroxidase functionality, thereby inhibiting growth and/or metastasis of the cancer in the subject.

[0021] Certain embodiments of the present disclosure provide a method of improving the susceptibility of a cancer to treatment with an anti-cancer agent in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that modulates peroxidase functionality.

[0022] Certain embodiments of the present disclosure provide a method of modulating endothelial cell migration in a biological system, the method comprising exposing the biological system to an effective amount of an agent that modulates peroxidase functionality, thereby modulating endothelial cell migration in the biological system.

[0023] Certain embodiments of the present disclosure provide a method of modulating endothelial cell proliferation in a biological system, the method comprising exposing the biological system to an effective amount of an agent that modulates peroxidase functionality, thereby modulating endothelial cell proliferation in the biological system.

[0024] Certain embodiments of the present disclosure provide a method of modulating endothelial tube formation in a biological system, the method comprising exposing the biological system to an effective amount of an agent that modulates peroxidase functionality, thereby modulating endothelial tube formation in the biological system.

[0025] Certain embodiments of the present disclosure provide a composition for modulating osteogenesis and/or angiogenesis, the composition comprising an effective amount of an agent that modulates peroxidase functionality.

[0026] Certain embodiments of the present disclosure provide an osteogenic composition, the composition comprising an effective amount of an agent that promotes peroxidase functionality.

[0027] Certain embodiments of the present disclosure provide an anti-osteogenic composition, the composition comprising an effective amount of an agent that inhibits peroxidase functionality.

[0028] Certain embodiments of the present disclosure provide a pro-angiogenic composition, the composition comprising an effective amount of an agent that promotes peroxidase functionality.

[0029] Certain embodiments of the present disclosure provide an anti-angiogenic composition, the composition comprising an effective amount of an agent that inhibits peroxidase functionality.

[0030] Certain embodiments of the present disclosure provide an anti-cancer composition, the composition comprising an effective amount of an agent that inhibits peroxidase functionality.

[0031] Certain embodiments of the present disclosure provide an anti-metastatic composition, the comprising an effective amount of an agent that inhibits peroxidase functionality.

[0032] Certain embodiments of the present disclosure provide a combination product, the combination product comprising the following components:

an agent that inhibits peroxidase functionality; and

an anti-cancer agent;

wherein the components are provided in a form for separate or combined administration to a subject in need thereof.

[0033] Certain embodiments of the present disclosure provide a method of identifying an agent for modulating osteogenesis and/or angiogenesis, the method comprising determining the ability of a candidate agent that modulates peroxidase functionality to modulate osteogenesis and/or angiogenesis and identifying the candidate agent as an agent for modulating osteogenesis and/or angiogenesis. [0034] Other embodiments are disclosed herein. BRIEF DESCRIPTION OF THE FIGURES

[0035] Certain embodiments are illustrated by the following figures. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the description.

[0036] Figure 1 shows that peroxidases promote HUVEC migration and proliferation.

[0037] Figure 2 shows induced HUVEC migration (A) and Invasion (B) by peroxidases after 24hrs. HUVEC migration is significantly greater in response to peroxidases vs vehicle control. This response can be attenuated using the specific irreversible peroxidase inhibitor 4-ABAH at 10μΜ and superoxide dismutase (SOD) lOU/ml.

[0038] Figure 3 shows that peroxidases promote 4T-1 tumour cell migration.

[0039] Figure 4 shows peroxidases induce endothelial cell tube formation on matrigel in vitro. The Figure shows induced HUVEC migration (A) and invasion (B) by peroxidases after 24hrs. HUVEC migration is significantly greater in response to peroxidases vs vehicle control. This response can be attenuated using the specific irreversible peroxidase inhibitor 4-ABAH at 10μΜ and superoxide dismutase (SOD) lOU/ml.

[0040] Figure 5 shows that peroxidases promote angiogenesis in vivo. Figure 4(A) shows images taken of representative excised matrigel plugs stimulated with PBS as negative control and the peroxidase enzymes, MPO, EPO, MPO/EPO and SBP at 5μg of total protein. Figure 4(B) shows peroxidase induced angiogenesis quantified using the Drabkin's reagent measure of hemoglobin.

[0041] Figure 6 shows that peroxidases induced an increase in tumour development and metastasis. (A) Representative images of non-invasive bioluminescent images after 23 days. (B) Peroxidases treatment of tumours lead to an increase in bioluminescence quantified as total flux (p/s) indicating an increase in tumour burden. (C) After 23 days, peroxidase treatment enhanced lung metastasis

[0042] Figure 7 shows histological staining of tumours. Representative images of H/E staining of tumours after 23 days.

[0043] Figure 8 shows myeloperoxidase (MPO) and eosinophil peroxidase (EPO) promote collagen I release by cultured human osteoblasts. Panel A shows ELISA detection of soluble collagen I in osteoblast-conditioned medium after 72 hours stimulation with MPO and EPO at the doses indicated. Ascorbic acid 2-phosphate (AA) at 100 μιηοΙ/Ε served as the positive control, whereas cells treated with Dulbecco's modified Eagle's medium (DMEM) alone (Unstim.) served as the baseline control. The levels of collagen I are expressed as fold change, normalized so the average values of Unstim were set to 1. Panel B shows viability of cultured osteoblasts after 72 hours stimulation as assessed using the alamarBlue dye assay. Cell viability was normalized so the average values of Unstim cells were set to 100% relative to each peroxidase dose. The data are pooled from five experiments each conducted using cells derived from different individual donors. Data are the mean ± SD of fifteen determinations for unstim, AA and each peroxidase dose. ' O.01, ⁱ P<0.001

[0044] Figure 9 shows that peroxidase enzymes SRB and HRB stimulate collagen I biosynthesis in normal human osteoblasts.

[0045] Figure 10 shows inhibition of peroxidase-induced collagen I release by 4 amino-benzoic acid hydrazide (4-ABAH). a ELISA detection of soluble collagen I levels in osteoblast-conditioned medium after cells were pre-treated for 30 minutes with 4-ABAH at the doses indicated, then stimulated by the addition of either 1.56 μg/mL myeloperoxidase (MPO), 1.56 μg/mL eosinophil peroxidase (EPO), or 100 μιηοι/L ascorbic acid 2-phosphate (AA) for a further 72 hours. Soluble collagen I levels are expressed as fold change, normalized to the average values of cells treated with Dulbecco's modified Eagle's medium alone (Unstim.). b Viability of osteoblasts after 72 hours treatment with 4-ABAH as assessed using the alamarBlue dye assay. Cell viability was normalized to the average values of cells treated with Dulbecco's modified Eagle's medium alone (Unstim.). Statistical significance was calculated by two-tailed Student's t test with the 4-ABAH-treated groups compared to the treatment group without 4-ABAH. Inhibition studies using 4-ABAH were independently performed three times. Data are the mean ± SD of triplicate determinations. *P<0.05, ' <0.01, *P<0.001

[0046] Figure 1 1 shows that peroxidase enzymes promote in vitro bone formation by culture human osteoblasts.

[0047] Figure 12 shows that peroxidase enzymes promote tissue-like bone regeneration in vitro.

[0048] Figure 13 shows that peroxidase enzymes regulate BMP-2 mRNA levels in cultured human osteoblasts.

[0049] Figure 14 shows peroxidases induce collagen I release without regulating collagen I al mRNA. Panel A shows ELISA detection of soluble collagen I levels in osteoblast-conditioned medium at the indicated time points upon stimulation with 1.56 μg/mL eosinophil peroxidase (EPO), 100 μηιοΙ/L ascorbic acid 2-phosphate (AA), or 10 ng/niL transforming growth factor (TGF)-P2. Cells treated with Dulbecco's modified Eagle's medium (DMEM) alone (Unstim.) served as the baseline control at each time point. The levels of soluble collagen I are expressed as μg/mL. Panel B shows quantitative real-time PCR analysis of the collagen I al mRNA expression (normalised to glyceraldehyde-3 -phosphate dehydrogenase) in osteoblasts at the indicated time points upon stimulation with 1.56 μg/mL eosinophil peroxidase (EPO), 100 μιηοΙ/L ascorbic acid 2-phosphate (AA), or lOng/mL transforming growth factor (TGF)- 2. Cells treated with DMEM alone (Unstim.) served as the baseline control at each time point. The levels of collagen I al transcript are expressed as fold change, normalized so the average values of Unstim at each time point were set to 1. Statistical significance was calculated by two-tailed Student's t test, with the various treatment groups compared to the DMEM alone (Unstim) group at each time point. Each time course experiment was independently performed three times. Data are the mean ± SD of triplicate determinations for each time point. * <0.05, ' <0.01, *P<0.001 [0050] Figure 15 shows inhibition of peroxidase-induced collagen I release by the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). a ELISA detection of soluble collagen I levels in osteoblast-conditioned medium after cells were pre-treated for 30 minutes with DMOG at the doses indicated, then stimulated by the addition of either 1.56 μg/mL myeloperoxidase (MPO), 1.56 μg/mL eosinophil peroxidase (EPO), or 100 μιηοι/L ascorbic acid 2-phosphate (AA) for a further 72 hours. Soluble collagen I levels are expressed as fold change, normalized to the average values of cells treated with Dulbecco's modified Eagle's medium (DMEM) alone (Unstim.). b Viability of osteoblasts after 72 hours treatment with DMOG as assessed using the alamarBlue dye assay. Cell viability was normalized to the average values of cells treated with DMEM culture medium alone (Unstim.). Statistical significance was calculated by two-tailed Student's t test with the DMOG-treated groups compared to the treatment group without DMOG. Inhibition studies using DMOG were independently performed twice over the same dose range. Data are the mean ± SD of triplicate determinations.. *P<0.05, ^† <0.01, *P<0.001.

[0051] Figure 16 shows eosinophil peroxidase regulates osteogenic gene expression. Quantitative real-time PCR analysis of the BMP-2, BSP, Wnt5a and FRZB mRNA expression (normalised to glyceraldehyde-3-phosphate dehydrogenase) in cultured osteoblasts following stimulation with 5 μg/mL eosinophil peroxidase (EPO) for 12 days. Cells treated with DMEM mineralization medium alone (Unstim.) served as the baseline control. Transcript levels are expressed as fold change, normalized so the average values of Unstim were set to 1. Statistical significance was calculated by two- tailed Student's t test with the EPO treatment groups compared to the mineralization medium alone (Unstim) group. Data are the mean ± SD of triplicate determinations. PO.001

[0052] Figure 17 shows localization of peroxidase distribution by indirect immunofluorescence. Cultured human osteoblasts were exposed to ^g/mL myeloperoxidase (MPO) or eosinophil peroxidase (EPO) in serum-free Dulbecco's modified Eagle's medium for either 20 minutes (surface) or 3 hours (intracellular). Cells were fixed, permeabilized (intracellular only) and stained for MPO and EPO localization, as described in Materials and Methods. Original magnification: x40 (Surface and Intracellular images). [0053] Figure 18 shows a MouseFix femoral fracture model. Immediately after fracture, mice were injected with 25 μg of SBP (or saline vehicle control) directly into the fracture site and then every second day until day 18. PET-CT imaging shows doubling of the callus size with SBP.

DETAILED DESCRIPTION

[0054] The present disclosure relates to methods, compositions and products for modulating osteogenesis and/or angiogenesis.

[0055] Certain disclosed embodiments provide methods, products, compositions, and uses thereof that have one or more advantages. For example, some of the advantages of certain embodiments disclosed herein include one or more of the following: identification of a new class of agents that modulate angiogenesis and/or osteogenesis; a new class of agents that may have utility in situations where modulation of angiogenesis and/or osteogenesis would have therapeutic benefit; use of a class of agents that may have utility as anti-cancer and/or anti-metastatic agents; use of a class of agents that may have utility to improve treatment of cancers with anti-cancer agents or chemotherapeutic agents; use of a class of agents that have the capacity to promote angiogenesis; use of a class of agents that have the capacity to inhibit angiogenesis; use of class of agents that have utility in research, and in particular for the study of angiogenesis, endothelial cell biology or bone formation; use of a class of agents that may have the capacity to promote angiogenesis in disorders relating to insufficient angiogenesis; use of a class of agents that may have the capacity to inhibit angiogenesis in disorders where inhibiting angiogenesis would be therapeutically beneficial; use of a class of agents that induce endothelial cell proliferation, migration and tube formation, processes which are essential for the formation of new blood vessels in vitro and in vivo; use of a class of agents that promote endothelial cell and tumour cell migration in vitro and potentiate metastasis in vivo; use of a class of agents that may have the capacity to promote osteoegenesis in disorders where promoting osteogenesis would be therapeutically beneficial; use of a class of agents that may have the capacity to inhibit osteoegenesis in disorders where inhibiting osteogenesis would be therapeutically beneficial; use of a class of agents that promote collagen deposition and mineralization of osteoblast cells; identification that exogenous peroxidises have a potent activity to promote angiogenesis and/or osteogenesis over endogenous peroxidases. Other advantages of certain embodiments of the present disclosure are disclosed herein.

[0056] The present disclosure is based upon the determination that peroxidase enzymes have hereto unrecognised activities apart from their usual recognised role as providing a mechanism for oxidative defence against invading micro-organisms. Specifically, the present disclosure is based on the recognition that peroxidases are modulators of osteogenesis and/or angiogenesis and as such are a target for new pro- angiogenic and anti-angiogenic therapies and also a target for new pro-osteogenic and anti-osteogenic therapies. Enzymes with peroxidase activity have been found to promote endothelial cell and tumour cell migration in vitro and potentiate metastasis in vivo. This establishes the connection between peroxidase deposition within and around tumours and the demonstration that these enzymes have a pro-tumourigenic role and a pro-metastatic role, establishes peroxidase enzymes as potential targets in the development of anti-tumour therapeutics. In addition, enzymes with peroxidase activity have been found to promote collagen deposition and mineralization of osteoblast cells, and to assist in bone healing, establishing that these enzymes are potential targets in the development of bone therapies.

[0057] Certain embodiments of the present disclosure provide a method of modulating osteogenesis and/or angiogenesis.

[0058] Certain embodiments of the present disclosure provide a method of modulating osteogenesis and/or angiogenesis in a subject, the method comprising administering to the subject an effective amount of an agent that modulates peroxidase functionality, thereby modulating osteogenesis and/or angiogenesis in the subject.

[0059] The term "peroxidase" refers to a molecule that has the capacity to promote the oxidation of various compounds using a peroxide (such as hydrogen peroxide) which is reduced in the process. The term covers peroxidases which are natural, non-natural, synthetic, recombinant, endogenous, or exogenous. Examples of peroxidases include a lactoperoxidase, a myeloperoxidase, an eosinophil peroxidase, and a glutathione peroxidase. Other peroxidases are contemplated. [0060] The term "peroxidase functionality" as used herein refers to a direct and/or indirect aspect of one or more of peroxidase activity, peroxidase function, peroxidase expression, and peroxidase associated signalling. In certain embodiments, peroxidase functionality is inhibited. In certain embodiments, peroxidase functionality is promoted.

[0061] The term "effective amount" as used herein refers to that amount of an agent that is sufficient to illicit the desired response or outcome. In certain embodiments, the effective amount is a therapeutically effective amount.

[0062] The term "therapeutically effective amount" as used herein refers to that amount of an agent that is sufficient to effect prevention and/or treatment, when administered to a subject. The therapeutically effective amount will vary depending upon a number of factors, including for example the specific activity of the agent being used, the severity of the disease, condition or state in the subject, the age, physical condition, existence of other disease states, and nutritional status of the subject.

[0063] The term "modulate", and variants thereof such as "modulating", as used herein refers to a promotion, inhibition and/or change of a property. In certain embodiments, the modulating comprises a promotion, increase, enhancement, or stimulation of a property. In certain embodiments, the modulating comprises an inhibition, decrease, reduction, retardation, delay or suspension of a property.

[0064] In certain embodiments, the agent comprises a drug, a small molecule, a protein, a polypeptide, a lipid, a carbohydrate, a nucleic acid, a DNA, a RNA, an oligonucleotide, a ribozyme, a biologic, an aptamer, a peptide, a cofactor, a ligand, a ligand mimetic, a receptor, an enzyme, a kinase, a phosphatase, a signalling molecule, a cytokine, a growth factor, a metal ion, a chelate, an antisense nucleic acid, a siRNA, a microRNA, an antibody, and an amino acid. Other types of molecules are contemplated.

[0065] The term "nucleic acid" as used herein refers to an oligonucleotide or a polynucleotide and includes for example DNA, RNA, DNA/RNA, a variant or DNA and/or RNA (for example a variant of the sugar-phosphate backbone and/or a variant of one or more bases, such as methylation), and may be single stranded, double stranded, non-methylated, methylated or other forms thereof. In certain embodiments, the nucleic acid is a non-naturally occurring nucleic acid, a naturally occurring nucleic acid, a nucleic acid of genomic origin, a mitochondrial nucleic acid, a nucleic acid of cDNA origin (derived from a mRNA), a nucleic acid derived from a virus, a nucleic acid of synthetic origin, a single stranded DNA, a double stranded DNA, an analogue of DNA and/or RNA, and/or a derivative, fragment and/or combination of any of the aforementioned. Examples of derivatives also include nucleic acids that have a blocking group at the 5' and/or 3' ends for example to improve stability, and/or nucleic acids fused to other molecules. Other types of nucleic acids are contemplated. Methods for producing nucleic acids are known and include for example nucleic acids produced by recombinant DNA technology or nucleic acids produced by chemical synthesis. Methods for use of nucleic acids to express proteins or polypeptides are known in the art.

[0066] In certain embodiments, the agent is an antisense nucleic acid, such as an antisense RNA. In certain embodiments, the agent is a small interfering RNA. In certain embodiments, the agent is a microRNA. Methods for producing and delivering antisense nucleic acids, microRNAs and siRNAs are known. For example, therapeutic nucleic acids for treating cancer are described in 'Nucleic Acid Therapeutics in Cancer" 2004 ed. Alan M. Gewirtz, Humana Press Inc.

[0067] In certain embodiments, the agent modulates one or more of peroxidase activity, peroxidase function, peroxidase expression, peroxidase associated signalling and peroxiredoxin functionality.

[0068] For example, the agent may change activity of a peroxidase, the agent may change localisation of a peroxidase, the agent may change the synthesis and/or degradation rates of a peroxidase, the agent may change the timing of peroxidase activity, the agent may change the ability of the peroxidase to interact with other species (such as a change in the ability to interact with a substrate), the agent may change the chemical composition of a peroxidase, and the agent may change signalling events associated with a peroxidase. In a similar manner, the agent may also change upstream or downstream effectors of peroxidase functionality. Other ways of altering peroxidase functionality are contemplated. [0069] In certain embodiments, the agent inhibits peroxidase functionality. Examples of agents that inhibit peroxidase functionality comprise an agent that decreases endogenous peroxidase expression, an antagonist of peroxidase activity, a ligand mimetic, a small molecule inhibitor, an antibody, an inhibitor of peroxidase internalization, and an inhibitor of peroxidase associated signalling. Other types of agents are contemplated.

[0070] Inhibitors of peroxidase are known in the art. Examples of peroxidase inhibitors include peptide base peroxidase inhibitors (for example as described in WO 201 1/044096) or MPO inhibitors of the 2-thioxanthines class of molecules, for example as described in Tiden AK et al (201 1) Journal of Biological Chemistry Oct 28; 286(43): 37578-89.

[0071] In certain embodiments, the agent promotes or increases peroxidase functionality. Examples of agents that promote or increase peroxidase functionality comprises an agent that increases endogenous peroxidase expression, an exogenous peroxidise, a synthetic peroxidise, a recombinant peroxidase, and an agent that increase peroxidase activity. Other types of agents are contemplated.

[0072] In certain embodiments, the agent comprises a peroxidase. Peroxidases may be obtained commercially or produced by a method known in the art. For example, a recombinant human peroxidase (EPO) may be produced as described in Ciaccio et al (2006) Biochem J. Apr 15 395 (Pt 2): 295-301.

[0073] In certain embodiments, the agent comprises an exogenous peroxidase. In certain embodiments, the agent comprises a recombinant or synthetic peroxidase.

[0074] In certain embodiments, the agent comprises a human peroxidase.

[0075] In certain embodiments, the agent comprises a non-human peroxidase, for example a plant peroxidase. In certain embodiments, the agent comprises a small molecule peroxidase mimetic. Examples of peroxidase mimetics include mimetics as described in WO 2008/100628. [0076] In certain embodiments, the agent is used to prevent and/or treat a disease, condition or state as described herein. In certain embodiments, the agent is used to prevent and/or treat a disease, condition or state that may benefit from the therapeutic promotion of angiogenesis. In certain embodiments, the agent is used to prevent and/or treat a disease, condition or state that may benefit from the therapeutic inhibition of angiogenesis. In certain embodiments, the agent is used to prevent and/or treat a disease, condition or state that may benefit from the therapeutic promotion of osteogenesis. In certain embodiments, the agent is used to prevent and/or treat a disease, condition or state that may benefit from the therapeutic inhibition of osteogenesis.

[0077] In certain embodiments, the subject is a human or animal subject. Methods for modulating osteogenesis and/or angiogenesis in a subject are as described. In certain embodiments, the subject is suffering from, or susceptible to, a disease, condition or state as described herein.

[0078] In certain embodiments, the subject is a mammalian subject, a livestock animal (such as a horse, a cow, a sheep, a goat, a pig), a domestic animal (such as a dog or a cat) and other types of animals such as monkeys, rabbits, mice and laboratory animals. Other types of animals are contemplated. Veterinary applications of the present disclosure are contemplated. Use of any of the aforementioned animals as animal models for angiogenesis or osteogenesis is also contemplated.

[0079] Subjects, suffering from or susceptible to various diseases, conditions and states are as described herein.

[0080] Certain embodiments of the present disclosure provide a method of modulating angiogenesis in a subject, the method comprising administering to the subject an effective amount of an agent that modulates peroxidase functionality, thereby modulating angiogenesis in the subject.

[0081] In certain embodiments, the agent modulates endothelial cell migration and/or endothelial cell tube formation. In certain embodiments, the agent promotes endothelial cell migration and/or endothelial cell tube formation. In certain embodiments, the agent inhibits endothelial cell migration and/or endothelial cell tube formation. [0082] In certain embodiments, the method is used to inhibit growth and/or metastasis of a cancer, to prevent and/or treat a disease condition or state associated with undesired and/or dysfunctional angiogenesis, to promote angiogenesis in disorders relating to insufficient angiogenesis and/or to improve the susceptibility of a cancer to treatment with an anti-cancer agent.

[0083] In certain embodiments, the angiogenesis comprises angiogenesis associated with a disease, condition or state as described herein.

[0084] In certain embodiments, the disease, condition or state is associated with undesired and/or dysfunctional angiogenesis in a subject.

[0085] In certain embodiments, the disease, condition or state is associated with uncontrolled or excessive angiogenesis in a subject.

[0086] Certain embodiments of the present disclosure provide a method of preventing and/or treating a disease condition or state associated with undesired and/or dysfunctional angiogenesis in a subject using an agent that modulates peroxidase functionality as described herein.

[0087] Certain embodiments of the present disclosure provide a method of preventing and/or treating a disease condition or state associated with uncontrolled and/or excessive angiogenesis in a subject using an agent that modulates peroxidase functionality as described herein.

[0088] The term "preventing", and related terms such as "prevention" and "prevent", refer to obtaining a desired pharmacologic and/or physiologic effect in terms of arresting or suppressing the appearance of one or more symptoms in the subject.

[0089] The term "treatment", and related terms such as "treating" and "treat", refer to obtaining a desired pharmacologic and/or physiologic effect in terms of improving the condition of the subject, ameliorating, arresting, suppressing, relieving and/or slowing the progression of one or more symptoms in the subject, a partial or complete stabilization of the subject, a regression of the one or more symptoms, or a cure of a disease, condition or state in the subject. [0090] In certain embodiments, the angiogenesis is associated with a cancer (e.g., primary, secondary or metastatic), an ocular disease (eg age-related macular degeneration), a diabetic retinopathy, a retinal vein occlusion, or rheumatoid arthritis, psoriasis, and endometriosis.

[0091] In certain embodiments, the cancer is a late stage cancer, a non-early stage cancer, a cancer that has progressed from the early stage, an established cancer, a metastatic cancer, or a cancer with a tumour diameter of greater than 0.5 cm, greater than 1 cm, or greater than 2 cm.

[0092] In certain embodiments, the angiogenesis comprises angiogenesis associated with a disease, condition or state selected from a cancer, multiple sclerosis, vascular malformations, obesity, psoriasis, warts, allergic dermatitis, primary pulmonary hypertension, asthma, cystic fibrosis, inflammatory bowel disease, peri-dontal disease, liver cirrhosis, endometriosis, arthritis, macular degeneration and osteomyelitis. Such conditions may benefit from the therapeutic inhibition of angiogenesis.

[0093] In certain embodiments, the angiogenesis comprises angiogenesis associated with a cancer. Examples of cancers (e.g., malignant, metastatic) include cancers from the bladder; brain; breast; colon; esophagus; gastrointestine; head; kidney; liver; lung; nasopharynx; neck; ovary; prostate; skin; stomach; testis; tongue; neuron or uterus. In addition, the cancer may for example be of the following histological type: neoplasm; malignant; carcinoma; carcinoma undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma; malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma; familial polyposis coli; solid carcinoma; carcinoid tumour; malignant; branchiolo-alveolar carcinoma; papillary carcinoma; squamous cell carcinoma; basal adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; ovarian stromal tumour, malignant; and roblastoma, malignant; Sertoli cell carcinoma; osteolytic and osteosclerotic bone cancers, Ewing's sarcoma and bone metastasis.

[0094] Certain embodiments of the present disclosure provide a method of preventing and/or treating a cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that modulates peroxidase functionality, thereby modulating preventing and/or treating the cancer in the subject.

[0095] In certain embodiments, the angiogenesis comprises angiogenesis associated with a disease, condition or stated selected from Alzheimer's disease, amyotrophic lateral sclerosis, diabetes, atherosclerosis, hypertension, Crohn's disease, lupus, preeclampsia, nephropathy, a chronic wound, ischemia and ischemic conditions, and coronary artery disease, and a diabetic ulcer. Other diseases, conditions or states are contemplated. Such conditions may benefit from the therapeutic promotion of angiogenesis.

[0096] Methods are known in the art for assessing the extent of angiogenesis in a subject. For example, the extent of angiogenesis may be assessed in situ in a patient or in a tumour by non-invasive techniques such as PET (Positron emission tomography), MRI (Magnetic Resonance Imaging) (Dynamic Contrast Enhanced, DCE-MRI) or CT (Computer Tomography) imaging. MRI or PET permit the use of angiogenic markers to be followed. Alternatively the extent may be assessed using a tumour biopsy or section and subsequent immune-histochemical analyses on endothelial cells to assess their activity and compare it to the activity of normal endothelial cells from a healthy subject or from endothelial cells from the patient but isolated at a different place in the body. Such immune-histochemical analyses may be done using pan-endothelial cell antibodies such as anti-CD31 and anti-CD34 to assess microvessel density. Tissue sections could be stained with markers for endothelial cells, combined with proliferation markers, to explore the ratio between tumour endothelial cells and tumour proliferating cells in the tissue. An example of an endothelial marker is CD31 or CD34.

[0097] In certain embodiments, the modulating angiogenesis comprises promoting angiogenesis. For example, an agent that promotes peroxidase functionality may be used to promote angiogenesis. Examples of agents that promote peroxidase functionality are as described herein.

[0098] In certain embodiments, the agent promotes peroxidase functionality. In certain embodiments, the agent promotes peroxidase functionality and the method comprises promoting angiogenesis in the subject.

[0099] In certain embodiments, the agent promotes peroxidase functionality and the subject is suffering from, or susceptible to, Alzheimer's disease, amyotrophic lateral sclerosis, diabetes, atherosclerosis, hypertension, Crohn's disease, lupus, preeclampsia, nephropathy, a chronic wound, ischaemia, coronary artery disease, and a diabetic ulcer.

[00100] Certain embodiments of the present disclosure provide a method of preventing and/or treating a disease condition or state in a subject selected from Alzheimer's disease, amyotrophic lateral sclerosis, diabetes, atherosclerosis, hypertension, Crohn's disease, lupus, preeclampsia, nephropathy, a chronic wound, ischaemia, coronary artery disease, and a diabetic ulcer, the method comprising administering to the subject a therapeutically effective amount of agent that promotes peroxidase functionality, thereby preventing and/or treating the disease, condition or state in the subject.

[00101] In certain embodiments, the modulating angiogenesis comprises inhibiting angiogenesis. For example, an agent that inhibits peroxidase functionality may be used to inhibit angiogenesis. Examples of agents that inhibit peroxidase functionality are as described herein.

[00102] In certain embodiments, the agent inhibits peroxidase functionality. In certain embodiments, the agent inhibits peroxidase functionality and the method comprises inhibiting angiogenesis in the subject. [00103] In certain embodiments, the agent inhibits peroxidase functionality and the subject is suffering from, or susceptible to, a cancer, multiple sclerosis, vascular malformations, obesity, psoriasis, warts, allergic dermatitis, primary pulmonary hypertension, asthma, cystic fibrosis, inflammatory bowel disease, peri-dontal disease, liver cirrhosis, endometriosis, arthritis, macular degeneration and osteomyelitis.

[00104] Certain embodiments of the present disclosure provide a method of preventing and/or treating a disease, condition or state in the subject selected from a cancer, multiple sclerosis, vascular malformations, obesity, psoriasis, warts, allergic dermatitis, primary pulmonary hypertension, asthma, cystic fibrosis, inflammatory bowel disease, peri-dontal disease, liver cirrhosis, endometriosis, arthritis, macular degeneration and osteomyelitis, the method comprising administering to the subject a therapeutically effective amount of agent that inhibits peroxidase functionality, thereby preventing and/or treating the disease, condition or state in the subject.

[00105] In certain embodiments, the agent inhibits peroxidase functionality and the subject is suffering from, or susceptible to, a cancer and/or metastasis of a cancer. In certain embodiments, the cancer comprises a cancer selected from a breast cancer, an ovarian cancer, endometrial cancer, or a prostate cancer. Examples of cancers are as described herein.

[00106] In certain embodiments, the agent inhibits peroxidase functionality and the subject is suffering from a cancer.

[00107] In certain embodiments, the subject is suffering from a late stage cancer, a non- early stage cancer, a cancer that has progressed from the early stage, an established cancer, a metastatic cancer, or a cancer with a tumour diameter of greater than 0.5 cm, greater than 1 cm, or greater than 2 cm. In certain embodiments, the subject is suffering from a solid cancer. In certain embodiments, the subject is suffering from a carcinoma. In certain embodiments, the subject is suffering from a sarcoma. In certain embodiments, the subject is suffering from a lymphoma. In certain embodiments, the subject is suffering from a germ cell cancer. In certain embodiments, the subject is suffering from a blastoma. In certain embodiments, the subject is suffering from a haematological cancer. In certain embodiments, the subject is suffering from a melanoma, a breast cancer, a prostate cancer, an ovarian cancer, lung cancer, a colorectal cancer, a gastric cancer, a pancreatic cancer, a bladder cancer, an oesophageal cancer, an urothelial cancer, a non-small cell lung cancer, head & neck cancer, a testicular cancer, an uterine cancer, a liver cancer, a renal cancer, a stomach cancer, a cerebral tumour, a malignant myeloma, a CML, an AML, or a lymphoproliferative tumour. In certain embodiments, the subject is suffering from a primary cancer, including a primary of any of the cancers described herein. In certain embodiments, subject is suffering from a secondary cancer, including a secondary cancer of any of the cancers described herein. In certain embodiments, the subject is suffering from a non- metastatic cancer including a non-metastatic cancer of any of the cancers described herein. In certain embodiments, the subject is suffering from a metastatic cancer, including a metastatic cancer of any of the cancers described herein. In certain embodiments, the subject is not suffering from a lung cancer.

[00108] In certain embodiments, the subject is susceptible to a cancer. In certain embodiments, the subject is susceptible to a solid cancer. In certain embodiments, the subject is susceptible to a carcinoma. In certain embodiments, the subject is susceptible to a sarcoma. In certain embodiments, the subject is susceptible to a lymphoma. In certain embodiments, the subject is susceptible to a germ cell cancer. In certain embodiments, the subject is susceptible to a blastoma. In certain embodiments, the subject is susceptible to a haematological cancer. In certain embodiments, the subject is susceptible to a melanoma, a breast cancer, a prostate cancer, an ovarian cancer, lung cancer, a colorectal cancer, a gastric cancer, a pancreatic cancer, a bladder cancer, an oesophageal cancer, an urothelial cancer, a non-small cell lung cancer, head & neck cancer, a testicular cancer, an uterine cancer, a liver cancer, a renal cancer, a stomach cancer, a cerebral tumour, a malignant myeloma, a CML, an AML or a lymphoproliferative tumour. In certain embodiments, the subject is susceptible to a primary cancer, including a primary of any of the cancers described herein. In certain embodiments, subject is susceptible to a secondary cancer, including a secondary cancer of any of the cancers described herein. In certain embodiments, the subject is susceptible to a non-metastatic cancer including a non-metastatic cancer of any of the cancers described herein. In certain embodiments, the subject is susceptible to a metastatic cancer, including a metastatic cancer of any of the cancers described herein. [00109] In certain embodiments, the subject has an increased risk or likelihood of suffering from a disease, condition or state as described herein.

[001 10] In certain embodiments, the subject has an increased risk or likelihood of suffering from a disease, condition or state selected from a cancer, multiple sclerosis, vascular malformations, obesity, psoriasis, warts, allergic dermatitis, primary pulmonary hypertension, asthma, cystic fibrosis, inflammatory bowel disease, peridental disease, liver cirrhosis, endometriosis, arthritis, macular degeneration and osteomyelitis.

[001 11] In certain embodiments, the subject has an increased risk or likelihood of suffering from a cancer. In certain embodiments, the subject has an increased risk or likelihood of suffering from a solid cancer. In certain embodiments, the subject has an increased risk or likelihood of suffering from a carcinoma. In certain embodiments, the subject has an increased risk or likelihood of suffering from a sarcoma. In certain embodiments, the subject has an increased risk or likelihood of suffering from a lymphoma. In certain embodiments, the subject has an increased risk or likelihood of suffering from a germ cell cancer. In certain embodiments, the subject has an increased risk or likelihood of suffering from a blastoma. In certain embodiments, the subject has an increased risk or likelihood of suffering from a haematological cancer. In certain embodiments, the subject has an increased risk or likelihood of suffering from a melanoma, a breast cancer, a prostate cancer, an ovarian cancer, lung cancer, a colorectal cancer, a gastric cancer, a pancreatic cancer, a bladder cancer, an oesophageal cancer, an urothelial cancer, a non-small cell lung cancer, head & neck cancer, a testicular cancer, an uterine cancer, a liver cancer, a renal cancer, a stomach cancer, a cerebral tumour, a malignant myeloma, a CML, an AML or a lymphoproliferative tumour. In certain embodiments, the subject has an increased risk or likelihood of suffering from a primary cancer, including a primary of any of the cancers described herein. In certain embodiments, subject has an increased risk or likelihood of suffering from a secondary cancer, including a secondary cancer of any of the cancers described herein. In certain embodiments, the subject has an increased risk or likelihood of suffering from a non-metastatic cancer including a non-metastatic cancer of any of the cancers described herein. In certain embodiments, the subject has an increased risk or likelihood of suffering from a metastatic cancer, including a metastatic cancer of any of the cancers described herein.

[001 12] In certain embodiments, the subject is undergoing treatment for a cancer as described herein. In certain embodiments, the subject is undergoing treatment with an anti-cancer agent or a chemotherapeutic agent as described herein.

[001 13] In certain embodiments, the subject is suffering from a cancer having inflammatory cell infiltration, such as neutrophil and/or eosinophil infiltration.

[001 14] In certain embodiments, the method further comprises administering to the subject an anti-cancer agent. In this regard, the administration of an agent that modulates peroxidase functionality may promote the efficacy of an anti-cancer agent.

[001 15] In certain embodiments, the method is used to improve the susceptibility of a cancer to treatment with an anti-cancer agent in a subject.

[001 16] Certain embodiments of the present disclosure provide a method of improving the susceptibility of a cancer to treatment with an anti-cancer agent in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that modulates peroxidase functionality.

[001 17] In certain embodiments, the agent inhibits peroxidase functionality.

[001 18] Certain embodiments of the present disclosure provide a method of improving the susceptibility of a cancer to treatment with an anti-cancer agent in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits peroxidase functionality.

[001 19] In certain embodiments, the method is used to inhibit growth and/or metastasis of a cancer in a subject.

[00120] Certain embodiments of the present disclosure provide a method of inhibiting growth and/or metastasis of a cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits peroxidase functionality, thereby inhibiting growth and/or metastasis of the cancer in the subject. [00121] In certain embodiments, the weight and/or volume of a tumour associated with the cancer is reduced upon administration of the agent. Methods for determining tumour weight and/or volume are known, and include for example imaging of a subject to determine these parameters in situ. In certain embodiments, the weight of the tumour associated with the cancer is reduced by 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. In certain embodiments, the volume of the tumour associated with a cancer is reduced by 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[00122] In certain embodiments, the weight and/or volume of a tumour associated with a cancer is reduced upon administration of the agent in combination with one or more other pharmacological agents, such as a chemotherapeutic agent or an anti-cancer agent. In certain embodiments, the weight of the tumour associated with a cancer using such combination treatment is reduced by 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. In certain embodiments, the volume of the tumour associated with a cancer is reduced by 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[00123] In certain embodiments, the weight and/or volume of a tumour associated with a metastatic cancer is reduced upon administration of the agent. Methods for determining tumour weight and/or volume are known, and include for example imaging of a subject to determine these parameters in situ. In certain embodiments, the weight of the tumour associated with a metastatic cancer is reduced by 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. In certain embodiments, the volume of the tumour associated with a metastatic cancer is reduced by 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[00124] In certain embodiments, the weight and/or volume of a tumour associated with a metastatic cancer is reduced upon administration of the agent in combination with one or more other pharmacological agents, such as a chemotherapeutic agent or an anticancer agent. In certain embodiments, the weight of the tumour associated with a metastatic cancer using such combination treatment is reduced by 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. In certain embodiments, the volume of the tumour associated with the metastatic cancer is reduced by 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[00125] In certain embodiments, the number of metastases is reduced is reduced upon administration of the agent. In certain embodiments, the number of metastases is reduced by 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. Methods for determining metastatic load or burden are known, and include for example imaging of a subject to determine this parameter in situ. In certain embodiments, the methods as described herein may be used to reduce the number of metastases in a subject. In certain embodiments, the number of metastases in the subject is reduced.

[00126] In certain embodiments, the number of metastases is reduced is reduced upon administration of the agent and an anti-cancer agent or a chemotherapeutic agent. In certain embodiments, the number of metastases is reduced by 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[00127] Certain embodiments of the present disclosure provide a method of modulating osteogenesis in a subject.

[00128] Certain embodiments of the present disclosure provide a method of modulating osteogenesis in a subject, the method comprising administering to the subject an effective amount of an agent that modulates peroxidase functionality, thereby modulating osteogenesis in the subject.

[00129] Examples of agents that modulate peroxidase functionality are as described herein.

[00130] In certain embodiments, the osteogenesis comprises osteogenesis associated with a disease, condition or state as described herein.

[00131] In certain embodiments, the subject is suffering from, or susceptible to, one or more of a bone related disorder, a bone fracture, dysfunctional and/or reduce bone mineralization, a bone spur, a spinal stiffening, osteoporosis, a cancer induced bone defect, a deficiency in bone grafting or union, a condition associated with inflammatory cells acting to promote osteogenesis, ankylosing spondylitis or heterotopic ossification.

[00132] Certain embodiments of the present disclosure provide a method of preventing and/or treating a disease, condition or state in a subject selected from a bone related disorder, a bone fracture, dysfunctional and/or reduce bone mineralization, a bone spur, a spinal stiffening, osteoporosis, a cancer induced bone defect, a deficiency in bone grafting or union, a condition associated with inflammatory cells acting to promote osteogenesis, ankylosing spondylitis and heterotopic ossification, the method comprising administering to the subject a therapeutically effective amount of an agent that modulates peroxidase functionality.

[00133] In certain embodiments, the agent promotes peroxidase functionality. Examples of agents that promote peroxidase are as described herein. In certain embodiments, the agent comprises a peroxidase. In certain embodiments, the peroxidase comprises an exogenous peroxidase.

[00134] In certain embodiments, the modulating of osteogenesis comprises promoting osteogenesis in the subject.

[00135] For example, an agent that promotes peroxidase functionality may be used to promote osteogenesis.

[00136] In certain embodiments, the agent promotes osteogenesis and the subject is suffering from, or susceptible to, one or more of a bone related disorder, a bone fracture, dysfunctional and/or reduce bone mineralization, a bone spur, a spinal stiffening, osteoporosis, a cancer induced bone defect, and a deficiency in bone grafting or union. Such conditions may benefit from the therapeutic promotion of osteogenesis.

[00137] Certain embodiments of the present disclosure provide a method of preventing and/or treating a disease, condition or state in a subject selected from a bone related disorder, a bone fracture, dysfunctional and/or reduce bone mineralization, a bone spur, a spinal stiffening, osteoporosis, a cancer induced bone defect, a deficiency in bone grafting or union, the method comprising administering to the subject a therapeutically effective amount of an agent that promotes peroxidase functionality.

[00138] Certain embodiments of the present disclosure provide a method of preventing and/or treating a fracture in a subject.

[00139] Certain embodiments of the present disclosure provide a method of preventing and/or treating a fracture in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that modulates peroxidase functionality, thereby preventing and/or treating the fracture.

[00140] Certain embodiments of the present disclosure provide a method of preventing and/or treating a fracture in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that promotes peroxidase functionality, thereby preventing and/or treating the fracture.

[00141] Certain embodiments of the present disclosure provide a method of modulating bone grafting in a subject.

[00142] Certain embodiments of the present disclosure provide a method of modulating bone grafting in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that modulates peroxidase functionality, thereby modulating bone grafting in the subject.

[00143] Certain embodiments of the present disclosure provide a method of promoting bone grafting in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that promotes peroxidase functionality, thereby modulating bone grafting in the subject.

[00144] In certain embodiments, the agent inhibits peroxidase functionality. Examples of agents that inhibit peroxidase functionality are as described herein. In certain embodiments, the agent comprises an inhibitor of peroxidase activity and/or peroxidase associated signalling.

[00145] In certain embodiments, the modulating of osteogenesis comprises inhibiting osteogenesis in the subject.

[00146] In certain embodiments, the agent inhibits peroxidase functionality and the subject is suffering from, or susceptible to, a condition associated with inflammatory cells acting to promote osteogenesis. In certain embodiments, the subject is suffering from, or susceptible to, ankylosing spondylitis or heterotopic ossification. Such conditions may benefit from the therapeutic inhibition of osteogenesis.

[00147] Certain embodiments of the present disclosure provide a method of preventing and/or treating a disease, condition or state in a subject associated with inflammatory cells acting to promote osteogenesis, ankylosing spondylitis or heterotopic ossification, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits peroxidase functionality, thereby preventing and/or treating the disease, condition or state.

[00148] In certain embodiment, the methods as described herein comprise administering to the subject an effective amount of an agent that modulates peroxidase functionality.

[00149] In certain embodiments, the agent as described herein is administered to the subject to produce a concentration in the subject of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1 uM or greater, 5 uM or greater, 10 uM or greater, 100 uM or greater, 500 uM or greater, 1 mM or greater, or 10 mM or greater.

[00150] In certain embodiments, the agent as described herein is administered to the subject in an amount ranging from one of the following selected ranges: 1 μg kg to 100 mg/kg; 1 μg/kg to 10 mg/kg; 1 μg/kg to 1 mg/kg; 1 μg/kg to 100 μg/kg; 1 μg/kg to 10μg/kg; 10 μg/kg to 100 mg/kg; 10 μg/kg to 10 mg/kg; 10 μg/kg to 1 mg/kg; 10 μg/kg to 100 μg/kg; 100 μg/kg to 100 mg/kg; 100 μg/kg to 10 mg/kg; 100 μg/kg to 1 mg/kg; 1 mg/kg to 10 mg/kg; and 10 mg/kg to 100 mg/kg body weight. The dose and frequency of administration may be determined by one of skill in the art. [00151] The agent as described herein may be administered to the subject in a suitable form. In this regard, the terms "administering" or "providing" include administering the agent, or administering a prodrug of the agent, or a derivative of the agent that will form a therapeutically effective amount of the agent within the body of the subject. The terms include for example routes of administration that are systemic (e.g., via injection such as intravenous injection, orally in a tablet, pill, capsule, or other dosage form useful for systemic administration of pharmaceuticals), and topical (e.g., creams, solutions, and the like, including solutions such as mouthwashes, for topical oral administration). Other forms of administration include delivery by way of a scaffold, such as a biomaterial scaffold including a scaffoled produced from collagen, hydroxyapatite, β-tricalcium phosphate or a combination thereof).

[00152] The agent may be administered alone or may be delivered in a mixture with other therapeutic substances and/or other substances that enhance, stabilise or maintain the activity of the agent. In certain embodiments, an administration vehicle (e.g., pill, tablet, implant, injectable solution, etc.) would contain the agent and/or additional substance(s).

[00153] The methods may also include combination therapy. In this regard, the subject is treated or given another drug or treatment modality in conjunction with the agent as described herein. This combination therapy can be sequential therapy where the subject is treated first with one and then the other, or the two or more treatment modalities are given simultaneously.

[00154] "Co-administering" or "co-administration" refers to the administration of two or more therapeutic substances together at one time. The two or more therapeutic substances can be co-formulated into a single dosage form or "combined dosage unit", or formulated separately and subsequently combined into a combined dosage unit, typically for intravenous administration or oral administration.

[00155] In certain embodiments, the agent is administered as an immediate release formulation. The term "immediate release formulation" is a formulation which is designed to quickly release a therapeutic substance in the body over a shortened period of time. In certain embodiments, the agent is administered as a sustained release formulation. The term "sustained release formulation" is a formulation which is designed to slowly release a therapeutic substance in the body over an extended period of time.

[00156] When administered to a subject, the effective dosage may vary depending upon the particular agent utilized, the mode of administration, the condition, and severity thereof, as well as the various physical factors related to the subject being treated. As discussed herein, suitable daily doses range from 1 μg/kg to 100 mg/kg, such as 200μg/kg/day. The daily dosages are expected to vary with route of administration, and the nature of the agent administered. In certain embodiments the methods comprise administering to the subject escalating doses of agent and/or repeated doses.

[00157] In certain embodiments, the agent is administered orally. In certain embodiments, the agent is administered via injection, such as intravenous injection. In certain embodiments, the agent is administered parenterally. In certain embodiments, the agent is administered by direct introduction to the lungs, such as by aerosol administration, by nebulized administration, and by being instilled into the lung. In certain embodiments, the agent is administered by implant. In certain embodiments, the certain embodiments, the agent is administered by subcutaneous injection, intraarticularly, rectally, intranasally, intraocularly, vaginally, or transdermally. In certain embodiments, the agent is administered by a biological or non-biological implant. In certain embodiments, the agent is administered incorporated in a matrix.

[00158] "Intravenous administration" is the administration of substances directly into a vein. In certain embodiments, the agent may also be administered intravenously. Compositions containing the agent as described herein suitable for intravenous administration may be formulated by a skilled person.

[00159] "Oral administration" is a route of administration where a substance is taken through the mouth, and includes buccal, sublabial and sublingual administration, as well as enteral administration and that through the respiratory tract, unless made through e.g. tubing so the medication is not in direct contact with any of the oral mucosa. Typical form for the oral administration of therapeutic substances includes the use of tablets or capsules. [00160] In certain embodiments, it may be desirable to administer the agent directly to the airways in the form of an aerosol. Formulations for the administration of aerosol forms are known.

[00161] In certain embodiments, the agent may also be administered parenterally (such as directly into the joint space) or intraperitoneally. For example, solutions or suspensions of these compounds in a non-ionised form or as a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxy-propylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations typically contain a preservative to prevent the growth of microorganisms.

[00162] In certain embodiments, the agent may also be administered by injection. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[00163] In certain embodiments, the agent may also be administered transdermally. Transdermal administrations are understood to include all administrations across the surface of the body and the inner linings of bodily passages including epithelial and mucosal tissues. Such administrations may be carried out using the inhibitor as described herein, or pharmaceutically acceptable salts thereof, in lotions, creams, foams, patches, suspensions, solutions, and suppositories (rectal and vaginal).

[00164] Transdermal administration may also be accomplished through the use of a transdermal patch containing the active compound and a carrier that is inert to the active compound, is non toxic to the skin, and allows delivery of the agent for systemic absorption into the blood stream via the skin. The carrier may take any number of forms such as creams and ointments, pastes, gels, and occlusive devices. The creams and ointments may be viscous liquid or semisolid emulsions of either the oil-in-water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may also be suitable. A variety of occlusive devices may be used to release the active ingredient into the blood stream such as a semi-permeable membrane covering a reservoir containing the active ingredient with or without a carrier, or a matrix containing the active ingredient.

[00165] In certain embodiments, the agent may also be administered by way of a suppository. Suppository formulations may be made from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository's melting point, and glycerin. Water soluble suppository bases, such as polyethylene glycols of various molecular weights, may also be used.

[00166] In certain embodiments, the agent may be administered or delivered by way of solid or semi-solid substrate, for example being incorporated into a matrix, a scaffold or a support, such as a biodegradable matrix or support. Methods for delivering agent vis scaffolds are known in the art. For example, a biomaterial scaffold including a scaffold produced from collagen, hydroxyapatite, β-tricalcium phosphate or a combination thereof may be used to deliver the agent. Methods for incorporating agents into substrates are known in the art.

[00167] In certain embodiments, the agent may be administered or delivered by was of an implantable composition. Methods for preparing implantable compositions are known in the art.

[00168] Additional numerous various excipients, dosage forms, dispersing agents and the like that are suitable for use in connection with the administration of the agent and/or the formulation into compositions, medicaments, or pharmaceutical compositions.

[00169] Formulations are known and described in, for example, Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, which is incorporated herein by reference in its entirety.

[00170] Certain embodiments of the present disclosure provide a composition for modulating osteogenesis and/or angiogenesis, the composition comprising an effective amount of an agent that modulates peroxidase functionality.

[00171] In certain embodiments, the composition is used to modulate osteogenesis and/or angiogenesis in vitro. In certain embodiments, the composition is used to modulate osteogenesis and/or angiogenesis ex vivo. In certain embodiments, the composition is used to modulate osteogenesis and/or angiogenesis in vivo. In certain embodiments, the composition is used to modulate osteogenesis and/or angiogenesis is in a biological system.

[00172] In certain embodiments, the composition comprises the agent alone or comprises one or more other therapeutic substances (such as an anti-cancer agent or a chemotherapeutic agent) and/or other substances that enhance, stabilise or maintain the activity of the agent.

[00173] In certain embodiments, the composition is an intravenous composition, an oral composition, an intratracheal composition, a composition for nebulized administration, a composition for aerosolized administration, a composition for instillation into the lung, a composition for parenteral administration, an implant, a composition for subcutaneous injection, a composition for intraarticular administration, a rectal composition, an intranasal composition, an intraocular composition, a vaginal composition, a transdermal composition, or a composition incorporated into a solid or semi solid substrate, such as a scaffold.

[00174] In certain embodiments, the agent is formulated in the composition so as to be an immediate release composition or formulation. The term "immediate release formulation" is a formulation which is designed to quickly release a therapeutic substance in the body over a shortened period of time. In certain embodiments, the agent is formulated in the composition so as to be a sustained release composition or formulation. The term "sustained release formulation" is a formulation which is designed to slowly release a therapeutic substance in the body over an extended period of time.

[00175] In certain embodiments, the agents as described herein may be used in a pharmaceutical composition. In certain embodiments, the composition is a pharmaceutical composition.

[00176] In certain embodiments, the agent may be used in a pharmaceutical composition for use in the methods as described herein. In certain embodiments, the agent may be used in a formulation or medicament, or used in the preparation of a formulation or medicament.

[00177] Certain embodiments of the present disclosure provide a pharmaceutical composition comprising an agent that modulates peroxidase functionality as described herein.

[00178] Certain embodiments of the present disclosure provide a pharmaceutical composition for modulating osteogenesis and/or angiogenesis, the composition comprising an effective amount of an agent that modulates peroxidase functionality and/or optionally one or more pharmaceutically acceptable additives, such as a carrier, a solvent, or an excipient.

[00179] For example, a pharmaceutical composition for intravenous use comprising an inhibitor of peroxidase functionality may be as follows: 10-5000 mg of peroxidase inhibitor in isotonic saline, optionally including one or more pharmaceutically acceptable additives and/or excipients.

[00180] In another example, a pharmaceutical composition for intravenous use comprising an agent that promotes peroxidase functionality may be as follows: 10-5000 mg of a recombinant human peroxidase in isotonic saline, optionally including one or more pharmaceutically acceptable additives and/or excipients.

[00181] In certain embodiments, the pharmaceutical composition or medicament further comprises one or more of a pharmaceutically acceptable carrier, excipient, vehicle and additive, as described herein.

[00182] In certain embodiments, the agent is present in a composition, formulation or medicament so as to produce a concentration of the agent in a subject of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1 uM or greater, 5 uM or greater, 10 uM or greater , 100 uM or greater, 500 uM or greater, 1 mM or greater, or 10 niM or greater. Other concentrations are contemplated.

[00183] In certain embodiments, the agent is present in a composition, formulation or medicament in an amount ranging from one of the following selected ranges: 1 μg kg to 100 mg/kg; 1 μg/kg to 10 mg/kg; 1 μg/kg to 1 mg/kg; 1 μg/kg to 100 μg/kg; 1 μg/kg to l(^g/kg; 10 μg/kg to 100 mg/kg; 10 μg/kg to 10 mg/kg; 10 μg/kg to 1 mg/kg; 10 μg/kg to 100 μg/kg; 100 μg/kg to 100 mg/kg; 100 μg/kg to 10 mg/kg; 100 μg/kg to 1 mg/kg; 1 mg/kg to 10 mg/kg; and 10 mg/kg to 100 mg/kg body weight. Other amounts are contemplated.

[00184] In certain embodiments, the agent is provided in a pharmaceutically acceptable carrier suitable for administering the composition to a subject. The carriers may be chosen based on the route of administration as described herein, the location of the target issue, the agent being delivered, the time course of delivery of the drug, etc. The term "pharmaceutically acceptable carrier" refers to a substantially inert solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. An example of a pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known in the art. Some examples of materials which can serve as pharmaceutically acceptable carriers include, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as TWEEN 80; buffering agents such as magnesium hydroxide and aluminium hydroxide; alginic acid; pyrogen-free water; isotonic saline; in a matrix, such as collagen, Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colouring agents, releasing agents, coating agents, sweetening, flavouring and perfuming agents, preservatives and antioxidants can also be present.

[00185] In certain embodiments, the agent may be administered or present in a pharmaceutical composition as a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" refers to acid addition salts or metal complexes which are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes include zinc, iron, and the like.

[00186] In certain embodiments, the composition (or formulation or medicament) comprises other therapeutic substances and/or substances that enhance, stabilise or maintain the activity of the active. In certain embodiments, the composition comprises an anti-cancer agent and/or a chemotherapeutic agent.

[00187] In certain embodiments, the composition is an oral composition. Oral formulations containing the agent are as described herein may comprise any conventionally used oral forms, including tablets, capsules, buccal forms, troches, lozenges and oral liquids, suspensions or solutions. Capsules may contain mixtures of the active compound(s) with inert fillers and/or diluents such as the pharmaceutically acceptable starches (e.g. corn, potato or tapioca starch), sugars, artificial sweetening agents, powdered celluloses, such as crystalline and microcrystalline celluloses, flours, gelatins, gums, etc. Useful tablet formulations may be made by conventional compression, wet granulation or dry granulation methods and utilize pharmaceutically acceptable diluents, binding agents, lubricants, disintegrants, surface modifying agents (including surfactants), suspending or stabilizing agents, including magnesium stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, xanthan gum, sodium citrate, complex silicates, calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, talc, dry starches and powdered sugar. Surface modifying agents include nonionic and anionic surface modifying agents. Representative examples of surface modifying agents include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidol silicon dioxide, phosphates, sodium dodecylsulfate, magnesium aluminium silicate, and triethanolamine. Oral formulations may utilize standard delay or time-release formulations to alter the absorption of the peptides. The oral formulation may also consist of administering the active ingredient in water or a fruit juice, containing appropriate solubilizers or emulsifiers as needed.

[00188] In certain embodiments, the composition comprises a composition for administration to the airways. In certain embodiments, it may be desirable to administer the agent directly to the airways in the form of an aerosol. Formulations for the administration of aerosol forms are known.

[00189] In certain embodiments, the composition comprises a composition for administration for parenteral or intraperitoneal composition. For example, solutions or suspensions of the agent in a non-ionised form or as a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxy- propylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations typically contain a preservative to prevent the growth of microorganisms.

[00190] In certain embodiments, the composition comprises an injectable composition. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[00191] In certain embodiments, the composition comprises an intraveneous composition Compositions containing the agent as described herein suitable for intravenous administration may be formulated by a skilled person.

[00192] In certain embodiments, the composition comprises a transdermal composition Transdermal administrations are understood to include all administrations across the surface of the body and the inner linings of bodily passages including epithelial and mucosal tissues. Such administrations may be carried out using the inhibitor as described herein, or pharmaceutically acceptable salts thereof, in lotions, creams, foams, patches, suspensions, solutions, and suppositories (rectal and vaginal).

[00193] Transdermal administration may also be accomplished through the use of a transdermal patch containing the active compound and a carrier that is inert to the active compound, is non toxic to the skin, and allows delivery of the agent for systemic absorption into the blood stream via the skin. The carrier may take any number of forms such as creams and ointments, pastes, gels, and occlusive devices. The creams and ointments may be viscous liquid or semisolid emulsions of either the oil-in-water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may also be suitable. A variety of occlusive devices may be used to release the active ingredient into the blood stream such as a semi-permeable membrane covering a reservoir containing the active ingredient with or without a carrier, or a matrix containing the active ingredient.

[00194] In certain embodiments, the composition comprises a suppository. Suppository formulations may be made from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository's melting point, and glycerin. Water soluble suppository bases, such as polyethylene glycols of various molecular weights, may also be used.

[00195] Additional numerous various excipients, dosage forms, dispersing agents and the like that are suitable for use in connection with the administration of the agent and/or the formulation into compositions, medicaments or pharmaceutical compositions.

[00196] Formulations are known and described in, for example, Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, supra.

[00197] Certain embodiments of the present disclosure provide an osteogenic composition.

[00198] Certain embodiments of the present disclosure provide an osteogenic composition, the composition comprising an effective amount of an agent that promotes peroxidase functionality.

[00199] Certain embodiments of the present disclosure provide an osteogenic pharmaceutical composition, the pharmaceutical composition comprising a therapeutically effective amount of an agent that promotes peroxidase functionality.

[00200] Certain embodiments of the present disclosure provide a method of preventing and/or treating one or more of a bone related disorder, a bone fracture, dysfunctional and/or reduced bone mineralization, a bone spur, spinal stiffening, osteoporosis, a cancer-induced bone defect, and a deficiency in bone grafting or union in a subject, the method comprising administering to the subject a therapeutically effective amount of a osteogenic composition as described herein.

[00201] Certain embodiments of the present disclosure provide an anti-osteogenic composition.

[00202] Certain embodiments of the present disclosure provide an anti-osteogenic composition, the composition comprising an effective amount of an agent that inhibits peroxidase functionality.

[00203] Certain embodiments of the present disclosure provide an anti-osteogenic pharmaceutical composition, the pharmaceutical composition comprising a therapeutically effective amount of an agent that inhibits peroxidase functionality.

[00204] Certain embodiments of the present disclosure provide a method of preventing and/or treating a condition associated with inflammatory cells acting to promote osteogenesis, ankylosing spondylitis or heterotopic ossification in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti- osteogenic composition as described herein.

[00205] Certain embodiments of the present disclosure provide a pro-angiogenic composition.

[00206] Certain embodiments of the present disclosure provide a pro-angiogenic composition, the composition comprising an effective amount of an agent that promotes peroxidase functionality.

[00207] Certain embodiments of the present disclosure provide a pro-angiogenic pharmaceutical composition, the pharmaceutical composition comprising a therapeutically effective amount of an agent that promotes peroxidase functionality.

[00208] Certain embodiments of the present disclosure provide a method of preventing and/or treating one or more of Alzheimer's disease, amyotrophic lateral sclerosis, diabetes, atherosclerosis, hypertension, Crohn's disease, lupus, preeclampsia, nephropathy, a chronic wound, ischaemia, coronary artery disease, and a diabetic ulcer in a subject, the method comprising administering to the subject a therapeutically effective amount of a pro-angiogenic composition as described herein.

[00209] Certain embodiments of the present disclosure provide an anti-angiogenic composition.

[00210] Certain embodiments of the present disclosure provide an anti-angiogenic composition, the composition comprising an effective amount of an agent that inhibits peroxidase functionality.

[00211] Certain embodiments of the present disclosure provide an anti-angiogenic pharmaceutical composition, the composition comprising a therapeutically effective amount of an agent that inhibits peroxidase functionality.

[00212] Certain embodiments of the present disclosure provide a method of preventing and/or treating one or more of cancer, growth and/or metastasis of a cancer, a disease condition or state associated with undesired and/or dysfunctional angiogenesis, and a disorder associated insufficient angiogenesis in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-angiogenic composition as described herein.

[00213] Certain embodiments of the present disclosure provide an anti-cancer composition as described herein. [00214] Certain embodiments of the present disclosure provide an anti-cancer composition, the composition comprising an effective amount of an agent that inhibits peroxidase functionality.

[00215] Certain embodiments of the present disclosure provide an anti-cancer pharmaceutical composition, the composition comprising a therapeutically effective amount of an agent that inhibits peroxidase functionality.

[00216] In certain embodiments, the anti-cancer composition further comprises an anticancer agent and/or a chemo therapeutic agent as described herein.

[00217] Certain embodiments of the present disclosure provide an anti-metastatic composition.

[00218] Certain embodiments of the present disclosure provide an anti-metastatic composition, the comprising an effective amount of an agent that inhibits peroxidase functionality.

[00219] Certain embodiments of the present disclosure provide an anti-metastatic pharmaceutical composition, the comprising a therapeutically effective amount of an agent that inhibits peroxidase functionality.

[00220] In certain embodiments, the anti-metastatic composition further comprises an anti-cancer agent and/or a chemotherapeutic agent as described herein.

[00221] Certain embodiments of the present disclosure provide a method of preventing and/or treating a cancer and/or metastasis of a cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an anticancer as described herein.

[00222] Certain embodiments of the present disclosure provide a method of preventing and/or treating a metastasis of a cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-metastatic as described herein. [00223] Examples of anti-cancer agents include alkylating agents (such as cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, and ifosfamide), anti-metabolites (such as azathioprine and mercaptopurine), plant alkaloids and terpenoids (such as vincristine, vinblastine, vinorelbine and vindesine), cell cycle inhibitors (such as podophyllotoxin), taxanes (such as paclitaxel), topoisomerase inhibitors (such as camptothecins, irinotecan and topotecan, amsacrine, etoposide, etoposide phosphate, and teniposide), cytotoxic antibiotics (such as actinomycin, doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin and mitomycin).

[00224] For example, an intravenous pharmaceutical composition for treatment of breast cancer comprising an anti-cancer agent and an agent that modulates peroxidase functionality may be as follows: 40-50 mg/kg body weight an anti-cancer agent (eg cyclophosphamide) and 100-5000 mg (typically 5-50 mg) of a peroxidase inhibitor in isotonic saline.

[00225] Certain embodiments of the present disclosure provide a combination product comprising an agent that modulates peroxidase functionality as described herein with another agent as described herein.

[00226] Certain embodiments of the present disclosure provide a combination product, the combination product comprising the following components:

an agent that modulates peroxidase functionality; and

an anti-cancer agent;

wherein the components are provided in a form for separate or combined administration to a subject in need thereof.

[00227] Agents that modulate peroxidase functionality, and anti-cancer agents are as described herein. In certain embodiments, the agent inhibits peroxidase functionality.

[00228] Certain embodiments of the present disclosure provide a combination product, the combination product comprising the following components:

an agent that inhibits peroxidase functionality; and

an anti-cancer agent; wherein the components are provided in a form for separate or combined administration to a subject in need thereof.

[00229] Certain embodiments of the present disclosure provide a method of modulating osteogenesis and/or angiogenesis in a biological system.

[00230] Certain embodiments of the present disclosure provide a method of modulating osteogenesis and/or angiogenesis in a biological system, the method comprising exposing the biological system to an effective amount of an agent that modulates peroxidase functionality, thereby modulating osteogenesis and/or angiogenesis in the biological system.

[00231] The term "biological system" as used herein refers to a cellular system and includes fore example one or more cells in vivo, ex vivo or in vitro; a tissue or organ in vivo or ex vivo, or an entire subject.

[00232] In certain embodiments, the biological system comprises one or more cells in vitro, one or more cells in culture, one or more cells ex vivo, a tissue or organ, or a human or animal subject. Other forms of biological systems are contemplated.

[00233] The term "exposing", and related terms such as "expose" and "exposure", as used herein refers to directly and/or indirectly contacting and/or treating a biological system with an agent that modulates peroxidase functionality.

[00234] Examples of agents are as described herein. In certain embodiments, the agent comprises a drug, a small molecule, a protein, a polypeptide, a lipid, a carbohydrate, a nucleic acid, a DNA, a RNA, an oligonucleotide, a ribozyme, a biologic, an aptamer, a peptide, a cofactor, a ligand, a ligand mimetic, a receptor, an enzyme, a kinase, a phosphatase, a signalling molecule, a cytokine, a growth factor, a metal ion, a chelate, an antisense nucleic acid, a siRNA, a microRNA, an antibody, and an amino acid.

[00235] In certain embodiments, the agent modulates one or more of peroxidase activity, peroxidase function, peroxidase expression, peroxidase associated signalling and peroxiredoxin functionality. [00236] For cells in vivo, an agent may be administered to a subject to expose cells to an agent, or another agent may be administered to a subject that results in the production of the agent in the subject, thereby exposing cells in vivo to the agent. In another example, one or more cells may be removed from a subject and contacted directly or indirectly with the agent, and cells then introduced back into the same or another subject to effect exposure to the agent. Examples of administration routes are as described herein.

[00237] For example for cells in vitro or ex vivo, the cells may be exposed for example to the agent in a tissue culture medium, or other suitable medium such as phosphate buffered saline. For cells in a tissue or organ, the cells may be exposed to the agent by way of perfusing the organ with the agent.

[00238] In certain embodiments, the biological system is exposed to a concentration of an agent of 0.1 nM or greater, 0.5 nM or greater, 1 nM or greater, 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 1 uM or greater, 5 uM or greater, 10 uM or greater , 100 uM or greater, 500 uM or greater, 1 mM or greater, or 10 mM or greater. Other concentrations are contemplated.

[00239] In certain embodiments, the biological system is exposed to an agent in an amount ranging from one of the following selected ranges: 1 μg/kg to 100 mg/kg; 1 μg/kg to 10 mg/kg; 1 μg/kg to 1 mg/kg; 1 μg/kg to 100 μg/kg; 1 μg/kg to ^g/kg; 10 μg/kg to 100 mg/kg; 10 μg/kg to 10 mg/kg; 10 μg/kg to 1 mg/kg; 10 μg/kg to 100 μg/kg; 100 μg/kg to 100 mg/kg; 100 μg/kg to 10 mg/kg; 100 μg/kg to 1 mg/kg; 1 mg/kg to 10 mg/kg; and 10 mg/kg to 100 mg/kg body weight. Other amounts are contemplated.

[00240] Certain embodiments of the present disclosure provide a method of modulating angiogenesis in a biological system, the method comprising exposing the biological system to an effective amount of an agent that modulates peroxidase functionality, thereby modulating angiogenesis in the biological system.

[00241] In certain embodiments, the agent modulates one or more of endothelial cell proliferation, endothelial cell migration and endothelial cell tube formation. [00242] In certain embodiments, the modulating angiogenesis in the biological system comprises inhibiting angiogenesis. Examples of angiogenesis are as described herein.

[00243] In certain embodiments, the agent inhibits peroxidase functionality. Examples of agents that inhibit peroxidase functionality are as described herein.

[00244] In certain embodiments, the method comprises inhibiting angiogenesis in the biological system and the agent inhibits peroxidase functionality.

[00245] Certain embodiments of the present disclosure provide a method of inhibiting angiogenesis in a biological system, the method comprising exposing the biological system to an effective amount of an agent that inhibits peroxidase functionality, thereby inhibiting angiogenesis in the biological system.

[00246] In certain embodiments, the biological system comprises a subject. In certain embodiments, the biological system comprises a subject suffering from, or susceptible to, a disease, condition or state as described herein.

[00247] In certain embodiments, the subject is suffering from, or susceptible to, multiple sclerosis, vascular malformations, obesity, psoriasis, warts, allergic dermatitis, primary pulmonary hypertension, asthma, cystic fibrosis, inflammatory bowel disease, peri-dontal disease, liver cirrhosis, endometriosis, arthritis and osteomyelitis.

[00248] In certain embodiments, the biological system comprises a subject suffering from, or susceptible to, a cancer and/or metastasis of a cancer. In certain embodiments, the cancer comprises a cancer selected from a breast cancer, an ovarian cancer, endometrial cancer, or a prostate cancer. Examples of cancers are as described herein.

[00249] In certain embodiments, the modulating angiogenesis in the biological system comprises promoting angiogenesis.

[00250] In certain embodiments, the agent promotes peroxidase functionality. Examples of agents that promote peroxidase functionality are as described herein. In certain embodiments, the agent comprises a peroxidase. In certain embodiments, the peroxidase comprises an exogenous peroxidase. [00251] In certain embodiments, the method comprises promoting angiogenesis in the biological system and the agent promotes peroxidase functionality.

[00252] Certain embodiments of the present disclosure provide a method of promoting angiogenesis in a biological system, the method comprising exposing the biological system to an effective amount of an agent that promotes peroxidase functionality, thereby promoting angiogenesis in the biological system.

[00253] In certain embodiments, the subject is suffering from, or susceptible to, Alzheimer's disease, amyotrophic lateral sclerosis, diabetes, atherosclerosis, hypertension, Crohn's disease, lupus, preeclampsia, nephropathy, a chronic wound, and coronary artery disease, and a diabetic ulcer. In certain embodiments, endothelial cell migration in the biological system is modulated. In certain embodiments, endothelial cell proliferation in the biological system is modulated. In certain embodiments, endothelial tube formation in the biological system is modulated.

[00254] Certain embodiments of the present disclosure provide a method of modulating endothelial cell migration in a biological system.

[00255] Certain embodiments of the present disclosure provide a method of modulating endothelial cell migration in a biological system, the method comprising administering to the biological system an effective amount of an agent that modulates peroxidase functionality, thereby modulating endothelial cell migration in the biological system.

[00256] In certain embodiments, the method comprises inhibiting endothelial cell migration and the agent inhibits peroxidase functionality.

[00257] In certain embodiments, the method comprises promoting endothelial cell migration and the agent promotes peroxidase functionality.

[00258] Certain embodiments of the present disclosure provide a method of modulating endothelial tube formation in a biological system.

[00259] Certain embodiments of the present disclosure provide a method of modulating endothelial tube formation in a biological system, the method comprising administering to the biological system an effective amount of an agent that modulates peroxidase functionality, thereby modulating endothelial tube formation in the biological system.

[00260] In certain embodiments, the method comprises inhibiting endothelial tube formation and the agent inhibits peroxidase functionality.

[00261] In certain embodiments, the method comprises promoting endothelial tube formation and the agent promotes peroxidase functionality.

[00262] Certain embodiments of the present disclosure provide a method of modulating endothelial cell proliferation in a biological system.

[00263] Certain embodiments of the present disclosure provide a method of modulating endothelial cell proliferation in a biological system, the method comprising administering to the biological system an effective amount of an agent that modulates peroxidase functionality, thereby modulating endothelial cell proliferation in the biological system.

[00264] In certain embodiments, the method comprises inhibiting endothelial cell proliferation and the agent inhibits peroxidase functionality.

[00265] In certain embodiments, the method comprises promoting endothelial cell proliferation and the agent promotes peroxidase functionality.

[00266] Certain embodiments of the present disclosure provide a method of modulating osteogenesis in a biological system.

[00267] Certain embodiments of the present disclosure provide a method of modulating osteogenesis in a biological system, the method comprising exposing the biological system to an effective amount of an agent that modulates peroxidase functionality, thereby modulating osteogenesis in the biological system.

[00268] In certain embodiments, the modulating osteogenesis in the biological system comprises inhibiting osteogenesis. Examples of osteogenesis are as described herein. [00269] In certain embodiments, the agent inhibits peroxidase functionality. Examples of agents that inhibit peroxidase functionality are as described herein.

[00270] In certain embodiments, the method comprises inhibiting osteogenesis in the biological system and the agent inhibits peroxidase functionality.

[00271] Certain embodiments of the present disclosure provide a method of inhibiting osteogenesis in a biological system, the method comprising exposing the biological system to an effective amount of an agent that inhibits peroxidase functionality, thereby inhibiting osteogenesis in the biological system.

[00272] In certain embodiments, the biological system comprises a subject. In certain embodiments, the biological system comprises a subject suffering from, or susceptible to, a disease, condition or state as described herein.

[00273] In certain embodiments, the agent inhibits peroxidase functionality and the subject is suffering from, or susceptible to, a condition associated with inflammatory cells acting to promote osteogenesis. In certain embodiments, the subject is suffering from, or susceptible to, ankylosing spondylitis or heterotopic ossification.

[00274] In certain embodiments, the modulating osteogenesis in the biological system comprises promoting osteogenesis.

[00275] In certain embodiments, the agent promotes peroxidase functionality. Examples of agents that promote peroxidase functionality are as described herein. In certain embodiments, the agent comprises a peroxidase. In certain embodiments, the peroxidase comprises an exogenous peroxidase.

[00276] In certain embodiments, the method comprises promoting osteogenesis in the biological system and the agent promotes peroxidase functionality.

[00277] Certain embodiments of the present disclosure provide a method of promoting osteogenesis in a biological system, the method comprising exposing the biological system to an effective amount of an agent that promotes peroxidase functionality, thereby promoting osteogenesis in the biological system. [00278] In certain embodiments, the agent promotes osteogenesis and the subject is suffering from, or susceptible to, one or more of a bone related disorder, a bone fracture, dysfunctional and/or reduce bone mineralization, a bone spur, a spinal stiffening, osteoporosis, a cancer induced bone defect, and a deficiency in bone grafting or union.

[00279] In certain embodiments, the method is used to modulate collagen production by osteoblasts, to modulate bone mineralization, to modulate bone remodelling, to prevent and/or treat a fracture, and to promote bone grafting. Other uses are contemplated.

[00280] Certain embodiments of the present disclosure provide a method of modulating collagen production by osteoblasts.

[00281] Certain embodiments of the present disclosure provide a method of modulating collagen production by osteoblasts in a biological system, the method comprising exposing the biological system to an effective amount of an agent that modulates peroxidase functionality, thereby modulating collagen production.

[00282] Certain embodiments of the present disclosure provide a method of modulating bone mineralization.

[00283] Certain embodiments of the present disclosure provide a method of modulating bone mineralization in a biological system, the method comprising exposing the biological system to an effective amount of an agent that modulates peroxidase functionality, thereby modulating bone mineralization.

[00284] Certain embodiments of the present disclosure provide a method of modulating remodelling of bone.

[00285] Certain embodiments of the present disclosure provide a method of modulating remodelling of bone in a biological system, the method comprising exposing the biological system to an effective amount of an agent that modulates peroxidase functionality, thereby modulating remodelling of bone.

[00286] Certain embodiments of the present disclosure provide a method of preventing and/or treating a fracture. [00287] Certain embodiments of the present disclosure provide a method of preventing and/or treating a fracture in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that modulates peroxidase functionality, thereby preventing and/or treating the fracture.

[00288] In certain embodiments, the agent promotes peroxidase functionality.

[00289] Certain embodiments of the present disclosure provide a method of preventing and/or treating a fracture in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that promotes peroxidase functionality, thereby preventing and/or treating the fracture.

[00290] Certain embodiments of the present disclosure provide a method of assisting a spinal fusion, healing of a fracture, and treating delayed unions and/or non-unions, the method comprising administering to the subject a therapeutically effective amount of an agent that modulates peroxidase functionality.

[00291] In certain embodiments, the agent promotes peroxidase functionality.

[00292] Certain embodiments of the present disclosure provide a method of assisting a spinal fusion, healing of a fracture, and treating delayed unions and/or non-unions, the method comprising administering to the subject a therapeutically effective amount of an agent that promotes peroxidase functionality.

[00293] Certain embodiments of the present disclosure provide a pharmaceutical composition comprising a modulator of peroxidase functionality for use any of the aforementioned methods.

[00294] Certain embodiments of the present disclosure provide a method of identifying a subject with cancer suitable for treatment with an agent that modulates peroxidase functionality.

[00295] Examples of cancers are as described herein. In certain embodiments, the cancer is a cancer associated with inflammatory cell infiltration into the cancer. [00296] Certain embodiments of the present disclosure provide a method of identifying a subject with cancer suitable for treatment with an agent that modulates peroxidase functionality, the method comprising identifying a subject with a cancer associated with inflammatory cell infiltration into the cancer.

[00297] In certain embodiments, the inflammatory cells comprise neutrophils, macrophages and/or eosinophils. Methods for identifying cancers with inflammatory cells infiltration are known in the art.

[00298] In certain embodiments, the method further comprises treating the subject so identified with an agent as described herein.

[00299] Certain embodiments of the present disclosure provide a method of treating a subject with a cancer, the method comprising:

identifying a subject suffering from a cancer with inflammatory cell infiltration; and

administering to the subject an agent that inhibits peroxidase functionality, thereby treating the cancer.

[00300] Certain embodiments of the present disclosure provide a method of identifying an agent for modulating osteogenesis and/or angiogenesis.

[00301] Certain embodiments of the present disclosure provide a method of screening for an agent for modulating osteogenesis and/or angiogenesis.

[00302] Certain embodiments of the present disclosure provide a method of identifying an agent for modulating osteogenesis and/or angiogenesis, the method comprising detemiining the ability of a candidate agent that modulates peroxidase functionality to modulate osteogenesis and/or angiogenesis and identifying the candidate agent as an agent for modulating osteogenesis and/or angiogenesis.

[00303] Certain embodiments of the present disclosure provide a method of identifying an agent for modulating osteogenesis and/or angiogenesis, the method comprising: determining the ability of a candidate agent to modulate peroxidase functionality; and

identifying the candidate agent as an agent for modulating osteogenesis and/or angiogenesis.

[00304] Methods for determining the ability of a candidate agent to modulate peroxidase functionality are known in the art. Methods for determining the ability of a candidate agent to modulate osteogenesis and/or angiogenesis are as described herein. Biological systems for use in determining the ability of a candidate agent to modulate osteogenesis and/or angiogenesis are described herein.

[00305] Certain embodiments of the present disclosure provide a method of identifying an agent for preventing and/or treating a disease, condition or state as described herein by determining the ability of a candidate agent that modulates peroxidase functionality to prevent and/or treat the disease, condition or state and identifying the candidate agent as an agent for preventing and/or treating the disease, condition or state.

[00306] Methods for determining the ability of a candidate agent to modulate peroxidase functionality are as described herein. Methods for determining the ability of a candidate agent to prevent and/or treat a disease, condition or state as described herein are known in the art. Suitable animal models are as described herein.

[00307] Certain embodiments of the present disclosure provide an agent identified using the methods as described herein.

[00308] Examples of test/candidate agents include a drug, a small molecule, a protein, a polypeptide, a lipid, a carbohydrate, a nucleic acid, an oligonucleotide, a ribozyme, a biologic, an aptamer, , a cofactor, a ligand, a ligand mimetic, a receptor, an enzyme, a kinase, a phosphatase, a cytokine, a growth factor, a metal ion, a chelate, an antisense nucleic acid, an antisense RNA, a microRNA, a siRNA, an antibody or antigen binding part thereof, an antagonist, an inhibitor, or a suppressor.

[00309] Standard techniques may be used for recombinant DNA technology, oligonucleotide synthesis, antibody production, peptide synthesis, tissue culture and transfection. Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), herein incorporated by reference.

[00310] The present disclosure is further described by the following examples. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.

EXAMPLE 1 - Methodologies

[00311] (i) Endothelial cell culture

[00312] Human Umbilical Vein Endothelial Cells (HuVECS) were seeded into 96 well plates at a density of 1.2xl0 ⁴ cells/well and cultured overnight in 20% fetal bovine serum/medium-200 supplemented with lx low serum growth supplements (LSGS- Invitrogen, containing: lOng/ml EGF, 3ng/ml FGF, lOmg/ml heparin, lmg/ml hydrocortisone), 1% non-essential amino acids, 1% L-glutamine, 1% sodium pyruvate, and 1% penicillin/streptomycin, at 37°C and 5%C0 ₂ . The cells were then starved overnight in 2% FCS-medium-200 without LSGS (EBM) and triplicate wells treated for 72h with various doses of proteins with peroxidase activity; Myeloperoxidase (MPO), Eosinophil Peroxidase (EPO), Soybean Peroxidase (SBP) in either 20%FCS-media-200 EBM or 2%FCS-media-200 EBM. After 72 hours the cells were assessed for viability/growth using an alamarBlue™ fluorescent dye assay. This assay measures the reduction of oxidized, blue nonfluorescent alamarBlue™ reagent to a pink fluorescent dye in the cell medium, such that the higher the amount of reduction, the greater the cell number and/or activity. Briefly, cells were bathed in a 10% alamarBlue™/phospate- buffered saline (PBS) solution for 30-60 minutes and fluorescence measured at wavelengths of 485nm excitation and 595nm emission using a FLUOstar Optima plate reader (BMG Labtech). Bars are the mean ± s.d. of triplicate determinations. [00313] (ii) Endothelial Cell migration/invasion

[00314] Cell migration was assayed using BD transwell chambers (8.0μΜ pore membrane). Cells were starved in 2%FCS media 200 EBM at no more than 70% confluency overnight. Cells (2xlO ^A 5cells) were suspended with ΙΟΟμΙ 2%FCS base medium (2%FCS Media-200 EBM) and planted into the upper chamber. The cells were then cultured at 37°C for 20 minutes to allow the cells to adhere. The lower chambers were then filled with 700 of proteins with peroxidase activity; Myeloperoxidase (MPO), Eosinophil Peroxidase (EPO), Soybean Peroxidase (SBP), at ^g/ml and together with and without inhibitors (4-ABAH 10μΜ and SOD lOU/ml) and cultured at 37°C. After 18 hours the cells in the upper chamber were washed with lxPBS and removed by scraping. The migrated cells to the underside of the porous membrane on the transwell were washed in lx PBS and fixed in 6: 1 ethanol/ acetic acid for 10 minutes. The transwell was then washed twice in lx PBS, then lx in distilled water and allowed to air dry. The transwell was then incubated in ^g/ml of DAPI fluorescent stain and migrated cells were counted in 5 random fields of view using a fluorescent microscope.

[00315] For invasion assay, the BD BioCoat Assay was used as per the kit instructions using the conditions as above.

[00316] (iii) In vitro tube formation

[00317] For tube formation assay, the BD Biocoat Angiogenesis Assay was used as per the kit instructions. Briefly, the BioCoat 96 well plate was thawed overnight at 4°C. Growth arrested HuVECS in 2%FCS medium 200-EBM were seeded into the BD BioCoat matrigel 96well plate at a density of 2x10 ^A 4 cells/well in ΙΟΟμΙ Cells were then stimulated with MPO, and EPO at 0.39 μg/ml, 3. ^g/ml, 12^g/ml, and Soybean Peroxidase (SBP), at 0.78μg/ml, 6.25μg/ml, 25μg/ml in triplicate wells. 2%FCS medium 200-EBM was used as a negative control and 2%LSGS medium 200 as a positive control. The plate was then incubated at 37°C for 6 hours where images were taken at 3 fields of view and the number of fully formed ring structures was quantified.

[00318] (iv) Matrigel Plug assay in vivo [00319] To examine the effect of peroxidases on angiogenesis in vivo, we used a Matrigel plug assay. 7 week old Balb/C mice were anaesthetised by Halothane gas. Each mouse was given a subcutaneous injection of growth factor reduced-phenol red free Matrigel (500μ1 injection) with a 27-gauge needle. Matrigel plus PBS served as the negative control, and Matrigel containing 5 μg of Peroxidases, MPO, EPO, MPO and EPO, SBP served as the test substances. After 1 1 days, the mice were humanely sacrificed and the matrigel plugs were carefully harvested. The plugs were photographed, weighed and then analysed by haemoglobin content or by histology. The Drabkins reagent method of determining haemoglobin content was employed. For histological analysis, we also embedded some of these plugs in paraffin for tissue sectioning and staining.

[00320] (iv) 4T-1 tumour growth and metastasis assay

[00321] Female Balb/C mice aged at 8 weeks (8 per group) were anaesthetised with halothane gas, and tumour cells lxlO cells in 50μ1 PBS with 1 :3 matrigel were injected into the 4 ^th mammary fat pad. Once the tumours had established (after 7 days), treatment with peroxidase enzymes (MPO, EPO, MPO/EPO, SBP at 5μg total in 20μ1 PBS were injected directly into the tumour weekly. Tumour growth was measured weekly using non-invasive bioluminescence imaging and tumour volume assessed by calliper measurements.

EXAMPLE 2 - Peroxidases promote HUVEC migration and proliferation.

[00322] The effect of peroxidases on endothelial cell migration and proliferation was determined. The data is shown in Figure 1. Panel A: Proliferation assay. HUVEC (3 x 10 ) cells were plated and stimulated with increasing concentrations of (i) EPO (0.05 - ^g/ml) and (ii) MPO (0.1 - 2μg/ml) on collagen coated wells. After 48 hrs endothelial proliferation was measured using alamarBlueTM dye assay. Panel B: Transwell migration assay. HUVEC (lxlO ⁵ ) were placed onto the membrane well of the transwell insert in the presence of treatment medium in the lower chamber. Cells treated with base culture media served as the vehicle control, and LSGS containing a mix of hEGF (lOng/ml) and bFGF (3ng/ml) served as the positive control. MPO and EPO (10μg/ml) were used as test mixed with and without peroxidase inhibitor (4-ABAH 10μΜ). Migrated cells stained with DAPI were counted from 5 fields of view in triplicate wells and results are represented as a fold change ± SEM over vehicle. Statistical significance was calculated by ANOVA (where indicated), and paired-Student's t-test (**p<0.001, *p<0.05). Data for endothelial proliferation are pooled from three separate experiments.

[00323] As can been, peroxidases stimulated endothelial cell proliferation in a dose dependent response, as compared to the vehicle control and the positive control VEGF. The peroxidases also stimulated endothelial cell migration as compared to the control, and the stimulation was dependent on peroxidase activity, as determined from the lack of stimulation in the present of 4-ABAH.

EXAMPLE 3 - Peroxidases Stimulate Endothelial cell migration and invasion

[00324] The effect of peroxidases on endothelial cell migration and invasion was investigated. Figure 2 shows induced HUVEC migration (A) and invasion (B) by peroxidases after 24 hrs. HUVEC migration is significantly greater in response to peroxidases vs vehicle control. This response can be attenuated using the specific irreversible peroxidase inhibitor 4-ABAH at 10μΜ and superoxide dismutase (SOD) lOU/ml.

[00325] These results clearly show that peroxidase enzymes have the capacity to promote migration and invasion of human endothelial cells in vitro. This is a precursor of angiogenesis.

EXAMPLE 4 - Peroxidases promote 4T-1 tumour cell migration

[00326] The effect of peroxidases on migration of tumour cells was investigates. The data is shown in Figure 3.

[00327] Transwell migration assay. 4T-1 cells (lxlO ⁵ ) were placed onto the membrane well of the transwell insert in the presence of treatment medium in the lower chamber. Cells treated with base culture media served as the vehicle control. MPO and EPO (10μg/ml) were used as test. Migrated cells stained with DAPI were counted from 5 fields of view in triplicate wells and results are represented as a fold change ± SEM over vehicle. Statistical significance was calculated by paired-Student's t-test (**p<0.001). [00328] These results clearly show that peroxidase enzymes have the capacity to promote migration of 4T-1 cells.

EXAMPLE 5 - Peroxidases Stimulate Endothelial tube formation on matrigel

[00329] Figure 4 shows that peroxidases induce endothelial cell tube formation on matrigel in vitro. (A) Images taken at 10X magnification of representative wells show the increase peroxidase induced HUVEC tube formation in comparison with vehicle control, (i) 5%FCS media- Vehicle Control, (ii) Soybean peroxidase (SBP) 25μg/ml, (iii) Myeloperoxidase (MPO) 12^g/ml, (iv) Eosinophil peroxidase (EPO) 12^g/ml, (v) Positive control with EGF+FGF growth factors. (B) HUVECs form significantly greater numbers of tubular rings on matrigel matrix in response to peroxidases vs vehicle control.

[00330] These results clearly show that peroxidase enzymes have the capacity to promote tube formation of human endothelial cells in vitro. This is a requirement for angiogenesis to occur.

EXAMPLE 6 - Peroxidases promote angiogenesis in vivo

[00331] Figure 5 shows that peroxidases promote angiogenesis in vivo. Figure 5(A) shows images taken of representative excised matrigel plugs stimulated with PBS as negative control and the peroxidase enzymes, MPO, EPO, MPO/EPO and SBP at 5μg of total protein. Figure 5(B) shows peroxidase induced angiogenesis quantified using the Drabkin's reagent measure of hemoglobin. Peroxidases induced in increase in hemoglobin to a significantly greater degree compared with vehicle control. The values represent the concentration of hemoglobin. (C) A representative histological assay shows an increase in CD31 positive cells (marker for endothelial cells) infiltrating the matrigel plug when compared to vehicle control.

[00332] These data confirm that peroxidases promote angiogenesis in vivo.

EXAMPLE 7 - Peroxidases promote tumouri gene sis and enhanced metastasis

[00333] Figure 6 shows that peroxidases induced an increase in tumour development and metastasis. (A) Representative images of non-invasive bioluminescent images after 23 days. (B) Peroxidases treatment of tumours lead to an increase in bioluminescence quantified as total flux (p/s) indicating an increase in tumour burden. (C) After 23 days, peroxidase treatment enhanced lung metastasis.

EXAMPLE 8 - Peroxidases promote tumourigenesis and enhanced metastasis

[00334] Figure 7 shows histological staining of tumours. (A) Representative images of H/E staining of tumours after 23 days. These results clearly show that peroxidases promote angiogenesis on the surface and within the tumour microenvironment providing more viable tumour regions within the necrotic core.

EXAMPLE 9 - Osteoblast methodologies

[00335] (i) Osteoblast cell culture

[00336] Osteoblasts were seeded into 96 well plates at a density of 1.2xl0 ⁴ cells/well and cultured for 5 days in 10% fetal bovine serum/Dulbecco's minimum essential media (10% FBS/DMEM) at 37°C and 5%C0 ₂ . The confluent cells were then starved overnight in serum-free DMEM and triplicate wells treated for 72h with various doses of proteins with peroxidase activity; Myeloperoxidase (MPO), Eosinophil Peroxidase (EPO), Soybean Peroxidase (SBP), and Horseradish Peroxidase (HRP) in serum-free DMEM. After 72 hours the culture media was collected for assessment of secreted, soluble collagen I protein and the cells assessed for viability/ growth using the alamarBlue™ fluorescent dye assay. This assay measures the reduction of oxidized, blue non-fluorescent alamarBlue™ reagent to a pink fluorescent dye in the cell medium, such that the higher the amount of reduction, the greater the cell number and/or activity. Briefly, cells were bathed in a 10% alamarBlue™/phospate-buffered saline (PBS) solution for 30-60 minutes and fluorescence measured at wavelengths of 485nm excitation and 595nm emission using a FLUOstar Optima plate reader (BMG Labtech). Cell viability was normalized so the average values of Unstim cells were set to 100% relative to each peroxidase dose. Bars are the mean ± s.d. of triplicate determinations.

[00337] (ii) Collagen I ELISA [00338] Soluble collagen I levels in osteoblast-conditioned media were measured by a direct coat ELISA method using a standard curve constructed from purified human collagen I extracted from placenta (BD Biosciences). Samples and standards (ΙΟΟμΙ/well) were added to a 96-well Maxisorp ELISA plate (Nunc) and left at 4°C overnight. The plate was then washed 3 times with PBS-tween 0.05% (PBS-T) and 2.5% bovine serum albumin (BSA)/PBS blocking solution added to each well and the plate incubated for 1 hour at RT. The plate was then washed 4 times with PBS-T and primary antibody (0.25μg/ml rabbit-anti-human-collagen I polyclonal; Rockland Immunochemicals) in 5% non-fat dairy milk added to each well for four hours at RT. After washing (4 x PBS-T), europium tagged anti-rabbit secondary antibody (0^g/ml 1% BSA/PBS; Wallac Oy) was added for 1 hour at RT. After a final wash (4 x PBS-T), Enhancement Solution (Wallac Oy) was added for at least 10 minutes and fluorescence measured at excitation 340nm and emission 615nm using a FLUOstar Optima plate reader (BMG Labtech). The collagen I concentration of each sample was determined from the standard curve ^g/ml) and normalized to control wells (DMEM only treated cells) with the mean+s.d. for triplicate wells calculated.

[00339] (iii) In vitro mineralization and Alizarin red staining

[00340] Primary human osteoblasts were seeded into 96 well plates at a density of 1.2xl0 ⁴ cells/well and cultured for 5 days in 10% fetal bovine serum/Dulbecco's minimum essential media (10% FBS/DMEM) at 37°C and 5%C0 ₂ . Triplicate wells were stimulated with various doses of proteins with peroxidase activity in osteogenic DMEM medium (DMEM supplemented with 5% FBS, ΙΟΟμΜ Ascorbic acid 2- phosphate, 10 ^"8 M dexamethasone and lOmM β -glycerophosphate) to assist bone crystal/nodule formation. Cells were maintained in culture for up to 21 days, with fresh medium ± peroxidase proteins changed every 5 days. To detect matrix mineralization, the Alizarin Red staining method was used. Cells were washed twice with PBS and then fixed with 10% phosphate-buffered formalin for 15min. The fixed cells were washed twice with distilled water and stained with 2% Alizarin Red S solution for 5 min. The excess dye was removed by repeated washing with distilled water, and mineralized matrix stained by Alizarin Red photographed using a Nikon Eclipse 50i microscope attached to a DS-L2 control unit (Digital Sight) and a DS-Fil digital camera (Nikon Corporation). Quantification of staining was achieved by eluting the Alizarin Red using 10% (w/v) cetylpiridinium chloride in lOmM phosphate buffer (pH 7.0) for 15min and reading the absorbance at 570nm using a FLUOstar Optima plate reader (BMG Labtech).

[00341 ] (iv) Quantification of mRNA levels by Real-time PCR

[00342] To evaluate the effect of peroxidases on mRNA expression by osteoblasts, total RNA was isolated using an RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RNA yield and purity were quantified by Nanodrop™ spectrophotometric measurement at 260nm (Nanodrop Technologies). Target gene mRNA expression was examined using real-time RT-PCR and normalised against glyceraldehydes-3-phosphate dehydrogenase (GAPDH). cDNA was synthesised by reverse transcription of ^g total RNA using random hexamer primers and Superscript III Reverse Transcriptase (Life Technologies). Real time RT-PCR was performed using TaqMan Array plates (Life Technologies) in the 96-well format where triplicate sets of gene-specific primers were aliquoted by the manufacturer. RT-PCR reactions were performed by combining TaqMan Fast Universal Master Mix (Life Technologies) with 50ng cDNA in a ΙΟμΙ final reaction volume. Thermal cycling conditions were as follows - Amplification occurred after initial denaturation at 95°C for lOmin followed by 40 cycles at 95°C for 15 sec, 60°C for 60 sec.

[00343] (v) INTEGRA Studies

[00344] INTEGRA® Dermal Regeneration Template (INTEGRA LifeSciences) was cut into 1cm x 1cm pieces using a sterile scalpel, the pieces transferred to a petri dish using sterile forceps and washed four times each with PBS followed by serum-free DMEM, to remove the alcohol storage medium and to equilibrate the porous scaffold for exposure to cells. After the last wash, the INTEGRA® pieces were transferred to the wells of a 24 well tissue culture plate (collagen layer facing up) immersed in media containing proteins with peroxidase activity (SBP at 25μg/ml in DMEM). This pre- treatment of the INTEGRA® with proteins with peroxidase activity was performed for up to 16 hours at 37°C in a C0 ₂ incubator. After the pre-incubation period, the media was replaced with fresh osteogenic DMEM medium (DMEM supplemented with 5% FBS, ΙΟΟμΜ Ascorbic acid 2-phosphate, 10 ^" M dexamethasone and lOmM β- glycerophosphate) (to assist bone crystal/nodule formation) containing 5xl0 ⁵ cells (either primary human osteoblasts or SaOS-2 human osteosarcoma) plus or minus proteins with peroxidase activity. After allowing 24 hours for the osteoblast cells to attach to the collagen matrix of the INTEGRA® scaffold, the INTEGRA® pieces were transferred to fresh 24 well plates to ensure any cells not attached to the matrix surface were removed. Fresh media containing proteins with peroxidase activity were added to the wells and the INTEGRA® pieces incubated for up to 28 days (post-treatment period) with the media changed every 7 days as necessary. At the end of the experiment the INTEGRA® pieces were harvested and fixed in 10% buffered formaldehyde before they were processed, embedded in paraffin wax, cut in cross-section and placed onto microscope slides for histological and immunohistochemical analysis.

[00345] Six to eight micron thick sections of the INTEGRA® were mounted on silane-coated slides and were dewaxed before rinsing rapidly in distilled water. Slides were immersed in 2% Alizarin Red solution for 5min at RT. Excess dye was drained off then slides were briefly rinsed with distilled water before immersing in acetone for 20 seconds, then 50% acetone/ 50% xylene for 20 seconds. Slides were then further dehydrated and mounted in DPX. Alizarin Red was used as a stain to specifically identify calcium deposition within the INTEGRA® scaffold. Calcium forms an alizarin red S-calcium complex in a chelation process and the reaction is birefringent.

EXAMPLE 10- Peroxidase enzymes stimulate collagen I biosynthesis in normal human osteoblasts

[00346] Type I collagen comprises approximately 80% of the total protein present in bone, where it plays an important structural role in determining the biomechanical competence of bone. When stimulated for 72 hours with either EPO or MPO in the absence of AA, cultured human osteoblasts demonstrated a dose-responsiveness to both mammalian peroxidases. Like AA, which was used as a control, the combined data from five adult donors showed the peroxidase enzymes increased the amount of collagen I released into the medium approximately six-fold, with highly significant differences detected as low as 98 ng/mL (Fig. 8A). Assessment of cell viability following stimulation indicated that neither the peroxidases nor AA had a significant impact on the number of cultured osteoblasts over the 72 hour stimulation period (Fig. 8B), indicating the increase in collagen I release is not due to an increase in cell number.

[00347] The ability of peroxidase enzymes to induce procollagen I secretion was not restricted to mammalian peroxidases, as the plant-derived peroxidase enzymes SBP and HRP were also potent stimulators of soluble procollagen I release (Figure 9). Both plant peroxidase enzymes were in the order of 50-fold more potent at stimulating a maximal collagen response when compared to the mammalian enzymes MPO and EPO. The peroxidase enzymes (either mammalian or plant-derived) were found to have no impact on the growth or viability of the cultured osteoblasts as assessed by alamarBlue staining.

EXAMPLE 1 1 - Peroxidase enzymatic activity is essential for collagen biosynthesis by normal human osteoblasts

[00348] The iron-containing heme catalytic domain is a highly conserved feature shared by both EPO and MPO. To determine whether peroxidase catalytic activity had a role in promoting collagen I production, osteoblasts were stimulated with either EPO or MPO in the presence of 4 amino-benzoic acid hydrazide (4-ABAH), an irreversible inhibitor of mammalian peroxidase enzymatic activity. A clear dose-dependent inhibition of collagen I release was observed over the 72 hour time period, with maximal inhibition achieved at the highest dose of 250 μιτιοΙ/L (Fig. 10A). In contrast, 4-ABAH over the same dose range had no impact on AA-induced collagen I release; confirming that 4- ABAH was inhibiting the peroxidase catalytic activity and not the cellular collagen biosynthetic pathway. Exposure of osteoblasts to increasing doses of 4-ABAH up to 250 μιηοΙ/L had no impact on cellular viability over the duration of the experimental period (Fig. 10B). These data confirm that the heme-containing catalytic domain plays an essential role in EPO and MPO-mediated collagen I release by osteoblasts.

EXAMPLE 12 - Peroxidase enzymes promote in vitro bone formation by culture human osteoblasts

[00349] Figure 1 1A shows NHB osteoblasts maintained in culture for 21 days under mineralization conditions in the presence of EPO in a dose range up to 12^g/ml. Cells were fixed and stained with Alizarin Red S dye for the detection of calcium phosphate (mineral) deposition. Light microscopy imaging clearly shows that EPO stimulated a dose-dependent increase in mineral deposition by the cultured human osteoblasts. Quantification of the Alizarin Red S staining indicates that the peroxidase enzyme EPO stimulated a greater than 9-fold increase in mineral deposition compared to cells maintained under mineralization conditions without peroxidase enzymes.

[00350] Figure 1 1B shows NHB osteoblasts maintained in culture for 21 days under mineralization conditions in the presence of SBP at 5μ /ηι1. Cells were fixed and stained with Alizarin Red S dye for the detection of calcium phosphate (mineral) deposition. Light microscopy imaging clearly shows that SBP stimulated a large increase in mineral deposition on the surface of the cell monolayer that was confirmed by Alizarin Red S staining.

[00351] These results clearly show that both mammalian and plant-derived peroxidase enzymes have the capacity to promote cultured human osteoblasts to deposit calcified mineral in vitro.

EXAMPLE 13 - Peroxidase enzymes promote tissue-like bone regeneration in vitro

[00352] Figure 12 shows that peroxidase enzymes promote tissue-like bone regeneration in vitro. The INTEGRA® Dermal Regeneration Template (DRT) was used as a pre-made three-dimensional scaffold to establish whether stimulation of osteoblastic cells with peroxidase enzymes could result in the generation of cell- associated mineralized extracellular matrix in vitro. After 28 days of culture in control media (Saline), normal human bone (NHB) osteoblasts deposited only a small amount mineral within the INTEGRA® scaffold as demonstrated by the Alizarin Red S staining. Most surprisingly however, when the INTEGRA® was incubated in media containing SBP at 25μg/ml, the osteoblasts deposited significantly more bone mineral throughout the interstices of the scaffold after 28 days as compared to control-treated scaffold. A similar response was also observed when the human osteosarcoma cell line SaOS-2 were allowed to mineralize the INTEGRA® scaffold over a 28 day period in the presence of SBP at 25μg/ml. These data clearly show that proteins with peroxidase activity have the ability to stimulate the production and calcification of marked amounts of bone by osteoblastic cells when cultured in a three-dimensional scaffold in vitro.

EXAMPLE 14 - Peroxidase enzymes regulate BMP-2 mRNA levels in cultured human osteoblasts

[00353] Bone Morphogenetic Protein-2 (BMP-2) is an important regulator of normal bone formation in vivo, and has been incorporated into collagen scaffolds for bone regeneration applications in the treatment of bony defects and healing of non-union fractures. To establish whether peroxidase enzymes could stimulate BMP-2 mRNA expression in cultured NHB osteoblasts, cells were maintained in culture for up to 21 days under mineralization conditions in the presence of SBP at 5μg/ml. BMP-2 mRNA levels were quantitated by RT-PCR and expressed as a fold change compared to cells cultured in the absence of peroxidase enzymes. As shown in Figure 13, the RT-PCR results clearly show that SBP stimulated a time-dependent increase in BMP-2 mRNA levels that was first evident after only 3-days exposure to peroxidase enzymes. BMP-2 mRNA levels peaked after 12 days of peroxidase stimulation and were still elevated after 21 days. These data suggest that peroxidase enzymes have the ability to promote new bone formation in vitro by regulating the expression of the important osteogenic factor BMP-2.

EXAMPLE 15 - Peroxidase enzymes regulate collagen biosynthesis and matrix mineralization by cultured human osteoblasts

[00354] 1. Materials and Methods

[00355] Peroxidases

[00356] Native human eosinophil peroxidase (EPO) was obtained from Cell Sciences (Canton, MA) and Lee Biosolutions Inc. (St. Louis, MO). Recombinant human myeloperoxidase (rhMPO) was purchased from R&D Systems (Minneapolis, MN).

[00357] Ethics statement

[00358] The use of all normal human donor-derived bone tissue was approved by the human ethics committees of the Royal Adelaide Hospital/University of Adelaide (Approval No. RAH1301 14). Human bone samples were obtained with informed written donor consent, as required and approved by the ethics committee.

[00359] Osteoblast Cell Culture

[00360] Normal human bone-derived osteoblasts were isolated from intertrochanteric trabecular bone samples from female donors undergoing primary hip replacement surgery, as described previously (Atkins GJ, Kostakis P, Pan B et al (2003) J Bone Miner Res 18: 1088-1098). Human osteoblasts were expanded in culture using Dulbecco's Modified Eagle's Medium (DMEM; high glucose with no AA), supplemented with 2mmol/L glutamine, 100 IU/mL penicillin, 100 μg/ml streptomycin, 20 mmol/L HEPES, and 10% fetal bovine serum (FBS; Invitrogen Life Technologies, Carlsbad, CA) in a 5% CC containing humidified atmosphere.

[00361] Collagen I Enzyme-Linked Immunosorbent Assay (ELISA)

[00362] To evaluate the effect of MPO and EPO on collagen I production, osteoblasts were seeded into 96-well plates (Nunc, Roskilde, Denmark) at a density of 1.2 x 10 ⁴ cells per well and cultured for 5 days in DMEM/10% FBS until reaching confluence. Cells were starved overnight in serum-free DMEM and then stimulated for an additional 72 hours in serum- free DMEM containing either ascorbic acid 2-phosphate at 100 μιηοι/L (Wako Chemical Industries, Osaka, Japan) as a positive control, or with the peroxidase proteins in the absence of ascorbic acid supplementation. At the end of the 72-hour treatment period, osteoblast-conditioned media was collected for measurement of secreted, soluble type I collagen by ELISA. Cell viability/growth was then assessed using the alamarBlue fluorescent dye assay (Invitrogen Life Technologies). Briefly, cells were incubated in a 10% alamarBlue/phosphate-buffered saline (PBS) solution for 30 minutes at 37°C and fluorescence measured at wavelengths of 530 nm excitation and 595 nm emission using a FLUOstar Optima plate reader (BMG Labtek Australia, Mornington, VIC).

[00363] The amount of soluble type I collagen in cell-conditioned medium was measured by a direct coat enzyme-linked immunosorbent assay method, using standard curves constructed from purified Type I human placental collagen (BD Biosciences Australia, North Ryde, NSW). Samples and standards (ΙΟΟμί per well) were added to a 96-well Maxisorp plate (Nunc) and left at 4°C overnight. The plate was then washed with PBS-Tween 0.05%, 2.5% bovine serum albumin (BSA)/PBS blocking solution added to each well and the plate incubated for 1 hour at room temperature. The plate was then washed with PBS-Tween 0.05% and primary antibody (0.25 μg/mL rabbit- anti-human-collagen I polyclonal; Rockland Immunochemicals, Limerick, PA) in 5% nonfat dairy milk added to each well for 3 hours at room temperature. After washing, europium-tagged anti-rabbit secondary antibody (0.5 μg/mL in 1% BSA/PBS; Perkin Elmer Life Sciences, Turku, Finland) was added for 1 hour at room temperature. After a final wash, Enhancement Solution (Perkin Elmer Life Sciences) was added and fluorescence measured at excitation 355 nm and emission 620 nm using a FLUOstar Optima plate reader (BMG Labtek Australia). The collagen content of each sample was determined from the standard curve ^g/mL), then normalised to DMEM-only treated cells.

[00364] Immunofluorescence Staining

[00365] Primary human osteoblasts (1.2 x 10 ⁴ cells per well) were seeded onto circular glass coverslip in DMEM supplemented with 2 mmol/L glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin, 20 mmol/L HEPES and 10% FBS (Invitrogen Life Technologies) and maintained in culture overnight. Cells were then treated with either saline (Control) or 1 μg of MPO or EPO in serum-free DMEM for up to 3 hours at 37°C. After peroxidase exposure, indirect immunofluorescence staining was performed to detect cellular distribution of both MPO and EPO. In brief, cells were fixed with 4% formaldehyde with 5% sucrose in PBS for 10 minutes. Cells were permeated with 0.25% Triton X-100 (Sigma- Aldrich) in PBS for 10 minutes to detect intracellular peroxidase uptake; nonspecific binding sites were blocked by using 3% BSA in PBS containing 0.1% glycine and a 1 : 10 dilution of nonimmune goat serum for 90 minutes. Cells were incubated for 1 hour with the primary antibodies [1 μg/mL rabbit anti-human MPO from Dako Australia, North Sydney, NSW (A0398); 1 : 1000 dilution of mouse anti-human EPO from Merck Millipore Australia, Bayswater, NSW (MAB1087)] in PBS containing BSA at 1 mg/mL and then counterstained with a 1 : 1000 dilution of Alexa Fluor 488-conjugated goat anti-mouse or goat anti-rabbit IgG (Cell Signaling Technologies, Beverly, MA) in PBS containing 1 mg/mL BSA. Coverslips were mounted on glass slides using Fluoromount (Sigma-Aldrich) and images captured using a fluorescence photomicroscope (Observer Zl ; Carl Zeiss Microscopy, Jena, Germany).

[00366] RNA isolation and Quantitative Real-Time PCR

[00367] For collagen I mRNA time-course studies, 6 x 10 ⁴ osteoblasts were seeded into T25 culture flasks in DMEM/10% FBS and maintained in culture for 5 days. On reaching confluence, cells were starved in serum-free DMEM overnight and then stimulated with a maximal dose of 10 ng/mL transforming growth factor (TGF)-p2, 100 μιηοι/L ascorbic acid 2-phosphate, 1.56 μg/mL EPO, or 1.56 μg/mL recombinant human MPO in serum- free DMEM. Total RNA was harvested at time points up to and including 48 hours using an RNeasy Mini Kit (Qiagen Australia, Chadstone, VIC) according to the manufacturer's instructions. RNA yield and purity were quantified by Nanodrop spectrophotometric measurement at 260 nm (Nanodrop Technologies, Thermo Fisher Scientific, Scoresby, VIC, Australia). Gene expression was examined using real-time PCR and normalized to the housekeeping gene glyceraldehyde 3- phosphate dehydrogenase (GAPDH). cDNA was synthesized by reverse transcription of 1 μg total RNA using random hexamer primers and Superscript III Reverse Transcriptase (Invitrogen Life Technologies). Quantitative real-time RT-PCR was performed using SYBR Green Fluor qPCR mastermix (Qiagen Australia) in a CFX96 Real-Time System (BioRad, Hercules, CA). Each reaction volume of 25 μΐ _^ contained cDNA templates, primer pairs and SYBR Green mastermix. Amplification occurred after initial denaturation at 95°C for 3 minutes, followed by 40 cycles at 95°C for 15 seconds, 60°C for 26 seconds, and 72°C for 10 seconds. The primer combination used for human GAPDH, collagen I al, BMP-2, BSP, Wnt5a and FRZB are shown in Table 1. Table 1 : qPC primer sequences

[00368] In vitro mineralization

[00369] Primary human osteoblasts were seeded into 96-well plates (Nunc) at a density of 1.2 x 10 ⁴ cells per well and cultured for 5 days in 10% fetal bovine serum/Dulbecco's minimum essential media (10% FBS/DMEM) at 37°C and 5%C0 ₂ . Triplicate wells were stimulated with either MPO or EPO in osteogenic DMEM medium [DMEM supplemented with 5% FBS, 100 μιηοι/L ascorbic acid 2-phosphate (Wako Chemical Industries), 10 ^"8 mol/L dexamethasone (Hospira Australia, Mulgrave, VIC) and 10 mmol/L β-glycerophosphate (Sigma-Aldrich)] to assist bone mineral formation. Cells were maintained in culture for up to 12 days, with fresh medium with or without EPO or MPO, changed every 4 days. To detect matrix mineralization, the Alizarin Red staining method was used. Cells were washed twice with PBS and then fixed with 10% phosphate-buffered formalin for 15 minutes. The fixed cells were washed twice with distilled water and stained with 2% Alizarin Red S solution (Sigma-Aldrich) for 5 minutes. The excess dye was removed by repeated washing with distilled water, and mineralized matrix stained by Alizarin Red photographed using a Nikon Eclipse 50i microscope attached to a DS-L2 control unit (Digital Sight, Nikon Europe, Amsterdam, The Netherlands) and a DS-Fil digital camera (Nikon Corporation, Tokyo, Japan).

[00370] 2. Results

[00371] Peroxidases regulate post-translational collagen biosynthesis

[00372] To investigate the mechanism by which peroxidase enzymes regulate collagen I production, we compared the effect of EPO to the time-dependent secretion of collagen I stimulated by either AA or TGF-p2. In the absence of AA, EPO increased the levels of collagen I detected in osteoblast-conditioned media by 12 hours (Fig. 14A). The amount of collagen I stimulated by the peroxidase enzyme increased with time, reaching maximal levels by 48 hours. The profile of collagen I release stimulated by EPO was comparable to that observed for AA, both in terms of magnitude and time dependency. In contrast, without AA supplementation, TGF-p2 failed to induce a significant increase in collagen I release over the same time frame (Fig. 14A). Quantitative real-time PCR analysis of collagen I al mRNA levels within these cells revealed that the robust increase in collagen I protein stimulated by EPO was not associated with a corresponding increase in collagen I mRNA levels (Fig. 14B). In contrast, TGF-P2 clearly induced a time-dependent increase in collagen I al mRNA, with a twofold increase detected at both the 24 and 48 hour time points. AA failed to induce a significant increase in collagen la mRNA expression over the 48 hour stimulation period.

[00373] AA is known to function as an enzymatic co-factor for prolyl hydroxylase to promote post-translational collagen biosynthesis. As the AA and peroxidase-mediated collagen response profiles were very similar, further studies were conducted to establish whether peroxidase enzymes were also capable of regulating post-translational collagen I biosynthesis in a prolyl hydroxylase-dependent manner. Cultured osteoblasts were stimulated with either MPO or EPO in the presence of dimethyloxalylglycine (DMOG), a cell-permeable structural analogue of a-ketoglutarate, which acts as a competitive inhibitor of prolyl hydroxylases. As a positive control, cells stimulated with AA were also treated with DMOG to confirm that the release of collagen I was dependent upon prolyl hydroxylase-mediated hydroxylation. DMOG blocked the AA and peroxidase- induced release of collagen I from cultured osteoblasts in a dose-dependent manner, with complete inhibition occurring at 100 μιηοι/L (Fig. 15 A). The inhibitory effect of DMOG was not associated with a loss in cell viability (Fig. 15B).

[00374] Eosinophil peroxidase promotes osteoblast matrix mineralization in vitro

[00375] EPO regulates osteogenic gene expression in vitro

[00376] To establish whether peroxidase enzymes could regulate the expression of genes associated with osteoblast differentiation and matrix mineralization, primary human osteoblasts were grown to confluence and stimulated with EPO under mineralizing conditions for 12 days. Quantitative real-time PCR analysis was performed to determine the effect EPO had on BMP-2, BSP, Wnt5a and FRZB gene expression. BMP-2 plays a fundamental role in regulating the regenerative capacity of osteoblastic cells, and is possibly the most extensively investigated cytokine that enhances skeletal repair. Relative to osteoblasts maintained in mineralization medium alone (Unstim), EPO stimulation resulted in a fourteen-fold increase in BMP-2 gene expression at a time when rapid mineralization of the cellular monolayer was occurring (Fig. 16). EPO was also found to induce the mRNA expression of BSP, an acidic noncollagenous bone ECM protein that facilitates mineralization by serving as a nucleation site for hydroxyapatite crystal formation. Wnt5 a- induced noncanonical signaling plays an essential role in skeletal development, and has been shown to promote osteoblast differentiation and mineralization. Our results show that EPO also induced a greater than twofold increase in Wnt5a expression, while at the same time down-regulating the expression of the secreted WNT inhibitor FRZB.

[00377] Cultured human osteoblasts rapidly bind and internalize peroxidase enzymes

[00378] To determine whether exposure of cultured osteoblasts to MPO and EPO could promote cell surface binding and internalization of these enzymes, immunofluorescence localization studies were performed (Fig. 16). The images clearly show that osteoblasts bind and rapidly internalize peroxidase enzymes. Both MPO and EPO accumulate on the cell surface within minutes of peroxidase exposure (Fig. 17; Surface), whereas no staining was detected in peroxidase-untreated cells, confirming the specificity of the EPO and MPO immunostaining (data not shown). After 3 hour of peroxidase exposure, there is intense intracellular MPO and EPO staining (Fig. 17; Intracellular). Both enzymes appear to localize to vesicle-like structures, with MPO adopting a distinct perinuclear distribution that is not evident with EPO staining.

[00379] 3. Discussion

[00380] The studies reported here provide evidence for the first time that mammalian peroxidase enzymes have the capacity to regulate osteoblastic cell function in vitro, including collagen I biosynthesis, osteogenic gene expression and matrix mineralization. These novel findings offer new insight into a potential mechanism, by which inflammatory cells are able to regulate osteogenesis at sites of normal and pathological bone formation.

[00381] Collagen I biosynthesis by osteoblasts is a critical early stage event required for the formation of new bone. AA is known to function as an enzymatic co- factor for prolyl hydroxylase to promote post-translational collagen biosynthesis. AA increases cellular secretion of collagen proteins by promoting efficient hydroxylation of peptidyl proline, leading to the assembly of a stabilized procollagen triple helical structure. Without hydroxylation, unfolded procollagen is thermally unstable and is retained within the cell and degraded. Our in vitro studies examining collagen I biosynthesis show that peroxidase enzymes elicit an overall response in cultured osteoblasts that is very similar to the effect we observed when stimulating these cells with AA. Exposure of osteoblasts to peroxidase proteins in the absence of AA supplementation stimulated a robust increase in collagen I secretion without a corresponding increase in collagen I mRNA levels. Our data showing the inhibition of peroxidase-induced collagen I release by the prolyl hydroxylase inhibitor DMOG, indicate that post-translational events are most likely to be affected by the peroxidases. Moreover, given that hydroxylation is an essential requirement for secretion of procollagen from the cell, we propose that peroxidase enzymes can also act at this point in the collagen I biosynthetic pathway to improve hydroxylation efficiency and the utilisation of translated protein. We believe this important observation has previously been overlooked by others because AA is routinely added to osteoblast culture medium when examining collagen biosynthesis. When combined with AA, the stimulatory effects of peroxidase enzymes on osteoblast collagen release are masked (data not shown), suggesting both agents target the same regulatory point in collagen biosynthesis. These data obtained using cultured human osteoblasts are entirely consistent with other studies that we have conducted and generated with primary fibroblasts derived from dermal, mammary and prostate tissue, indicating the mechanism by which peroxidase enzymes regulate collagen I biosynthesis appears to be highly conserved across different mesenchymal-derived cell types.

[00382] We have demonstrated by immunofluorescence localization studies that cultured osteoblasts can also rapidly bind and internalize both MPO and EPO. Once internalized, these catalytically active enzymes are able to consume hydrogen peroxide (H ₂ O ₂ ) as an essential cofactor for the generation of multiple intracellular reactive oxidant species. Cellular H ₂ O ₂ is predominantly produced through dismutation of superoxide that is formed in a reaction catalysed by NADPH oxidase. H ₂ O ₂ is lipid soluble and can readily diffuse across cellular membranes and is known to oxidise the Fe bound to the prolyl hydroxylase enzyme to Fe , leading to inhibition of prolyl hydroxylase enzymatic activity and substrate hydroxylation. In support of this concept, supplementation of rabbit calvarial osteoblast cultures with exogenous H ₂ O ₂ has previously been reported to suppress collagen I expression by these cells. Moreover, 10- day chick embryo tibiae exposed to elevated H ₂ O ₂ levels in an organ culture system show a marked inhibition of collagen synthesis as measured by a decrease in ³ H- hydroxyproline incorporation into total collagen. Importantly, our findings indicate the peroxidase heme-containing catalytic domain plays an essential role in MPO and EPO- mediated collagen biosynthesis in osteoblasts. It is therefore conceivable that one mechanism by which the peroxidase enzymes promote osteoblast collagen biosynthesis is by catalytically depleting intracellular H ₂ O ₂ levels, thereby negating the inhibitory effect H ₂ O ₂ has on prolyl hydroxylase enzymatic activity.

[00383] In addition to stimulating collagen I biosynthesis, we have also demonstrated that EPO is capable of promoting osteoblast matrix mineralization in vitro. Concomitant with mineralization, EPO was found to regulate the expression of several key osteogenic effector genes associated with osteoblast differentiation and matrix mineralization, including BMP-2, BSP, Wnt5a and FRZB. BMP-2 has previously been reported to stimulate BSP and Wnt5a mRNA and protein levels in cultured osteoblasts, raising the possibility that EPO indirectly regulates matrix mineralization via induction of BMP-2 which acts as an autocrine intermediate to regulate additional downstream effector genes including BSP and Wnt5a.

[00384] While eosinophils are known to be recruited to sites of bone fracture in the immediate period following injury, the capacity of this inflammatory cell type to influence new bone formation has not been contemplated. Recent data however, shows that in patients suffering chronic rhinosinusitis, a clear association exists between elevated mucosal eosinophilia and increased osteitic bone formation in the paranasal sinuses, suggesting a causal link between eosinophil infiltration and pathological osteogenesis. Moreover, in an IL-5 overexpression transgenic mouse model where eosinophils comprise more than 60% of circulating white blood cells, extensive eosinophilia and ectopic bone formation were observed in the spleen, together with increased cancellous bone growth in skeletal long bones compared to wild type litter mates and it has been suggested that the release of latent TGF-P2 during the process of degranulation is one potential mechanism, by which activated eosinophils could stimulate osteoblasts to elicit new bone formation. Eosinophil peroxidase is the most abundant secretory granule proteins released along with latent TGF-β during eosinophil degranulation. Our data showing eosinophil-derived EPO regulation of osteoblast collagen biosynthesis and matrix mineralization provide an alternative mechanistic explanation to account for the pro-osteogenic properties of eosinophils.

[00385] Heterotopic ossification (HO) is a debilitating condition characterized by the formation of ectopic bone in soft tissue such as muscle following traumatic injury. Inflammatory cell involvement is now recognised to be an important contributing factor in HO progression, although the mechanism or identity of the specific factors released by these inflammatory cells at the injury site remains poorly defined. Based on our data showing that inflammatory cell-derived peroxidase enzymes can regulate osteoblast functionality in vitro, consideration should be given to the potential role that peroxidase enzymes such as MPO and EPO may have in promoting ectopic bone formation in HO.

[00386] In conclusion, our novel in vitro findings suggest that the peroxidase enzymes MPO and EPO are likely to regulate multiple cellular processes involved in new bone formation, including osteoblast collagen I biosynthesis, osteogenic gene regulation and bone matrix mineralization. Our data also highlight a potential mechanism to link pathological bone formation at sites where elevated tissue eosinophilia is a feature.

EXAMPLE 16 - Murine femoral fracture model

[00387] A standardized femoral fracture model using a flexible plate (MouseFix) was used to examine the effect soybean peroxidase (SBP) has on periosteal callus formation and subsequent bone repair.

[00388] Mice (C57B1/6 aged 11 to 12 weeks) were anesthetized, the left hind limb was shaved, and a longitudinal incision (approximately 12 mm) was made along the lateral aspect of the thigh. The lateral fascia and muscles were longitudinally split, and the middle third of the femoral shaft was made accessible to apply the fracture fixation implants. In this area, the surrounding muscles were bluntly dissected from the bone without stripping the periosteum. The flexible plate was attached to the lateral aspect of the femoral shaft using four bicortically placed, head-locking screws. A well-defined, full-thickness osteotomy was generated beneath the flexible plate using a saw guide, resulting in a fracture gap width of 0.22 mm in the midfemoral shaft. Muscles and skin layers were closed with sutures. Purified and lyophilized SBP (BioResearch Products, North Liberty, IA, USA) was reconstituted in saline to a concentration of 125ug/mL, and animals injected with a bolus dose of 25 ug of SBP in 200ul (or saline vehicle control) directly into the fracture site (using palpation to determine plate position and subsequently guide injection) at the time of surgery and then every second day until harvest 18 days after fracture. Positron emission tomography-computed tomography (PET-CT) scanning post-surgery at Day 18 was used to monitor bone formation at the fracture site.

[00389] The murine femoral fracture model is as described in Raggatt LJ, WuUschleger ME, Alexander KA, Kaur S, Maugham ML, Gregory LS, Steck R, and Pettit AR (2014) "Fracture healing via periosteal callus formation requires macrophages for both initiation and progression of early endochondral ossification." Am J Pathol 184£12}:3192-3204). [00390] Fractures in this model heal via periosteal endochondral callus formation. The validity and reproducibility of this system as an experimental fracture model has previously been established.

[00391] In this model, mice were injected with 25 μg of SBP every 3 days over the fracture site until day 18. PET-CT imaging was performed on day 18 and bones were harvested 21 days post-surgery for histological assessment. Treatment with SBP resulted in a robust callus formation on the periosteal surface, shown here by PET-CT imaging, which was approximately double the size of the vehicle PBS injected animals (Figure 18).

[00392] These results, and the other in-vitro and in-vivo results described herein, demonstrate that peroxidases participate in bone dynamics, with prominent regulatory and effector functions.

EXAMPLE 17- Treatment of breast cancer

[00393] Subjects suffering from breast cancer may be identified by known clinical characteristics.

[00394] Treatment of human patients with breast cancer may be undertaken by administration twice daily of a formulation including a peroxidase inhibitor (eg 5-50 mg), typically by iv administration in isotonic saline, or by oral administration in tablet form and further including for example one or more lactose, maize starch, sucrose and purified talc.

[00395] Treatment with the peroxidase inhibitor may be for a defined intervention period (for example 8 -12 weeks) or be maintained indefinitely. Serum and/or plasma levels of the agent may be determined at various intervals and any adverse effects monitored. Dose or frequency adjustments can be made based on the serum concentrations, clinical symptoms and any adverse effects.

[00396] It is envisaged that treatment with the peroxidase inhibitor, such as a small molecule inhibitor, will provide significant improvement in the clinical characteristics of the patients compared with a placebo, including reduced growth of the primary tumour and reduced metastasis.

EXAMPLE 18 - Peroxidases in new bone formation

[00397] To determine whether peroxidase enzymes can promote new bone formation in vivo, a well-established mouse calvarial defect model may be used, for example as described in Behr B. et al (2012) TISSUE ENGINEERING: Part A Volume 18, Numbers 9 and 10, 1079-1086, where a small 3mm diameter piece of skull bone is surgically removed. Normally, this size defect in the skull is large enough that it is unable to heal naturally over the lifetime of the animal, with only 10% of the defect diameter closing over an 8 week period post-surgery. By placing a collagen sponge (which will act as a scaffold) into the defect ± peroxidase enzymes, it will be possible to establish whether peroxidase enzymes stimulate local bone cells that surround the defect to migrate into the scaffold and lay down new bone to heal the defect.

[00398] Through the use of MicroCT in vivo imaging, the formation of newly formed bone may be measured within the defect over an 8 week period to track the progress of our treatments. At the end of the 8 week experimental period, the mice will be humanely killed and histological analyses performed to assess the extent and quality of healing that has been stimulated by the peroxidase enzymes. It is anticipated that treatment with exogenous peroxidises will promote bone formation.

[00399] A suitable methodology is as follows: Balb/C female mice will be anaesthetized using an anaesthetic cocktail comprising Ketamine hydrochloride (80mg/kg) and Xylazine (lOmg/kg) administered via intra-peritoneal (i.p.) injection. Once the mouse is fully anaesthetized and non-responsive to paw pinching, the hair on the scalp will be removed by mechanical shaving. The surgical site will be sterilised with betadine and alcohol (x3). A single midline sagittal incision in the scalp of the animal will be made using a scalpel to expose the parietal bone (skull). The pericranium membranous material is removed from the right parietal bone by blunt scraping with a scalpel blade. A unilateral 3mm full-thickness defect in the right non-suture parietal bone will be created using a sterile diamond-coated trephine drill bit attached to an electrical drill. The parietal bone is <0.3mm thick, so extreme caution will be taken not to disturb the underlying dura mater membrane. A 3mm piece of sterile collagen sponge (scaffold) with or without peroxidase enzymes will carefully be placed into the defect and the wound site sealed by suturing the skin closed.

[00400] At day 3, week 3 and week 6 post-surgery, all animals within each group will be anaesthetized (Ketamine hydrochloride 80mg/kg and Xylazine lOmg/kg administered via i.p. injection) and bone morphometric parameters quantitated by microCT imaging.

[00401] At day 3, week 3 and week 6 post-surgery, all animals within each group will be anaesthetized (Ketamine hydrochloride 80mg/kg and Xylazine lOmg/kg administered via i.p. injection) and bone morphometric parameters quantitated by microCT imaging.

EXAMPLE 19 - Peroxidases in the treatment of tibial non-unions

[00402] Modulators of peroxidase functionality may have applications, for example, in in humans and animals for the treatment of spinal fusion, fracture healing, delayed unions and non-unions.

[00403] For example, an exogenous peroxidase may be used in the treatment of tibial non-unions in humans as follows:

[00404] Human patients with tibial non-unions may be treated by insertion of an intramedullary rod, accompanied by exogenous peroxidase in a type I collagen carrier. For example, the carrier may contain 0.5-50 mg (typically 1-10 mg) of exogenous peroxidase formulated with 1 g of particulate type-1 bone collagen (for example as described in Pluhar G.E et al (2006) J Bone Joint Surg Br vol. 88-B no. 7, 960-966) Patients may be followed at frequent intervals over a 24 month period using a variety of criteria. Assessment criteria may include the severity of pain at the fracture site, the ability to walk with full weight-bearing, the need for surgical re-treatment of the nonunion during the course of the treatment, plain radiographic evaluation of healing, and physician satisfaction with the clinical course.

[00405] It is anticipated that treatment with peroxidase will result in healed fractures or an improvement in one or more of the above clinical characteristics.

[00406] Although the present disclosure has been described with reference to particular embodiments, it will be appreciated that the disclosure may be embodied in many other forms. It will also be appreciated that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

[00407] Also, it is to be noted that, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context already dictates otherwise.

[00408] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[00409] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[00410] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.

[00411] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.

[00412] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.

[00413] Future patent applications may be filed on the basis of the present application, for example by claiming priority from the present application, by claiming a divisional status and/or by claiming a continuation status. It is to be understood that the following claims are provided by way of example only, and are not intended to limit the scope of what may be claimed in any such future application. Nor should the claims be considered to limit the understanding of (or exclude other understandings of) the present disclosure. Features may be added to or omitted from the example claims at a later date.

[00414] Although the present disclosure has been described with reference to particular examples, it will be appreciated by those skilled in the art that the disclosure may be embodied in many other forms.