WO2015054718A1 - Bone formation - Google Patents

Abstract

Use of APC for inducing or promoting bone formation.

Description

Bone formation

Field of the invention

The invention relates to methods and compositions for inducing bone formation.

Background of the invention

Reference to any prior art in the 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 Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

It is estimated that up to 2.2 million bone grafting procedures are performed annually [Giannoudis, P.V., H. Dinopoulos, and E. Tsiridis, Bone substitutes: An update. Injury, 2005. 36(3, Supplement): p. S20-S27.]. As a result of injury or tumour resection, the loss of large quantities of bone tissue can overwhelm the body's natural bone healing capacity, leading to non-union. Together with infection, poor bone healing associated with major bone loss remain key challenges for orthopaedic medicine. Non-union results in recurrent surgical procedures and long in-hospital stays which is challenging for both patients and surgeons [Panagiotis, M., Classification of non-union. Injury, 2005. 36(4, Supplement): p. S30-S37; Zeckley, C, et al., The Aseptic Femoral and Tibial Shaft Non-Union in Healthy Patients - An Analysis of the Health-Related Quality of Life and the Socioeconomic Outcome. The Open Orthopaedics Journal, 2011. 5: p. 193-7.]. Two of the key causes that lead to non-union are an insufficiency of biological factors required for repair, and infection of the bone (osteomyelitis) [Zeckley, C, et al., The Aseptic Femoral and Tibial Shaft Non-Union in Healthy Patients - An Analysis of the Health-Related Quality of Life and the Socioeconomic Outcome. The Open Orthopaedics Journal, 2011. 5: p. 193-7; Schindeler, A., et al., Bone remodeling during fracture repair: The cellular picture. Seminars in Cell & Developmental Biology, 2008. 19(5): p. 459-466.J

Insufficient biological factors can result from a large bone defect size, lack of biological growth factors (which can be further depleted by wound debridement) as well as damaged or reduced blood supply. Current treatments to restore osteogenic factors and an appropriate microenvironment include bone grafting [Pape, H., A. Evans, and P. Kobbe, Autologous bone graft: properties and techniques. Journal of orthopaedic trauma, 2010. 24(Suppl 1): p. S36-40.], bone transport [Lavini, F., C. DalPOca, and P. Bartolozzi, Bone transport and compression- distraction in the treatment of bone loss of the lower limbs. Injury, 2010. 41(11): p. 1191-1195; Bobroff, G., S. Gold, and D. Zinar, Ten year experience with use of Ilizarov bone transport for 5 tibial defects. Bulletin (Hospital for Joint Diseases (New York, N.Y.)), 2003. 61(3-4): p.101-7.], addition of growth factors and tissue engineering approaches. Nevertheless, all of these methods have limitations and there is an ongoing search for more effective agents.

Recombinant human Bone Morphogenetic Proteins (rhBMPs), while clinically effective in promoting new bone, have significant associated complications such as inflammation and

10 induction of bone resorption [Granholm, S., et al., Osteoclast progenitor cells present in significant amounts in mouse calvarial osteoblast isolations and osteoclastogenesis increased by BMP-2. Bone, 2013. 52(1): p. 83-92; Ritting, A.W., E.W. Weber, and M.C. Lee, Exaggerated Inflammatory Response and Bony Resorption From BMP-2 Use in a Pediatric Forearm Nonunion. The Journal of Hand Surgery, 2012. 37(2): p. 316-321.]. Other complications of high

15 dose BMP use are increasingly reported [Woo, E.J., Recombinant human bone morphogenetic protein-2: adverse events reported to the Manufacturer and User Facility Device Experience database. The Spine Journal, 2012. 12(10): p. 894-899; Carragee, E.J., E.L. Hurwitz, and B.K. Weiner, A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned. The Spine Journal, 2011. 11(6): p. 471-

20 491.].

Adjunctive therapies that allow a reduction of rhBMP dose are seen as one approach to the complications associated with high dose BMPs. Work has shown that combinations of rhBMP-2 with anti-resorptives (such as bisphosphonates) can increase the net bone produced [Little, D.G., et al., Manipulation of the Anabolic and Catabolic Responses With OP-1 and 25 Zoledronic Acid in a Rat Critical Defect Model. Journal of Bone and Mineral Research, 2005.

20(11): p. 2044-2052; Yu, N., et al., Use of BMPs and bisphosphonates in improving bone fracture healing. Frontiers in Bioscience (Elite Edition), 2012. 1(4): p. 2647-53.].

There is a need for new approaches to bone formation in therapeutic applications.

There is also a need to find agents that are synergistic with BMP that may reduce cost, 30 ameliorate side effects, and potentially increase efficacy of BMP-based interventions. There is also a need for anabolic agents for bone formation that can be used synergistically with anti-catabolic agents.

Summary of the invention

The invention seeks to address one or more of the above mentioned needs or limitations, or to provide an alternative approach to bone formation and in one embodiment provides a use of activated protein C (APC) or analogues thereof or compositions including same for inducing or promoting bone formation.

In another embodiment there is provided APC or analogues thereof or compositions including same for use in inducing or promoting bone formation. In another embodiment there is provided APC or analogues thereof or compositions including same for the manufacture of a medicament for use in inducing or promoting bone formation. The medicament may take the form of a composition, formulation, scaffold or matrix described below.

In another embodiment there is provided a method for inducing or promoting bone formation including:

- providing an individual requiring bone formation,

- providing an amount of APC or analogue thereof effective for inducing or promoting bone formation to the individual, thereby inducing bone formation in the individual. Typically the APC or analogue thereof is provided to a site or region of bone or bone - related tissue in which bone formation is required.

In other embodiments there is provided a use of APC or analogues thereof or compositions including same for inducing anabolism of bone.

In another embodiment there is provided APC or analogues thereof or compositions including same for use in inducing anabolism of bone. In another embodiment there is provided APC or analogues thereof or compositions including same for the manufacture of a medicament for use in inducing anabolism of bone. The medicament may take the form of a composition, formulation, scaffold or matrix described below. In another embodiment there is provided a method for inducing anabolism of bone including:

- providing an individual requiring induction of bone anabolism,

- providing an amount of APC or analogue thereof effective for inducing anabolism of bone to the individual, thereby inducing anabolism of bone in the individual.

Typically the APC or analogue thereof is provided to a site or region of bone or bone - related tissue in which anabolism of bone is required.

In other embodiments there is provided a use of APC or analogues thereof or compositions including same for remodelling of bone tissue. In another embodiment there is provided APC or analogues thereof or compositions including same for use in remodelling of bone tissue.

In another embodiment there is provided APC or analogues thereof or compositions including same for the manufacture of a medicament for use in remodelling of bone tissue. The medicament may take the form of a composition, formulation, scaffold or matrix described below.

In another embodiment there is provided a method for remodelling of bone tissue including:

- providing an individual requiring remodelling of bone tissue,

- providing an amount of APC or analogue thereof effective for inducing remodelling of bone tissue to the individual, thereby remodelling of bone tissue in the individual. Typically the APC or analogue thereof is provided to a site or region of bone or bone - related tissue in which remodelling of bone tissue is required.

In other embodiments there is provided a use of APC or analogues thereof or compositions including same for increasing the volume of bone tissue. In another embodiment there is provided APC or analogues thereof or compositions including same for use in increasing the volume of bone tissue.

In another embodiment there is provided APC or analogues thereof or compositions including same for the manufacture of a medicament for use in increasing the volume of bone tissue. The medicament may take the form of a composition, formulation, scaffold or matrix described below.

In another embodiment there is provided a method for increasing the volume of bone tissue including:

- providing an individual requiring increased bone tissue volume,

- providing an amount of APC or analogue thereof effective for increasing the volume of bone tissue to the individual, thereby increasing the volume of bone tissue in the individual.

Typically the APC or analogue thereof is provided to a site or region of bone or bone - related tissue in which increased volume of bone tissue is required.

The above described methods or uses may be applied to strengthen bone, to repair a bone defect, or to other clinical outcome in which bone formation is necessary.

In the above described methods or uses, the APC or analogue thereof may be provided to the individual or site or region of bone or bone -related tissue in the form of a composition, formulation, scaffold or matrix described below.

In another embodiment there is provided a composition including:

- APC or analogue thereof; - a bone morphogenic protein (BMP).

In certain embodiments the APC or analogue thereof and BMP in the composition may be present in synergistically effective amounts.

In another embodiment there is provided a kit including: - APC or analogue thereof;

- BMP.

Typically the kit further includes written instructions for use of the kit in a method described above.

In another embodiment there is provided a composition including: - APC or analogue thereof;

- a bone re-sorption compound.

In another embodiment there is provided a kit including: -APC or analogue thereof;

- a bone re-sorption compound. Typically the kit further includes written instructions for use of the kit in a method described above.

In another embodiment there is provided a scaffold or matrix for use in a method described above, the scaffold or matrix including a composition as described above.

In another embodiment there is provided a formulation for topical application to bone for use in a method described above, the formulation including a composition as described above.

In another embodiment there is provided an injectable formulation for use in in a method described above, the injectable formulation including a composition as described above. In the above described embodiments a fragment of APC having activity in inducing or promoting bone formation may be used as an alternative or in addition to APC or analogue thereof. Further, protein C may be used as an alternative or in addition to APC.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

Fig 1. Receptor expression in MG-63 cells. (A) IHC using goat anti-EPCR, rabbit anti- PAR1 or mouse anti-PAR2. Negative control IgGs above. (B) RT-PCR showing expression of EPCR, PARI and PAR2 with a β-actin (ACTB) control.

Fig 2. APC stimulates MG-63 growth and differentiation as evidenced by cell counting (A) and measurement of matrix mineral by Alizarin red S staining (B). Mean ± S.E (n=4). Cells were grown in 400μΜ L-ascorbic acid and lOmM β- glycerophosphate for 8 days. At= antagonist. ** P<0.01. Fig 3. APC enhances rhBMP-2 induced bone formation. Ectopic bone was increased as seen by XR and 3D reconstruction (A). Quantification by microCT showed significant increases in bone volume (B) and tissue volume (C) of the nodule. Osteoclast number (TRAP stain) was slightly enhanced by APC treatment (D) and vascularity by CD31 IHC was substantively increased (E) (*P<0.05, **P<0.01). Cell counting averaged from 5 fields of view. Total 3 groups, 10 per group.

Fig 4. MG-63 cells express EPCR PARI and PAR2 as shown by (A) immunocytochemical detection and RT PCR

Fig 5. Effect of APC on pellets via μCT analysis. Figure shows representative pellet X- rays, 3D μCT reconstructed models and cross sectional compilation of 20 slices. CTAn analysis was carried out for bone volume (A) and tissue volume (B) as well as the ratio of bone volume to tissue volume (BV/TV) (C). Statistical analysis was carried out by one way ANOVA and Newman-Keuls post-test. Data represented as mean ±S.E (n=10). ** Denotes PO.01, *** denotes PO.001 between treatment and control. Figs 6 and 6a. Legend for PARI and PAR2 KO mice experiments. Recombinant BMP-2 ± APC were infused and implanted into 15 WT, 10 PARI KO and 10 PAR2 KO mice. Scaffolds were allowed to form ectopic bone pellets over 3 wks, fixed in 4% PFA overnight and imaged via X- ray. Femur present under pellets to provide a confirmation of the correct scale. Fig 7. Expression of CD31 (marker for endothelial cell, blood vessels) in bone pellets.

CD31 positive cells were stained by IHC in bone pellets and evaluated by ScanScope analysis, similar and graphed. Statistical analysis was conducted by one way ANOVA and Newman-Keuls post-test. Data represented as mean ±S.E (n=10). Blue scale bar indicates 300μπι. Black scale bar indicates 200μπι. Arrowhead indicate staining. * Denotes P<0.05 Fig 8. Expression of EPCR in bone pellets. EPCR positive cells were stained by IHC in bone pellets and evaluated by ScanScope analysis, similar and graphed. Statistical analysis was conducted by one way ANOVA and Newman-Keuls post-test. Data represented as mean ±S.E (n=10). Blue scale bar indicates 300μπι. Black scale bar indicates 200μηι. Arrowhead indicate staining. Fig 9. Expression of PARI in bone pellets. PAR 1 positive cells were stained by IHC in bone pellets and evaluated by ScanScope analysis, similar and graphed. Statistical analysis was conducted by one way ANOVA and Newman-Keuls post-test. Data represented as mean ±S.E (n=10). Blue scale bar indicates 300μπι, black scale bar indicates 200μιη. Arrowhead indicate staining. Fig 10. Expression of PAR2 in bone pellets. PAR2 positive cells were stained by IHC in bone pellets and evaluated by ScanScope analysis, similar and graphed. Statistical analysis was conducted by one way ANOVA and Newman-Keuls post-test. Data represented as mean ±S.E (n=10). Blue scale bar indicates 200μιη. Black scale bar indicates ΙΟΟμηι. Arrowhead indicate staining. Fig. 11. Amino acid sequence of 3K3 A-APC.

Detailed description of the embodiments

As described herein the inventor has found that activated protein C (APC) can be used to produce bone tissue in vivo. Specifically, the inventor has demonstrated that APC induces an anabolic response in vivo. The outcome is de novo production of bone. The anabolic response is induced by APC and without requirement for other bone morphogenic protein. Further the inventor has found that the response does not require, nor involve a concomitant catabolic response that would involve bone re-sorption.

A. Definitions The term "comprise" and variations of the term, such as "comprising", "comprises" and

"comprised', are not intended to exclude further additives, components, integers or steps.

"Bone" generally refers to a mineralized tissue primarily comprising a composite of deposited calcium and phosphate in the form of hydroxyapatite, collagen (primarily Type I collagen) and bone cells such as osteoblasts, osteocytes and osteoclasts, as well as to bone marrow tissue. Bone is a vascularised tissue.

Bone is generally in the form of "compact bone" or "spongy bone". From a gross anatomical perspective there are clear differences between compact and spongy bone. Specifically, compact bone generally represents a dense area of bone tissue that does not contain cavities, whereas spongy bone contains numerous interconnecting cavities defined by complex trabeculae. Otherwise, under a microscope, the trabeculae of spongy bone, and compact bone have the same basic histologic structure.

"Long bones" are generally bones in which compact bone is found at the diaphysis, which is the cylindrical part of the bone, whereas the spongy bone is found at the epiphyses, i.e. the bulbous ends of a bone. Examples of long bones include humerus, radius, ulnar, tibia, fibular and femur.

"Short bones" are generally bones where there is usually a core of spongy bone completely surrounded by compact bone. Examples include the bones of the hand.

"Flat bones" generally have 2 layers of compact bone called plates separated by a layer of spongy bone. Examples of flat bones include parietal, frontal, occipital and temporal bones of the skull, the mandible and maxilla.

"Endochondral ossification" generally refers to production of bone within cartilage tissue, as generally occurs in fetal skeletal system development. This bone production generally occurs at a primary ossification centre at the diaphyses, and then at a secondary ossification centre at the epiphyses. Endochondral ossification is generally required for formation of long and short bones.

"Intramembranous ossification" is another important process for development of the fetal skeletal system, although unlike endochondral ossification, intramembranous ossification generally refers to production of bone that does not occur within cartilage. Intramembranous ossification is generally required for formation of flat bones. Intramembranous ossification is also an essential process during the natural healing of bone fractures

"Subchondral bone" is generally bone located below cartilage, and therefore generally provides support for a cartilaginous articular surface. "Bone-related tissue" generally refers to tissue that is either supported by bone (for example articular tissue) or tissue that is connected to bone, for example, a ligament or tendon. Generally, bone -related tissue is cartilaginous.

"Inducing or promoting bone formation" generally refers to an anabolic process the end result of which is bone. Generally this does not involve a catabolic process that leads to re- modelling of bone. However, the bone arising from inducing or promoting bone formation in accordance with the invention may be remodelled with or without clinical intervention. In certain embodiments the induction or promotion of bone formation involves a process that more closely resembles intramembranous ossification. As described herein and exemplified in the examples, the process generally involves the proliferation and differentiation of osteoblasts and the mineralisation of calcium. The process may or may not require the presence of cartilaginous tissue.

A "bone defect" is generally a structural disruption of bone requiring repair. A defect can assume the configuration of a "void', which is understood to mean a three-dimensional defect such as, for example, a gap, cavity, hole or other substantial disruption in the structural integrity of a bone or joint. A defect can be the result of accident, disease, surgical manipulation, and/or prosthetic failure. The defect may be a void having a volume incapable of endogenous or spontaneous repair. Generally, these are capable of some spontaneous repair, albeit biomechanically inferior. Other defects susceptible to repair include, but are not limited to, nonunion fractures; bone cavities; tumor resection; fresh fractures (distracted or undistracted); cranial/facial abnormalities; periodontal defects and irregularities; spinal fusions; as well as those defects resulting from diseases such as cancer, arthritis, including osteoarthritis, and other bone degenerative disorders such as osteochondritis dessicans.

"Repair" generally refers to new bone formation which is sufficient to at least partially fill a void or structural discontinuity at a defect. Repair does not, however, mean, or otherwise necessitate, a process of complete healing or a treatment which is 100% effective at restoring a defect to its pre-defect physiological/structural/mechanical state.

When a bone is fractured, the damaged blood vessels produce a localized haemorrhage with formation of a blood clot. Destruction of bone matrix and death of bone cells adjoining the fracture may also occur. During repair, the blood clot, the remaining cells, and the damaged bone matrix may be removed by macrophages. The periosteum and the endosteum around the fracture respond with intense proliferation of osteoprogenitor cells, which form a cellular tissue surrounding the facture and penetrating between the extremities of the fractured bone. Immature bone is then formed by endochondral ossification of small cartilage fragments that appear in the connective tissue of the fracture. Bone is also formed by means of intramembranous ossification. Repair progresses in such a way that irregularly formed trabeculae of immature bone temporarily unite the extremities of the fractured bone forming a "bone callus". Normal stress imposed on the bone during repair and during return to activity serves to remodel the bone callus, influencing its structure, and the primary bone tissue of the callus is therefore gradually reabsorbed and replaced by lamellar bone, resulting in restoration of the original bone structure and function. "Activated protein C" ("APC) is a serine protease having a molecular weight of about 56 kD that plays a central role in physiological anticoagulation. The inactive precursor, protein C, is a vitamin K-dependent glycoprotein synthesised by the liver and endothelium and is found in plasma. Activation of protein C occurs on the endothelial cell surface and is triggered by a complex formed between thrombin and thrombomodulin. Another endothelial specific membrane protein, endothelial protein C receptor (EPCR), has been shown to accelerate this reaction more than 1000-fold.

The 'analogue' in the phrase "APC or an analogue thereof', refers to an "APC analogue". An APC analogue is generally a compound that may act via the endothelial protein C receptor (EPCR) and the protease activated receptor - 1 (PAR-1), or the PAR-1 and protease activated receptor -3 (PAR-3), to minimise apoptosis, or to increase cell survival in stressed or injured cells. As described further herein, APC analogues generally have a sequence that is homologous to human protein C sequence.

B. Bone formation

It will be understood that the invention applies to the induction or promotion of bone formation. In one embodiment, there is provided a method for inducing or promoting bone formation. The method includes the following steps:

- providing an individual requiring bone formation,

- providing APC or APC analogue to the individual, thereby inducing bone formation in the individual.

The individual may require bone formation for the purpose of remedying or repairing a bone defect. The bone defect may be a fracture, such as a non-union fracture or a fresh fracture (distracted or undistracted). Thus in one embodiment there is provided a method for repairing a bone fracture including the following steps:

- providing an individual requiring repair of bone fracture,

- providing a APC or APC analogue to the individual, thereby repairing a bone fracture in the individual.

In another embodiment the individual requires bone formation for the filling of a void in bone tissue. The void may generally be a three dimension defect such as a gap, cavity or whole arising from disease, surgical manipulation and/or prosthetic failure. The void may have a volume incapable of endogenous or spontaneous repair. For example the void may be twice the diameter of the subject bone. Thus in another embodiment there is provided a method for filling a void in bone including the following steps:

- providing an individual having a void in a bone,

- providing a APC or APC analogue to the individual, thereby repairing filling the void in the bone. Typically the APC or APC analogue is provided to a site or region of bone or bone - related tissue in which bone formation is required. In this embodiment, the APC or APC analogue is provided by local administration of APC or analogue to the site or region of bone or bone related tissue. Local administration generally requires direct contact of the site or region of bone or bone -related tissue with the APC or analogue. The APC or APC analogue may be provided for direct contact with a site or region of bone or bone -related tissue by applying APC in the form of a composition, formulation, scaffold or matrix described below to the site or region of bone or bone related tissue.

The APC or APC analogue may be applied to bone only, or to bone and bone -related tissue.

The APC or APC analogue may be applied, to periosteum only, or endosteum only, or to both periosteum and endosteum.

The APC or APC analogue may be applied to compact bone only, or spongy bone only, or to both spongy and compact bone. Where the objective is to repair a defect in the form of a fracture, the APC or APC analogue may be applied by direct contact to the bone at the site of the fracture, including to one or more of the periosteum, endosteum, or callus. In this embodiment the APC or analogue thereof may be provided on or below the periosteum.

Where the objective is to fill a void in bone, for example a gap, cavity, hole or other, the bone APC may be provided on or below the periosteum.

In one embodiment, the method is for formation of intramembranous bone, or formation of spongy bone, or both.

As described herein, the invention further provides for inducing the anabolism of bone. Specifically, as exemplified herein, the inventors have found improvements in bone formation seen with APC treatment result from a mechanism primarily involving bone anabolism. The finding is significant as few other biological factors have been found to have this function. Bone anabolism is particularly required where there is a clinical need to increase bone density, or to increase bone volume. Thus in one embodiment there is provided a method for inducing anabolism of bone including: - providing an individual requiring induction of bone anabolism,

- providing an amount of APC or analogue thereof effective for inducing anabolism of bone to the individual, thereby inducing anabolism of bone in the individual. In one embodiment, the individual may require treatment to increase bone density. For example, the individual may have a form of osteoporosis.

In another embodiment, the invention provides for increase in volume of bone tissue. In this embodiment, the outcome of anabolism may be increases in any one or more dimensions of bone. This treatment may be particularly relevant where the intention is to improve the volume of an improperly formed bone. Thus there is provided a method for increasing the volume of bone tissue including:

- providing an individual requiring increased bone tissue volume,

- providing an amount of APC or analogue thereof effective for increasing the volume of bone tissue to the individual, thereby increasing the volume of bone tissue in the individual.

In the above described embodiments the APC or analogue thereof may be applied to subchondral bone, i.e. adjacent bone related tissue, or it may be contacted with bone to permit bone formation in the absence of cartilaginous tissue.

The APC or APC analogue may be applied to a long bone, short bone or flat bone. In one embodiment, the method may involve the administration of a further compound for influencing bone production. The compound may be one that is anabolic, in the sense that it is involved in new bone production, or catabolic, in the sense of causing bone re-sorption.

In one embodiment, the composition for use in the method includes APC or APC analogue and a further bone morphogenic protein (BMP). In this embodiment, the further BMP may be provided with the APC or analogue to provide an effect that is greater than the sum contribution of APC or APC analogue and BMP to bone production. The application is particularly useful for providing for desired bone outcome without providing for unwanted side effects arising from dosage of APC or APC analogue or BMP. Examples of BMP include mammalian osteogenic protein-1 (OP-1, also known as BMP-7, and the Drosophila homolog 60A), osteogenic protein-2 (OP-2, also known as BMP-8), osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A or CBMP-2A, and the Drosophila homolog DPP), BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-5, BMP-6 and its murine homolog Vgr-1, BMP-9, BMP-10, BMP11, BMP-12, GDF3 (also known as Vgr2), GDF8, GDF9, GDF10, GDF11, GDF12, BMP-13, BMP-14, BMP- 15, GDF-5 (also known as CDMP-1 or MP52), GDF-6 (also known as CDMP-2), GDF-7 (also known as CDMP-3), the Xenopus homolog Vgl and NODAL, UNIVIN, SCREW, ADMP, and NEURAL.

In one embodiment, the method or composition of the invention may include a compound for re-sorption of bone tissue with APC or APC analogue . Such a compound may be useful for re-modelling bone tissue arising from use of the APC or analogue according to the invention. The re-sorption compound may be bisphoshonate, such as for example, zoledronic acid. Thus in another embodiment there is provided a method for remodelling of bone tissue including:

- providing an individual requiring remodelling of bone tissue,

- providing an amount of APC or analogue thereof effective for inducing remodelling of bone tissue to the individual, thereby remodelling of bone tissue in the individual. For the treatment of fracture, a composition containing from O.lmg to lOmg/mL of APC or analogue thereof, preferably from 2.5 to 5.0 mg/ml of APC or analogue thereof may be prepared in sterile water. The composition is preferably prepared as an injectable composition.

The composition is generally injected into the site of the injury. In one embodiment it is preferable to inject directly into the soft tissue adjacent to the fracture. In another embodiment it could be administered by intra osseous injection. This could be performed in saline, injectable ceramic, or other high viscosity carrier.

Preferably the injection permits APC or analogue to be delivered to at least one, and more preferably, one or more opposing surfaces formed from the fracture. Generally it is preferable to achieve an even application of the APC or analogue across all of the relevant opposing surfaces. Clinically the preferred method would be to apply via surgical means only a single time with or without other agents. Follow up doses by percutaneous injection or topical application could be applied, in the absence of osteogenic drivers such as rhBMP-2. Follow up dosing could be a preferred method for preventing or treating bone infection. Alternatively an implant could be used that allows for sustained in vivo dosing using APC. One example of this could be the use of sucrose acetate isobutyrate.

Injections, including follow up injections, may be made more than once a week, and typically twice a week i.e. 'biweekly'. The injections may be administered for a period of about three to four weeks. In one embodiment, a bolus of APC or analogue thereof may be delivered by injection of

APC or analogue more or less immediately after fracture

In another embodiment, the APC or analogue thereof may be applied in the form of a putty, paste, sponge or scaffold. Acellular collagen sponges or other bioresorbable carriers may be preferred. This could include a carboxymethylcelulose, a collagen putty or a high viscosity carrier medium such as sucrose acetate isobuyrate. It could also be delivered via polymer scaffolds, including PLLA, PLGA, PGA, PCL. It could also be applied topically or by direct injection.

In one embodiment, APC or analogue thereof could be applied into the fracture at the time of fracture or prior to casting for closed fractures. For open fractures it could be introduced to the fracture gap after debridement of the wound area. For wounds were infection is suspected it could be injected adjacent to the healing fracture or into the intra osseous space as mentioned above.

The outcome of the treatment may be observed by reference to CD31 and TRAP staining of the fracture site. Generally the expression of these molecules is expected. Further a callus may be formed, although soft tissue is unlikely to have formed by the 3 week end point. Preferably the treatment should lead to normal progression of endochondral bone healing. This involves a cartilaginous soft callus being progressively replaced by woven bone, which is then remodelled into lamellar/cortical bone. In alternative embodiments the composition for application to fracture may include anabolic or anti-catabolic factors. These could in include osteogenic BMPs including but not limited to BMP-2, BMP4, BMP-6, BMP-7 and BMP-9, other members of the TGF and GDF families including TGFpi and Myostatin, and other growth factors including IGFs, PDGF, and FGFs. The preferred embodiments include rhBMP-2 or rnBMP-7. The composition may also include combination with antibodies to Sclerostin or RANKL, or with anti-catabolic agents including Cathepsin K inhibitors and bisphosphonates (e.g. Pamidronate, Zoledronic Acid).

The effective amount of the APC or APC analogue may be expected to vary depending upon the circumstances in which bone formation is required. It would be well within the skill of persons skilled in the art to adjust the amount appropriately to obtain optimal results. It is, however, expected that generally the effective amount of the agent will be in the range of 0.1 to 100000 g per kg of body weight, more preferably between 1 and 10000 μg per kg of body weight, and most preferably between about 10 and 1000 μg.

In certain embodiments the APC or APC analogue may be provided in doses of from about 0.5mg to 50mg, preferably from 0.5 to 20 mg, more preferably from 1 to 15 mg.

C. Formulations

The APC and/ or protein C utilised in the present invention may be obtained by purification from a suitable source (eg blood taken from humans or other animals) or produced by standard recombinant DNA techniques such as is described in, for example, Maniatis, T. et al., Molecular Cloning: a laboratory manual, Second Edition, Cold Spring Harbor Laboratory Press.

Recombinant APC or protein C may incorporate modifications (eg amino acid substitutions, deletions, and additions of heterologous amino acid sequences), thereby forming APC analogues which may, for example, enhance biological activity or expression of the respective protein. One example is 3K3A-APC by ZZ Biotech which is a genetically engineered variant of the APC and which has reduced anti-coagulant activity. Specifically, 3K3A-APC has KKK191/193AAA mutation. This mutation may correspond to loop 37 of APC. Another example of an APC analogue contains the RR229/230AA mutation corresponding to the calcium loop of APC. Another example of an APC analogue contains the RR306/312AA mutation corresponding to the autolysis loop of APC. Another APC analogue contains RKRR306/314AAAA corresponding to the autolysis loop of APC. Each of these examples of APC analogues have reduced anticoagulant activity as compared with activity of native APC. However, each of them has related APC function in terms of binding to EPCR and PAR-1 or PAR-3. In a preferred embodiment, the methods of the invention utilise the 3 3A-APC analogue

(KKK191/193AAA) for the various applications described herein including for inducing or promoting bone growth, for increasing anabolism of bone, for increasing bone volume, for remodelling of bone, or for fracture repair, or correcting a defect or void in bone tissue. The amino acid sequence of 3K3 A-APC is shown in SEQ ID No: 1. APC analogues generally have a sequence that is homologous to human protein C sequence. Percentage identity between a pair of sequences may be calculated by the algorithm implemented in the BESTFIT computer program (Smith & Waterman. J. Mol. Biol. 147:195- 197, 1981 ; Pearson, Genomics 11 :635-650, 1991). Another algorithm that calculates sequence divergence has been adapted for rapid database searching and implemented in the BLAST computer program (Altschul et al., Nucl. Acids Res. 25:3389-3402, 1997). In comparison to the human sequence, the protein C polynucleotide or polypeptide may be only about 60% identical at the amino acid level, 70% or more identical, 80% or more identical, 90% or more identical, 95% or more identical, 97% or more identical, or greater than 99% identical.

Conservative amino acid substitutions (e.g., Glu Asp, Val/lle, Ser/Thr, Arg/Lys, Gln/Asn) may also be considered when making comparisons because the chemical similarity of these pairs of amino acid residues are expected to result in functional equivalency in many cases. Amino acid substitutions that are expected to conserve the biological function of the polypeptide would conserve chemical attributes of the substituted amino acid residues such as hydrophobicity, hydrophilicity, side-chain charge, or size. In comparison to the human sequence, the protein C polypeptide may be only about 80% or more similar, 90% or more similar, 95% or more similar, 97% or more similar, 99% or more similar, or about 100% similar. Functional equivalency or conservation of biological function may be evaluated by methods for structural determination and bioassay. The codons used may also be adapted for translation in a heterologous host by adopting the codon preferences of the host. This would accommodate the translational machinery of the heterologous host without a substantial change in chemical structure of the polypeptide.

Recombinant forms of protein C can be produced with a selected chemical structure (e.g., native, mutant, or polymorphic). As an illustration, a gene encoding human protein C is described in U.S. Patent 4,775,624 and can be used to produce recombinant human protein C as described in U.S. Patent 4,981 ,952. Human protein C can be recombinantly produced in tissue culture and activated as described in U.S. Patent 6,037,322. Natural human protein C can be purified from plasma, activated, and assayed as described in U.S. Patent 5,084,274. The nucleotide and amino acid sequence disclosed in these patents may be used as a reference for protein C.

The APC and /or protein C may also be glycosylated by methods well known in the art and which may comprise enzymatic and non-enzymatic means.

Suitable functional fragments of an APC may be produced by cleaving purified natural APC or recombinant APC with well known proteases such as trypsin and the like, or more preferably, by recombinant DNA techniques or peptide/polypeptide synthesis. Such functional fragments may be identified by generating candidate fragments and assessing biological activity by, for example, assaying for activation of MMP-2, promotion of repair of a wounded endothelial monolayer and /or angiogenesis in chicken embryo chorio-alantoic membrane (CAM) in a manner similar to that described in the examples provided herein. Preferably, functional fragments will be of 5 to 100 amino acids in length, more preferably, of 10 to 30 amino acids in length. The functional fragments may be linear or circularised and may include modifications of the amino acid sequence of the native APC sequence from whence they are derived (eg amino acid substitutions, deletions, and additions of heterologous amino acid sequences). The functional fragments may also be glycosylated by methods well known in the art and which may comprise enzymatic and non-enzymatic means.

Suitable APC mimetic compounds (ie compounds which mimic the function of APC) may be designed using any of the methods well known in the art for designing mimetics of peptides based upon peptide sequences in the absence of secondary and tertiary structural information. For example, peptide mimetic compounds may be produced by modifying amino acid side chains to increase the hydrophobicity of defined regions of the peptide (eg substituting hydrogens with methyl groups on aromatic residues of the peptides), substituting amino acid side chains with non-amino acid side chains (eg substituting aromatic residues of the peptides with other aryl groups), and substituting amino- and /or carboxy-termini with various substituents (eg substituting aliphatic groups to increase hydrophobicity).

Alternatively, the mimetic compounds may be so-called peptoids (ie non-peptides) which include modification of the peptide backbone (ie by introducing amide bond surrogates by, for example, replacing the nitrogen atoms in the backbone with carbon atoms), or include N- substituted glycine residues, one or more D-amino acids (in place of L-amino acid(s)) and /or one or more a-amino acids (in place of β-amino acids or γ-amino acids). Further mimetic compound alternatives include "retro-inverso peptides" where the peptide bonds are reversed and D-amino acids assembled in reverse order to the order of the L-amino acids in the peptide sequence upon which they are based, and other non-peptide frameworks such as steroids, saccharides, benzazepinel,3,4-trisubstituted pyrrolidinone, pyridones and pyridopyrazines. Suitable mimetic compounds may also be designed /identified by structural modelling/ determination, by screening of natural products, the production of phage display libraries, minimised proteins, SELEX (Aptamer) selection, combinatorial libraries and focussed combinatorial libraries, virtual screening/ database searching, and rational drug design techniques well known in the art. Medicaments and delivery systems (ie gels, sponges, gauzes and meshes) according to the present invention, may contain one or more other active compounds or substances such as other molecules involved in the protein C pathway (eg protein S, EPCR, factor V/V a or factor VIII/ Villa); antimicrobial agents such as chlorhexidine, povidine-iodine and ciprofloxacin; anticoagulants such as heparin or anuthrombin III; steroids such as dexamethasone; inhibitors of inflammation; cardiovascular drugs such as calcium channel blockers; cytokines /growth factors such as epidermal growth factor; local anaesthetics such as bupivacaine; antitumor drugs such as taxol; polyclonal, monoclonal or chimeric antibodies, or functional derivatives or fragments thereof such as antibodies to regulate cell proliferation.

Further, where the medicaments and delivery systems according to the present invention utilise protein C, the medicaments and delivery systems may also include a suitable amount of an agent for activating the protein C (eg thrombin, kallikrein and/ or thrombomodulin). Medicaments according to the present invention preferably include an amount of the agent in the range of 0.01 to 1000 μ£ per g of medicament, in admixture with a pharmaceutically-acceptable carrier.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Examples

Example 1 Human osteoblasts express APC receptors

In vitro experiments were performed in the MG-63 human osteoblast-like cell line. Immuno-cytochemical staining showed robust expression of the APC receptors, EPCR, PARI 5 and PAR2 (Fig 1 A). Gene expression was confirmed by RT-PCR (Fig IB).

Example 2 PAR antagonists can prevent the effects of APC

APC can stimulate osteoblast proliferation and differentiation. At 48 hours post treatment, APC increases MG-63 cell count by 20% (PO.05) (Fig 2A), and matrix mineralization at day 8 (Fig 2B). Treatment with either PARI or PAR2 antagonists suppressed 10 the proliferative effect of APC (data not shown) as well as the effects on matrix mineral (Fig 2B). These data are consistent with PARI/2 being the canonical signalling pathways for APC.

Example 3 APC promotes ectopic bone formation in mice

A critical proof-of-principle that APC would be a beneficial bone healing drug was to test it in an in vivo bone formation model. We utilized a mouse ectopic bone formation model where

15 10μg rhBMP-2 is implanted into a hind limb muscle pouch in C57BL5 mice and bone forms over 3 weeks. Addition of 25μg APC significantly increased bone volume (BV) by +73% (Fig 3B, P<0.01) and increased tissue volume by +104% (Fig 3C, P<0.01) compared to controls as quantified by microCT (Skyscan 1174). Subsequent analysis was performed to better understand the underlying mechanism. It was hypothesized that APC was increasing bone anabolism rather

20 than suppressing catabolism, and consistent with this hypothesis we saw increased rather than decreased osteoclast number (-20% increase) by TRAP stain (Fig 3D, P<0.05). The increases in osteoclast number may suggest that APC treatment could show synergy with anti-resorptive treatments, such as bisphosphonates. Consistent with APC being a pro-angiogenic factor, significant increases in CD31+ cells were seen at 3 weeks (Fig 4E, P<0.01), in the groups co- 5 treated with both 10μg and 25μg APC.

Example 4 Synergy between APC and anti-catabolic treatments

Hypothesis & Approach: Based on a substantive body of evidence that an optimal bone healing response can be obtained by modulating the anabolic and catabolic responses we trial addition of a bisphosphonate to a rhBMP-2/APC formulation. Previous work has repeatedly demonstrated synergy between rhBMP-2 and bisphosphonates in ectopic bone formation as well as other more advanced models.

Moreover, our preliminary data above reveals an increase in osteoclast number with APC treatment in our BMP-induced ectopic bone assay that accompanied increases in bone anabolism. Thus, we hypothesise that the anabolic effects of APC may be attenuated by concomitant increases in resorption. We add the potent BP, Zoledronic acid (ZA), as an additional factor to control any excessive resorption.

Experimental design & methods: The ectopic bone formation model is performed as previously described with rhBMP-2 and APC. Ten μ rhBMP-2 (Medtronic) ± 50 μg APC (Lilly) is delivered by collagen sponge (Medtronic) and implanted into the hind limb of C57BL6 mice. Starting at d4 post-op, mice receive saline or 0.02 mg kg ZA twice weekly; this has been previously found by our group to be an effective dosing regimen. This design has 4 groups (rhBMP-2, rhBMP-2+APC, rhBMP-2+ZA, rhBMP-2+APC+ZA) with n=8 per group needed for statistical power total 32 mice).

For this and all other studies, animals receive anaesthesia during surgical procedures (ketamine 75mg/kg, xylazine 4mg/kg and/or isofluorane), pre- and/or post-operative analgesia as required (0.1 mg kg buprenorphine), and regular monitoring by trained animal staff. All animal experiments undergo review from the institutional animal ethics committee prior to being initiated. Animals are ordered from the Animal Resources Centre (ARC, Perth).

Outcomes & Statistics: Specimens are harvested at week 3 for XR, quantitation of bone volume and other parameters of microarchitecture by microCT (Skyscan 1174), and histological staining for osteoclasts (TRAP) and blood vessels (CD31+ IHC). All methods are reliable and established. For these animal studies (and other experiments described 4.2 and 4.3), group comparisons are performed using a Kruskal Wallis test, and post-hoc inter-group comparison by Mann- Whitney U test. Values of P<0.05 considered statistically significant.

Significance: These experiments confirm that APC can enhance osteoclastic bone resorption while increasing bone formation. This understanding allows exploration of the use of anticatabolic drugs, whether bisphosphonates or alternative emerging anti-resorptives, to work in synergy with APC and/or rhBMPs. In addition, combination with bisphosphonates could be translated to other conditions such as osteonecrosis of the hip, where bone resorption is associated with collapse of the hip and poor outcomes.

Example 5 Sheep study using the combined treatment formulation

Hypothesis & Approach: We upscale to a size relevant to human translation in our final experiments and utilise old adult Merino sheep. We hypothesize that our combined treatment will repair a 30mm diameter femoral defect in 12 wks and lead not only to full regeneration but complete remodelling of the defect (e.g 100% of torsional strength compared to non-operated contralateral tibia of the sheep).

Experimental design & methods: A 30mm defect is created in the tibia of sheep and held by a 10-hole Synthes LCP locking plate and 6 screws, with the plate placed dorsolaterally. Similar models have been published, and despite non-union in controls, plate fixation was able to maintain position over 1 year. We use the optimal treatment determined from above experiments (APC + rBMP-2 (2 mg) ± ZA) and upscale to 800 μg APC, which is the high dose currently used topically to treat human chronic wounds, and 2mg rBMP_2. There is 2 study groups at each of two timepoints (3 months and 12 months), each requiring 8 sheep (i) control group with sponge alone, (ii) optimised treatment group, for a total of 32 sheep. Two positive control groups, namely autograft and scaffold-BMP, as well as negative group (empty defect) have been already operated and analysed.

Outcomes: Radiographs are performed at 3 months and 12 month, at harvest. Tibia are scanned with a Stratec XCT Research SA+ QCT scanner (Stratec, Germany), which provides volumetric bone mineral density (vBMD), cortical thickness and cross-sectional area of newly formed bone. Three-dimensional reconstructions are made using GTVol software (Skyscan, Belgium). Bone histology and histomorphometry are used to measure to examine for bone, cartilage and fibrous tissue at the implant site. Significance: This work will demonstrate the utility of APC in a large animal model, which will help guide future clinical trials. Example 6 -PAR-1 and PAR-2 Knock out mice studies

To further understand whether or not EPCR and PARs were implicated in the ectopic bone formation, we conducted histology and staining of bone pellets for EPCR, PARI and PAR2. Staining of EPCR was throughout the pellet, yet there was no difference between control and ^g or 25μg APC (11.4% decrease P=0.48, 2.2% decrease P=0.88 respectively).

PARI staining was also similar to EPCR in bone pellets and there was no significant difference between control and APC treatment with 3.6% increase in 10μg treatment group (P=0.83) and 14.8% increase in 25μg treatment group (P=0.42). Similarly, PAR2 staining was also not significantly different between control and APC treatment with 4.8% decrease in ^g treatment group (P=0.74) and 15.3% increase in 25μg treatment group (P=0.18).

Claims

1. A method of inducing formation of bone in an individual including the step of

- providing an individual requiring bone formation,

- providing a therapeutically effective amount of APC or APC analogue to the individual, thereby inducing bone formation in the individual.

2. The method of claim 1 wherein the individual has a bone defect in which bone formation is required.

3. The method of claim 2 wherein the defect is a fracture or void.

4. The method of claim 3 wherein the defect is a fracture.

5. The method of claim 4 wherein the APC or APC analogue is administered directly to bone at the site of the fracture.

6. The method of claim 5 wherein the APC or APC analogue is administered directly to the bone callus.

7. The method of claim 6 wherein the APC or APC analogue is administered by injection.

8. The method of claim 6 wherein the APC or APC analogue is administered in the form of a gel, putty or paste.

9. The method of claim 8 wherein the APC or APC analogue is administered to a periosteal surface.

10. A method of any one of the preceding claims wherein the APC analogue is 3K3A

-APC.

11. A composition including:

- APC or APC analogue

- a bone morphogenetic protein (BMP).

12. The composition of claim 11 wherein the APC or APC analogue and BMP are provided in synergistically effective amounts.

13. The composition of claim 12 wherein the BMP is selected from the group consisting of: osteogenic protein- 1 (OP-1, also known as BMP-7), osteogenic protein-2 (OP-2, also known as BMP-8), osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A or CBMP- 2A), BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-5, BMP-6, BMP-9, BMP- 10, BMPl l, BMP-12, GDF3 (also known as Vgr2), GDF8, GDF9, GDF10, GDFl l, GDF12, BMP-13, BMP-14, BMP-15, GDF-5 (also known as CDMP-1 or MP52), GDF-6 (also known as CDMP-2), GDF-7 (also known as CDMP-3), NODAL, UNIVIN, SCREW, ADMP, and NEURAL.

14. The composition of claim 13 wherein the BMP is BMP-2.

15. The composition of claim 14 wherein the BMP -2 is human BMP-2.

16. The composition of claim 13 wherein the BMP and APC or APC analogue are presented in a ratio of BMP to APC of 5: 1 on a weight per weight basis.

17. The composition of any one of the preceding claims, further including a bone resorption compound.

18. The composition of claim 17 wherein the bone re-sorption compound is a bisphosphonate.

19. The composition of claim 18 wherein the bisphosphonate is zoledronic acid.

20. A kit including:

- APC - BMP further including written instructions for use of the kit in bone formation.

21. A kit according to claim 20 wherein the APC and BMP are provided in a form enabling APC to be administered before or after BMP is administered.

22. A kit according to claim 21 wherein the APC and BMP are provided in a form enabling APC to be co-administered with BMP.

23. A composition including: - APC - a compound for re-sorption of bone.

24. The composition of claim 23 wherein the APC and bone re-sorption compound are provided in amounts enabling anabolic and catabolic responses.

25. The composition of claim 24 wherein the compound for re-sorption of bone is a bisphosphonate.

26. The composition of claim 25 wherein the bisphosphonate is zoledronic acid.

27. A kit including -APC

- bone re-sorption compound further including written instructions for use of the kit in bone formation.

28. The kit of claim 27 wherein the APC and bone re-sorption compound are provided in a form enabling APC to administered before or after the bone re-sorption compound is administered.

29. The kit of claim 28 wherein the APC and bone re-sorption compound are provided in a form enabling APC to be co-administered with the bone re-sorption compound.