WO2007012841A2

WO2007012841A2 - Compositions comprising monobutyrin

Info

Publication number: WO2007012841A2
Application number: PCT/GB2006/002778
Authority: WO
Inventors: Andrew Harrison; Ed Margerrison; Robert Morgan
Original assignee: Smith & Nephew, Plc
Priority date: 2005-07-28
Filing date: 2006-07-25
Publication date: 2007-02-01
Also published as: WO2007012841A3; GB0515477D0

Abstract

The invention relates to compositions/medicaments, devices and methods for treating musculo-skeletal tissue. The pharmaceutical composition for promoting musculo-skeletal tissue repair, regeneration and/or formation comprises monobutyrin and/or derivatives, fragments and/or analogues thereof, and a second agent capable of promoting musculoskeletal tissue repair, regeneration and/or formation.

Description

COMPOSITION

This application claims the benefit of U.K. Provisional Application 0515477.8, filed July 28, 2005 titled "Composition" and the entire contents of which is hereby incorporated by reference.

The invention relates generally to the field of musculo-skeletal biology and is concerned with the provision of methods, pharmaceutical compositions/ medicaments and devices for promoting musculo-skeletal tissue regeneration, repair and formation which comprise such compositions.

BACKGROUND OF THE INVENTION

Bone

Bone healing is required in situations of traumatic injury (e.g. fracture), total joint arthroplasty and where bone has been compromised as a result of surgical procedure. In many situations, the healing either does not take place leading to, for example, a non union, or occurs at a sub optimal rate resulting in a loss of function. Additionally, there are many comorbidities known to impair bone healing including diabetes and osteoporosis. Certain drug therapies such as steroids are also known to impair bone repair when it is needed. It is also apparent that the functioning of many currently used orthopaedic implants is sub-optimal. An example is a cementless tibial tray used in total knee arthroplasty, wherein the bone ingrowth to allow fixation of the implant is often insufficient to allow joint stability.

Vertebrate bone, as a tissue providing mechanical support for the body, undergoes constant remodelling through the formation and resorption of bone mediated, it is widely thought, by the activities of osteoblasts and osteoclasts respectively. Bone remodelling comprises a complex and highly organised interaction between cells and the extracellular matrix (ECM). The remodelling process is, however, adaptive in response to requirements of growth or habitual activity. In a normal healthy adult skeleton, the rate of bone formation approximates with the rate of bone resorption, through a process known as remodelling. Bone resorption or formation is not, though, a generalised feature of the entire skeleton simultaneously but occurs in discrete sites which may be surrounded by areas of quiescent bone. Where resorption occurs excessively, several clinical problems can occur either at a specific locality or more extensively throughout the skeleton.

For example, osteoporosis is a disease that is characterised by abnormalities in the amount and architectural arrangement of bone tissue. Osteoporosis is a major clinical condition that can lead to fractures of bone following only minimal trauma. Osteoporosis results from a shift in the balance of bone resorption and formation towards resorption so that there is net bone loss. In addition to the distress to sufferers, the direct hospital costs of osteoporosis have been estimated, in the U.S. only, to approach $13 billion and in the UK to approach £750 million. The term Osteoporosis' in fact refers to a group of conditions that are associated with loss of bone tissue and an accompanying architectural abnormality that occurs in cancellous bone space. When the condition develops in post-menopausal women it is referred to as postmenopausal osteoporosis. Fractures occur commonly in the hip, spine and distal radius and are considered in many countries to be a major public health problem (Lindsay R (1993), Clinical Rheumatology Osteoporosis; V.7, No.3). While genetics, diet and life-style appear to be factors in the pathogenesis of the disease, loss of ovarian function is an important determinant, at least in postmenopausal osteoporosis.

One reason for the low bone formation in osteoporosis is a reduced number of active osteoblasts. Agents capable of increasing the number of these cells would therefore have utility in conditions characterised by low bone mass.

Other osteoporotic-associated disease states include steroid induced osteoporosis, idiopathic juvenile osteoporosis, and posttransplantation osteoporosis where bone resorption is a secondary indication of disorder.

In the disease known as Paget's disease, there is excessive osteoclastic resorption of bone which results in excessive osteoblastic bone formation leading to disorganised bone structure.

Long term bed rest or disability for reasons that may not necessarily be directly related to diseases of the bone can lead to bone loss and danger of fracture on remobilisation or rehabilitation.

In cancer, formation of primary and secondary tumours often cause resorption and/or formation and subsequent increased liability to fracture or loss of function.

Tumour-induced osteolysis may also lead to pathologically raised serum calcium levels, which are believed to increase significantly morbidity in cancer patients.

Several approaches have been taken to treat low bone mass which are based on the use of anti-resorptive agents such as bisphosphonates that reduce or inhibit bone loss but none of these approaches are entirely satisfactory since the subsequent increase in bone formation occurs slowly.

The use of bisphosphonates to inhibit bone resorption is also not ideal since the degree of side effects is regarded by some as unacceptably high and its use is not well tolerated by a significant proportion of the population.

Oestrogen and other hormone replacements have a history of use for postmenopausal osteoporosis, either alone or in combination with other therapeutics. However suggestions of an increased risk of endometrial and breast cancer, as well as the continuation of menstrual bleeding, which is often unwelcome in the elderly female section of the population who form the majority of sufferers of osteoporosis, has provided a need for an alternative approach.

Other treatments for osteoporosis employing agents which affect osteoclast function have been used e.g. calcitonin or parathyroid hormone but with limited success.

As well as diseases and conditions which affect the rate of bone regeneration, physical knocks and accidents may also cause bone fractures.

The rate of bone fractures in the United States alone, is estimated at 6 millions individuals per year.

The most well established method for bone repair is the mechanical one, and this typically involves hard implants and hardware, such as plates, pins and screws. Within the category of hard implants, there exist an array of plastics, organic-based synthetic cements and metal prostheses. There are two major considerations and concerns in using mechanical hardware and implants. The first relates to the effectiveness of the physiological integration of the hardware into the body systems, while the second is that of the long-term durability of the non-biological material which has been implanted. Despite these problems, mechanical implants are very popular, and, while not comprising living bone tissue, make significant contributions assisting in the bone reconstruction.

When a bone is completely fractured, a significant proportion of fractures require medical intervention beyond simple immobilisation

(casting). A major problem in such instances is the lack of proximity of the two bone ends. This results in an inappropriate and prolonged repair process, which may prevent recovery. The average length of time for the body to repair a fracture is 25 - 100 days, for moderate load-bearing, and one year for complete repair. Thus, both simple fractures and medically complicated breaks would benefit from novel therapeutic modalities which accelerate and/or complete the repair process. The same is true for those bone diseases (referred to as osteoporosis or osteopenias) which result in a thinning of the bone the primary symptom of which is an often-debilitating fracture.

Some examples of previous attempts using biological stimuli of bone regeneration include:

1. Autologous bone grafts. These grafts are harvested from the patient undergoing therapy. Their use is limited for two main reasons: firstly, material is present in only a finite amount, and secondly, the procedure of harvesting the material leads to significant morbidity.

2. Allogeneic bone sources. These are widely used in orthopaedic surgery and come in a variety of forms (including demineralised bone matrix). Concern has been raised in the use of such material owing to, for example, the potential for disease transmission, and also the efficacy of the material following processing which may include, for example, freeze drying which is believed to reduce the potential for host bone integration.

3. Synthetic bone grafts. These materials generally do not suffer from the limitations described above, although their use has been limited by their inability to overcome a biological impairment to bone healing. In this capacity, they represent a largely inert scaffold over/ through which bone can regenerate in certain limited indications.

4. Other osteogenic stimuli. Again, there are a variety of osteogenic stimuli known in the art. The best known and widely researched stimuli are the bone morphogenic proteins (BMPs) which have received limited clinical success for two main reasons: firstly, BMPs are known to lead to the differentiation of precursor cells into osteoblastic cells, and in many situations, this step may not be the rate limiting step of repair. Secondly, their limited clinical efficacy has been at the expense of using super-physiological doses, and the safety profile of these agents has been called into question.

There is therefore a need for osteogenic stimuli which can overcome specific biological obstacles to bone healing, without the potential for associated morbidity of autograft, lack of efficacy of allograft and synthetic scaffolds, and potential super-physiological doses of other osteogenic stimuli.

Cartilage

Cartilage has a limited capacity for self repair. The cartilage of the body can be damaged by physical knocks. Damaged cartilage is prone to further degeneration, i.e. osteoarthritis.

The disease osteoarthritis (OA) which is characterised by the destruction of articular cartilage can also occur without any minor injury. It affects at least 16 million Americans and is symptomatic in 80% of the population over 75 years of age. With an ageing population its relevance is increasing and becoming more of a burden on healthcare services.

A major constituent of cartilage is collagen.

Collagen is one of the most abundant animal proteins in nature. It is present in all types of multicellular animals, including humans, where it is estimated to account for about 30% of the total human body protein. Collagen constitutes the fibrillar component of the soft connective tissues (e.g., skin, ligament, and tendon) and is the major component of the organic matrix of calcified tissues such as bone and dentine. In addition to its structural significance, collagen plays an important role in development and wound healing, and has been implicated in ageing and some disease processes.

There are several genetically distinct types of collagen, which are referred to as types I, II, III, and so forth. Type Il collagen is the major collagen of cartilage. It is synthesised by chondrocytes as a procollagen molecule with noncollagenous aminopropeptide and carboxypeptide extensions. These two extensions are removed by specific peptideases before type Il collagen is incorporated into fibrils. By the term cartilage we mean any cartilage of the animal or human body including but not limited to: articular, hyaline, meniscal and yellow-elastic cartilage.

Current and proposed cartilage regeneration therapies such as mosaic plasty, autologous chondrocyte implantation and tissue engineering. have the associated problems as discussed above for bone regeneration therapies.

There is therefore a need for chondrogenic stimuli which can overcome specific biological obstacles to cartilage healing, without the potential for associated morbidity of autograft, lack of efficacy of allograft and synthetic scaffolds, and potential super-physiological doses of other chondrogenic stimuli.

It is a feature of the invention to provide compositions for promoting musculo-skeletal tissue (such as bone and cartilage) repair, regeneration and formation.

It is a feature of the invention to provide a method for promoting musculo-skeletal tissue repair, regeneration and formation.

It is a feature of the invention of the invention to provide a scaffold to aid musculo-skeletal tissue repair, regeneration and formation.

Surprisingly we have found that monobutyrin acts as a stimulus for promoting musculo-skeletal tissue repair, regeneration and formation.

Monobutyrin has surprisingly been found to be osteogenic. Monobutyrin has surprisingly been found to be chondrogenic.

Monobutyrin is also referred to by those skilled in the art by the following, non-exhaustive list of synonyms:

EINECS 209-165-5, Glycerol-alpha-mono-n-butyrate, NSC 8451 , alpha-Monobutyrin, 2,3-Dihydroxypropyl butyrate, Butanoic acid, 2,3-dihydroxypropyl ester.

Monobutyrin exists in three isomeric forms, having the chemical structures as illustrated in Figures 1-3. Monobutyrin spontaneously isomerises into these forms. For example at ambient room temperature monobutyrin isomerises to approximately 45%:10%:45% of R-l-monobutyrin: 2- monobutyrin:S-1- monobutyrin

Monobutyrin has been previously described as being pro- angiogenic, and has been used for the treatment of tumours and some other disorders. We have surprisingly found that the molecule has a direct osteogenic effect (on MG-63 osteoblast-like cells and primary osteoblasts) in the absence of any angiogenic effect and therefore any effects seen on bone in vivo are due to a previously unknown mechanism of action.

BRIEF SUMMARY OF THE INVENTION

According to an aspect of the invention there is provided a pharmaceutical composition for promoting musculo-skeletal tissue repair, regeneration and/or formation, the composition comprising;

(i) monobutyrin and/or derivatives, fragments and/or analogues thereof, (ii) a second agent capable of promoting musculoskeletal tissue repair, regeneration and/or formation and;

(iii) a pharmaceutically acceptable carrier.

In this application the term "pharmaceutical composition" and "medicament" are to be taken as equivalent meaning.

The active compound monobutyrin is an ester composed of glycerol and butyric acid, and can be made simply in a laboratory setting by known means to the skilled person.

Embodiments of the invention include where the musculoskeletal tissue is bone, cartilage, synovium, muscle, tendon, ligament or meniscus.

It is envisaged that the present invention can be used to treat bone repair, or induce bone growth without a large concentration of monobutyrin. The concentration of monobutyrin in the composition is preferably from about 100μg/ml to 5mg/ml and even more preferably from about 10μg/ml to 100μg/ml.

It is envisaged that the present invention can be used to treat cartilage repair, or induce cartilage growth without a large concentration of monobutyrin. The concentration of monobutyrin in the composition is preferably from about 100μg/ml to 5mg/ml and even more preferably from about 10μg/ml to 100μg/ml.

Embodiments of the invention include where the composition stimulates osteogenesis in cells selected from the group consisting of embryonic stem cells, adult stem cells, osteoblastic cells, preosteoblastic cells and skeletal progenitor cells derived from bone, bone marrow or blood.

Embodiments of the invention include where the composition stimulates chondrogenesis in cells selected from the group consisting of embryonic stem cells, adult stem cells, chondrocytes, fibroblasts and skeletal progenitor cells derived from bone, bone marrow or blood.

The tissue to be repaired, formed or regenerated may be in vivo or in vitro.

In embodiments of the invention the second agent is a bone anabolic agent.

In further embodiments of the invention the second agent is selected from the group consisting of bone morphogenetic factors, cartilage derived morphological protein, epidermal growth factor, fibroblast growth factor, platelet derived growth factor, parathyroid hormone, insulin-like growth factor, sodium fluoride, bisphosphonates, calcium carbonate, prostaglandins, vitamin D, vitamin K, oestrogen and mixtures thereof.

Embodiments of the invention include where the second agent and the pharmaceutically acceptable carrier are the same. For example, where calcium phosphate is the second agent and the pharmaceutically acceptable carrier.

Although it is envisaged that this invention will benefit bone fracture repair (such as, non-union fractures, osteoporotic fractures) it may be used to treat other clinical conditions and diseases where the tissue healing response has been impaired by physiological abnormalities or injury.

Clinical conditions and diseases characterised by musculo- skeletal tissue loss which may benefit from this invention include, but are not restricted to; osteoporosis, arthritis, diabetes, cancer, Paget's disease, Schϋllers disease, renal bone dystrophy, brachypodism,

Hunter-Thompson chondrodysplasia, a spinal deformation, bone dysplasia, scoliosis, peridonatal disease, osteomalacia or fibrous osteitis,

Embodiments of the invention include where osteoporosis is steroid-induced osteoporosis, idiopathic juvenile osteoporosis, postmenopausal osteoporosis, post-traumatic osteoporosis, senile osteoporosis.

Embodiments of the invention include where the arthritis is osteoarthritis or rheumatoid arthritis.

Embodiments of the invention include where the cancer is osteosarcoma or myeloma.

Embodiments of the present invention include where bone loss is induced by steroid use.

Pharmaceutical compositions of the invention can be prepared according to methods well known and called for by accepted pharmaceutical practice.

Pharmaceutical compositions suitably comprise the composition of the invention together with a pharmaceutically acceptable carrier and are suitably in unit dosage form. Pharmaceutical compositions of the invention are suitable for administration either locally or systemically.

Pharmaceutical compositions of the invention are suitable for administration via the oral, parenteral, topical or intravenous routes.

Embodiments of the invention include where the pharmaceutical compositions are delivered from both aqueous and solid media.

Formulations will include dip coating of solid substrates with the pharmaceutical composition, mixing the pharmaceutical composition with aqueous solutions, powder coating and integration of the pharmaceutical composition into resorbable substrates among others.

Embodiments of the invention include where the pharmaceutical composition additionally comprises excipients, preservatives, solubilisers, buffering agents, albumin, lubricants, fillers, stabilisers and mixtures thereof.

It is envisaged that the composition is formulated in a form selected from the group consisting of a liquid solution, liquid emulsion, liquid suspension, coated capsules, pills, tablets, suppositories, lyophilized powders, transdermal patches, gels, ointments, lotions, creams and sprays.

In preferred embodiments of the invention the composition is provided as a liquid solution, emulsion or suspension encapsulated within a biodegradable vesicle selected from liposomes, microspheres and nanospheres, thereby enabling temporally- controlled release of the composition. Alternatively the composition is formulated in a biodegradable film, biodegradable coating or biodegradable matrix. Suitable pharmaceutical carriers include, for example, but are not restricted to, synthetic bone grafts (such as ceramic calcium phosphates), allogeneic bone grafts (such as demineralised bone matrix), autogenic bone grafts, gels (e.g. Hyaluranon), both injectable and non-injectable forms, synthetic polymers (such as polyhydroxyacids) and natural polymers (such as collagen), surgical fixation means (such as surgical screws, surgical pins, surgical rods or surgical plates).

The composition can be mixed with or onto the surface of an appropriate carrier, or could be covalently linked to a carrier to allow specific release kinetics into the site of action.

In embodiments of the invention monobutyrin and/or the second agent will be released from carrier over a time period that spans a minimum of the first 3 days of fracture up to 14 days post fracture.

The first 24-48 hours of fracture repair involve harsh conditions of tissue breakdown and turnover to prepare the fracture site. These conditions are likely to catabolise any active agent present at this time. By releasing monobutyrin at beyond 3 days post fracture, where there is a proliferation and differentiation of the fracture repair cell types, proliferation and differentiation will be enhanced during the early stages of fracture repair.

For example, compositions comprising monobutyrin can be formulated into/onto a number of different carriers, including:

• Ceramics; calcium sulphate powder can be spray dried with a composition comprising monobutyrin, and the resultant ceramic can be dry powder compacted into an appropriate shape for implantation. The compositions comprising monobutyrin can also been adsorbed onto the surface of tricalcium phosphate ceramics for subsequent implantation.

• Hydrogels: compositions comprising monobutyrin can be admixed to hyaluronic acid to provide an injectable formulation.

• Polymers: compositions comprising monobutyrin can be admixed into a polymer with an appropriate solvent, or could be covalently linked to a polymer to allow sustained/ delayed release of the active ingredient.

• Others: compositions comprising monobutyrin can be admixed with saline or other similar liquids/ solutions.

In further embodiments of the invention the monobutyrin and/or the second agent is coupled to a "bone-seeking" substance such as a tetracycline or bisphosphonates to improve target specificity as known by those skilled in the art.

Function manipulating agents of the invention may be manufactured according to any appropriate method of choice.

Pharmaceutical compositions of the invention can comprise a composition of the present invention in the form of a pro-drug which can be metabolically converted to the active form of the invention agent by the recipient host.

Pharmaceutical compositions of the present invention may also be used in conjunction, e.g. simultaneously, sequentially or separately with other therapies. According to a further aspect of the invention there is provided an implantable orthopaedic device comprising a composition comprising monobutyrin and/or derivatives, fragments and/or analogues thereof.

Embodiments of the invention include, for example where the implantable orthopaedic device is a prosthetic implant (such as an endoprosthesis e.g a hip implant), surgical screw (such as a bone screw), surgical nail (such as a intramedullary nail), surgical pin (such as a cartilage anchor pin), surgical rod, surgical plate, bone filler, autologous tissue graft, allogenic tissue graft or a scaffold.

Further embodiments of the invention include where the composition further comprises a second agent capable of promoting musculoskeletal tissue repair, regeneration and/or formation.

The second agent capable of promoting musculoskeletal tissue repair, regeneration and/or formation is preferably selected from the group consisting of bone morphogenetic factors, cartilage derived morphological protein, epidermal growth factor, fibroblast growth factor, platelet derived growth factor, parathyroid hormone, insulin-like growth factor, sodium fluoride, bisphosphonates, calcium carbonate, prostaglandins, vitamin D, vitamin K, oestrogen and mixtures thereof.

In embodiments of the invention the composition is applied to a surface of the device, for example a bone-contacting surface or a cartilage-contacting surface.

Aptly the pharmaceutical composition of the invention is present as a layer, for example as a coating on a surface (e.g the bone- contacting surface or cartilage-contacting surface) of the device. Suitably, medical devices according to the invention may be prepared by absorbing the composition onto, for example, the titanium oxide or other surface of a metallic surface or of a polymer surface, e.g. bone screw, by incorporating the composition into a carrier material and coating the carrier onto the medical device.

In an embodiment of this aspect of the invention, the pharmaceutical composition can be powder coated onto calcium sulphate powder and compacted into bone graft substitute materials, for example JAX™ (Smith & Nephew Inc, Community Trade Mark No. E2052611) to allow delivery of the composition into bone voids. Calcium phosphate can also be dipped coated into the pharmaceutical composition to achieve the same end.

In an embodiment of this aspect of the invention, the bone- contacting surface or the cartilage-contacting surface) has been 'derivatised' or modified such that the composition of the invention is directly bonded, aptly by covalent bonds, to the surface.

In further embodiments of the invention the composition is impregnated within the device.

In further embodiments of the invention there is provided an artificial scaffold material for promoting musculo-skeletal tissue formation, the scaffold having operatively coupled thereto the composition of the invention.

In further embodiments of the invention there is provided an artificial scaffold material for promoting bone formation, the scaffold having operatively coupled thereto the composition of the present invention. In further embodiments of the invention there is provided an artificial scaffold material for promoting cartilage formation, the scaffold having operatively coupled thereto the composition of the present invention.

The scaffold of the invention may in the form of a three dimensional matrix or layer, for example, a continuous film, or gel. The matrix structure may be manufactured from fibres or a suitable material which is then textile processed (e.g. braided, knitted, woven or non-woven, melt-blown, felted, hydro-entangled) and further manipulated into a desired three dimensional shape. The matrix structure may also assume other forms, e.g. sponges or foams.

Suitable scaffold materials are preferably biodegradable/resorbable and are not inhibitory to cell growth or proliferation. Typically the materials should not elicit an adverse reaction from the patients' body and should be capable of sterilisation by for example ethylene oxide treatment.

In embodiments of the invention typically the material is osteoconductive.

Suitable materials therefore include, for example, biodegradable polyesters such as polylactic acid (PLA), polyglycolic acid (PGA), polydioxanone, polyhydroxyalkanoates, e.g. poly hydroxbuty rate (ICI) and hyaluronic acid derivatives, e.g. HYAFF (Fidia), hydrophilic polyurethanes, polyetherpolyester, polyethylene oxide, polyetherpolyamide, carboxymethylcellulose, ethylene-vinyl acetate copolymers, polybutadiene, styrene-butadiene-styrene block copolymers and the like. Other scaffold materials are collagen based e.g. cross-linked collagen/elastin material, cross-linked collagens manufactured from acid-soluble type I bovine collagen sources, collagen gels, (for example those sold under the trade names COLLASTAT and COLETICA). Collagen from natural or recombinant sources may be used.

Modified or chimeric recombinant fibrillar collagens (herein "modified collagen") are also provided which incorporate a composition from the present invention and features that promote its assembly, stability and use as a biomaterial. The modified collagen may be used as a scaffold material described supra. Approaches include use of the C-terminal globular domain from type I collagen to promote triple helix formation; the removal or alteration of the collagenase cleavage site to suppress degradation; the inclusion of additional lysines to promote cross-linking and the alternation of N- terminal globular domain cleavage site to promote the retention of the N-terminal domain in the mature fibre. For example, the chordin/SOG sequence of collagen Ha could be substituted for the protein/polypeptide function manipulating agent. Analogous domain shuffling approaches may be used to incorporate a composition of the present invention into other extracellular matrix components (e.g. fibronectin link protein or collagen IV) or ECM binding molecules or sequences (e.g. heparin binding domains). See, for example, WO 97/08311, the entire content of which are incorporated herein by reference.

In other specific embodiments, there is provided a bone substitute material, for example a bone void filler, comprising a composite material comprising any one of the above scaffold materials and a crystalline phase (e.g. an apatite such as hydroxyapatite) incorporating the composition of the invention. The crystalline phase is an osteoinductive or osteoinductive phase.

In other specific embodiments, there is provided a cartilage substitute material, comprising a composite material comprising any one of the above scaffold materials and a crystalline phase (e.g. an apatite such as hydroxyapatite) incorporating the composition of the invention.

In other specific embodiments of the invention the composition of the present invention is delivered as a scaffold in the form of a gel. Typically the gel will comprise thrombin, fibrinogen and Factor XIII or another transglutaminase to cross-link the gel.

Suitably the composition is bound to a solid matrix and implanted to the desired orthopaedic site.

The composition of the present invention, preferably bound to a solid matrix, has the advantage over the prior art that excess monobutyrin produced naturally in the body are not wasted. Excess monobutyrin may be utilised or excreted from the body.

Listed below are some of the advantages that the invention has over current bone grafts:

• Autologous bone grafts. No donor site morbidity is associated with the use of monobutyrin.

• Allogeneic bone sources: As a synthetic material, there is no potential for disease transmission • Synthetic bone grafts: We have demonstrated in a critical sized segmental defect that the bone repair is enhanced over and above that which can be attained with the use of a synthetic void filler. Specifically, we can demonstrate improved cortical bridging, a greater linear bone growth rate, and a higher bone density.

• Other osteogenic stimuli: Monobutyrin in our uses is applied at extremely low concentrations (0.03% w/w) compared with e.g. BMPs (typically dosed at mg/ml levels). Two key advantages here are that the cost of such treatment will be significantly lower (there is no need to produce a recombinant product), and that we are dosing at near physiological levels and the associated toxicological profile will be significantly better.

Another advantage of this therapy is that it takes advantage of a dual mode of action: the direct osteogenic effect of monobutyrin has been described above, but it also has a known angiogenic effect which will assist in the repair of bone tissue where there is vascular compromise for example in diabetes.

Bone may form through a process of endochondral ossification through which cartilage is laid down first and is then mineralised. In this way bone forms through cartilage formation and therefore any treatment that is found to heal bone can be presumed to stimulate cartilage formation and it can also be assumed that the converse is true.

It is envisaged that the present invention can be used to treat cartilage repair, or induce cartilage growth without a large concentration of the growth factor being needed. Using large concentrations of growth factors has been a problem to date as this suffers from the disadvantage that large concentration of the growth factor as noted above, can cause a shift in biological equilibrium possibly making the growth factor less potent.

According to a further aspect of the invention there is provided a composition comprising monobutyrin or derivatives, fragments and/or analogues thereof and a second agent capable of promoting musculo-skeletal tissue repair, regeneration and/or formation for use as a medicament.

Embodiments of the invention include where the musculo- skeletal tissue is bone, cartilage, synovium, muscle, tendon, ligament or meniscus.

Embodiments of the invention include where the second agent is selected from the group consisting of bone morphogenetic factors, cartilage derived morphological protein, epidermal growth factor, fibroblast growth factor, platelet derived growth factor, parathyroid hormone, insulin-like growth factor, sodium fluoride, bisphosphonates, calcium carbonate, prostaglandins, vitamin D, vitamin K, oestrogen and mixtures thereof.

According to a further aspect of the invention there is provided a method to promote musculoskeletal tissue repair, regeneration and/or formation in a subject, the method comprising the step of administering to the subject a composition comprising monobutyrin and/or derivatives, fragments and/or analogues thereof.

According to a still further aspect of the invention there is provided the use of monobutyrin or derivatives, fragments and/or analogues thereof in the manufacture of a medicament for the treatment of diseases or clinical conditions that may be alleviated by the promotion of muscle-skeletal tissue repair, regeneration and/or formation.

Although it is envisaged that this invention will benefit bone fracture repair (such as, non-union fractures, delayed fractures, mal- union fractures or osteoporotic fractures) it may be used to treat other clinical conditions and diseases where the tissue healing response has been impaired by physiological abnormalities or injury.

Clinical conditions and diseases characterised by musculoskeletal tissue loss which may benefit from this invention include, but are not restricted to; osteoporosis, arthritis, diabetes, cancer, Paget's disease, Schϋllers disease, renal bone dystrophy, brachypodism, Hunter-Thompson chondrodysplasia, a spinal deformation, bone dysplasia, scoliosis, peridonatal disease, osteomalacia or fibrous osteitis,

Embodiments of the invention include where osteoporosis is steroid-induced osteoporosis, idiopathic juvenile osteoporosis, post- menopausal osteoporosis, post-traumatic osteoporosis, senile osteoporosis.

According to further aspects of the invention there are provided animal models useful in the investigation of musculo-skeletal tissue disorders. For example, the role of monobutyrin in the skeletal system may be investigated using non-human mammalian, e.g. mouse.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The invention will now be described by way of example only with reference to the following examples, tables and drawings:

Figure 1 shows a chemical structure of monobutyrin - the R-I- monobutyrin (chemical structure 1).

Figure 2 shows a chemical structure of monobutyrin - the 2- monobutyrin (chemical structure 2).

Figure 3 shows a chemical structure of monobutyrin - the S-1- monobutyrin (chemical structure 3). Figure 4 shows a significant increase in alkaline phosphatase expression at day 3 only in primary human osteoblasts stimulated with 500 and 100O u g/ml monobutyrin.

Figure 5 illustrates the bone graft substitute JAX™.

DETAILED DESCRIPTION OF THE INVENTION

Example 1 : ΛΛG-63 cells (osteoblast-like cells)

MG-63 cells were cultured in Eagle's minimal essential media with 10% foetal calf serum at 37⁰C and 5% CO₂. For experimental purposes cells were seeded at 5x10³ cells per well in 96 well plates. All cells were cultured with ascorbic acid (50μg/ml) as standard. Cells were stimulated with either monobutyrin or BMP-2. Monobutyrin was dosed at 0.1%, 0.05% or 0.01 %v/v, either once at day 1 or daily for up to 5 days. BMP was dosed once only on day 0 at 100ng/well. Cells were lysed at 1 , 3 and 5 days after the first dose and assayed for alkaline phosphatase using a standard 96 well format p-nitrophenyl phosphate (pNPP) assay. Briefly, cells were lysed in 200μl of 0.2M carbonate buffer containing 0.1% Triton X-100 and freeze thawed three times. 50μl of each cell lysate was added to 50μl of pNPP working solution (40mg pNPP in 9ml 0.2M carbonate buffer pH10.2, 1ml 10OmM MgCI₂ , 20ml H₂O). Colorimetric changes (absorbance), reflecting the level of alkaline phosphatase activity in each sample, were assessed at 405nm using a Multiskan plate reader. Absorbance levels were compared to a standard curve of p-nitrophenol (pNP) concentrations to allow quantitation of alkaline phosphatase activity.

Cells showed a significant increase in alkaline phosphatase expression at 3 and 5 days at all concentrations of monobutyrin used (Details are in Table 1). 0.01% monobutyrin was less effective than the other two concentrations. These data show that monobutyrin has a direct osteogenic effect.

Table 1

[MB] AA Control BMP control Doses # Days Alkaline Fold over Fold (nmol/ml/μg) (nmol/ml/μg) Culture Phosphatase AA over d (nmol/ml/μg) BMP

0.10% 0.13+/- 0.11 0+/-0 1 1 1.24+/- 0.26 9.5 N/A

0.05% 0.13+/- 0.11 0+/-0 1 1 0.77 +/- 0.39 5.9 N/A

0.01% 0.13+/- 0.11 0+/-0 1 1 0.54 +/- 0.2 4.2 N/A

0.10% 1.1 +/-0.23 0.48+/- 0.13 1 3 15.33+/- 1.92 13.9 31.9

0.05% 1.1 +/-0.23 0.48+/- 0.13 1 3 20.6 +/- 3.46 18.7 42.9

0.01% 1.1 +/-0.23 0.48+/- 0.13 1 3 14.49 +/- 3.83 13.2 30.2

0.10% 1.04+/- 0.33 1.31 +/-0.22 1 5 11.81 +/-2.61 11.4 9.0

0.05% 1.04+/- 0.33 1.31 +/-0.22 1 5 6.32+/- 1.56 6.1 4.8

0.01% 1.04+/- 0.33 1.31 +/-0.22 1 5 3.69+/- 1.01 3.5 2.8

0.10% 5.41 +/-1.35 4.19+/- 1.1 3 3 44.25 +/- 5.38 8.2 10.6

0.05% 5.41 +/-1.35 4.19+/- 1.1 3 3 52.69 +/-19.61 9.7 12.6

0.01% 5.41 +/-1.35 4.19+/- 1.1 3 3 19.94+/- 4.06 3.7 4.8

0.10% 1.47+/- 0.24 0.48 +/- 0.27 5 5 16.1 +/-3.78 11.0 33.5

0.05% 1.47+/- 0.24 0.48 +/- 0.27 5 5 10.04 +/-2.46 6.8 20.9

0.01% 1.47+/- 0.24 0.48 +/- 0.27 5 5 3.12+/- 0.78 2.1 6.5

10 Example 2: Primary human osteoblasts

Primary human osteoblasts were obtained from TCS Cellworks. Cells were cultured in Dulbeccos modified essential media with 10% foetal calf serum, 50 g/ml ascorbic acid and 1x10^"8 M

15 dexamethasone as standard at 37°C and 5% CO₂. For experimental purposes cells were seeded into 96 well plates at 5x10³ cells per well. Cells were stimulated once with a dose range of monobutyrin (100, 500 and 1000 g/ml) or BMP-2 at 1 g/ml. Cells were subsequently cultured for 3, 7 or 9 days further before lysis for assaying for alkaline

20 phosphatase as described above. Alkaline phosphatase levels were assayed for as described above. Analysis showed a significant increase in alkaline phosphatase expression at day 3 only in those cells stimulated with 500 and 100O u g/ml monobutyrin (as shown in the Figure 4).

Example 3: Rabbit Segmental Defect

Monobutyrin has been formulated onto both calcium sulphate Jax™ and collagen sponge for in-vivo delivery purposes. Calcium sulphate was dry spray powder coated with monobutyrin before pressing into Jax™. Collagen sponge was soaked in an aqueous solution of monobutyrin. A known dose range of monobutyrin was used in both studies.

A critical size (15mm) segmental defect was performed in the rabbit ulna and packed with either the monobutyrin formulated JAX™ granules or the monobutyrin formulated collagen sponge. Carrier only acted as negative controls. Animals were returned to normal load bearing and assessment of bone filling was performed at 12 weeks.

Both formulations showed a statistically significant difference in bone fill between one of the doses of monobutyrin and the negative controls. The scoring system and data from the collagen sponge formulation is shown below.

Radiograph Scoring Criteria

1. Growth from proximal and distal ends of the ulna: • No growth 0

• Growth from one end only 2

• Growth from both ends 4

• Growth from both ends and that meet 6

2. Defect fill by new bone:

• No fill 0

• >0% and <25% fill 1

• >25% and <50% fill 2 • >50% and 75% fill 3

• >75% and up to 100% fill 4

3. Scorers week blinded and scored the 12-week X-ray from each animal in each treatment group based upon the above criteria. The two scores were added together to give a final score out of 10 for each X-ray. An average (mean) score for each animal from the 3 scorers in each treatment group was calculated +/- SD shown in the tables below.

Table 2: Individual scores for each animal from each scorer

A mean of the mean scores above was then calculated, as shown in Table 3:

Table 3. Comparison of mean scores for the negative control and monobutyrin treatment

Multiple Comparisons and Tukey-Kramer based analyses showed that the difference between the two groups shown in table 1 was significant (p<0.05). It is therefore concluded that monobutyrin is able to enhance bone healing in the rabbit ulna segmental defect.

In view of the foregoing, it will be seen that the several advantages of the invention are achieved and attained.

The embodiments were chosen and described in order to best explain the principles of the invention and its practical application to thereby enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated.

As various modifications could be made in the constructions and methods herein described and illustrated without departing from the scope of the invention, it is intended that all matter contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative rather than limiting. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims appended hereto and their equivalents.

Figure 5 illustrates the bone graft substitute JAX™, which is an example of an implantable orthopaedic device according to the present invention. A bone graft is a procedure whereby material is placed in a bone void to provide an environment for and/or induce new bone growth.

Claims

1. A pharmaceutical composition for promoting musculoskeletal tissue repair, regeneration and/or formation, the composition comprising; i. monobutyrin and/or derivatives, fragments and/or analogues thereof, ii. a second agent capable of promoting musculoskeletal tissue repair, regeneration and/or formation and; iii. a pharmaceutically acceptable carrier.

2. A pharmaceutical composition according to claim 1 , wherein the musculoskeletal tissue is bone, cartilage, synovium, muscle, tendon, ligament or meniscus.

3. A pharmaceutical composition according to claim 1 or 2, wherein the second agent is selected from the group consisting of bone morphogenetic factors, cartilage derived morphological protein, epidermal growth factor, fibroblast growth factor, platelet derived growth factor, parathyroid hormone, insulin-like growth factor, sodium fluoride, bisphosphonates, calcium carbonate, prostaglandins, vitamin D, vitamin K, oestrogen and mixtures thereof.

4. A pharmaceutical composition according to any of claims 1 to 3, wherein the composition stimulates osteogenesis in cells selected from the group consisting of embryonic stem cells, adult stem cells, osteoblastic cells, preosteoblastic cells and skeletal progenitor cells derived from bone, bone marrow or blood.

5. A pharmaceutical composition according to any of claims 1 to 6, wherein the concentration of monobutyrin is from about 100μg/ml to 5mg/ml.

6. A pharmaceutical composition according to claim 5, wherein the concentration of monobutyrin is from about 10μg/ml to 100μg/ml.

7. A pharmaceutical composition according to any of claim 1 to 6, wherein the composition additionally comprises excipients, preservatives, solubilisers, buffering agents, albumin, lubricants, fillers, stabilisers and mixtures thereof.

8. A pharmaceutical composition according to any of claim 1 to 7, wherein the composition is formulated in a form selected from the group consisting of a liquid solution, liquid emulsion, liquid suspension, coated capsules, pills, tablets, suppositories, lyophilized powders, transdermal patches, lotions and creams.

9. A pharmaceutical composition according to claim 8, wherein the composition is provided as a liquid solution, emulsion or suspension encapsulated within a vesicle selected from liposomes, microspheres and nanospheres.

10. A pharmaceutical composition according any of claims 1 to 9, wherein the composition is formulated in a controlled release form selected from a biodegradable vesicle, biodegradable film, biodegradable coating or biodegradable matrix.

11. A pharmaceutical composition according to claim 10, wherein the film, coating or matrix is applied to a surface of an implantable orthopaedic device.

12. A pharmaceutical composition according to claim 11 , wherein the device is a prosthetic implant.

13. A pharmaceutical composition according to claim 10, wherein the device is a surgical fixation means,

14. A pharmaceutical composition according to claim 10, wherein the device is a bone filler.

15. A pharmaceutical composition according to claim 10, wherein the device is autologous or allogenic graft tissue.

16. A pharmaceutical composition according to claim 10, wherein the device is a scaffold.

17. An implantable orthopaedic device comprising a composition comprising monobutyrin and/or derivatives, fragments and/or analogues thereof.

18. An implantable orthopaedic device according to claim 17, wherein the device is a prosthetic implant, surgical screw, surgical pin, surgical nail, surgical rod, surgical plate, bone filler, autologous tissue graft, allogenic tissue graft or a scaffold.

19. An implantable orthopaedic device according to claim 17 or 18, wherein the composition further comprises a second agent capable of promoting musculo-skeletal tissue repair, regeneration and/or formation.

20. An implantable orthopaedic device according to claim 19, wherein the second agent is selected from the group consisting of bone morphogenetic factors, cartilage derived morphological protein, epidermal growth factor, fibroblast growth factor, platelet derived growth factor, parathyroid hormone, insulin-like growth factor, sodium fluoride, bisphosphonates, calcium carbonate, prostaglandins, vitamin

D, vitamin K, oestrogen and mixtures thereof.

21. An implantable orthopaedic device according to any of claims 17 to 20, wherein the composition is applied to a surface of the device.

22. An implantable orthopaedic device according to any of claims 17 to 20, wherein the composition is impregnated within the device.

23. A method to promote musculo-skeletal tissue repair, regeneration and/or formation in a subject, the method comprising the step of administering to the subject a composition comprising monobutyrin and/or derivatives, fragments and/or analogues thereof.

24. A method according to claim 23, wherein the tissue is bone, cartilage, synovium, muscle, tendon, ligament or meniscus.

25. A method according to claim 23 or 24, wherein the subject has osteoporosis, arthritis, diabetes, cancer, Paget's disease, Schϋllers disease, renal bone dystrophy, brachypodism, Hunter-Thompson chondrodysplasia, a spinal deformation, bone dysplasia, scoliosis, peridonatal disease, osteomalacia or fibrous osteitis,

26. A method according to claim 25, wherein the osteoporosis is steroid-induced osteoporosis, idiopathic juvenile osteoporosis, post-menopausal osteoporosis, post-traumatic osteoporosis, senile osteoporosis.

27. A method according to claim 25, wherein the arthritis is osteoarthritis or rheumatoid arthritis

28. A method according to claim 25, wherein the cancer is osteosarcoma or myeloma,

29. A method according to claim 25, wherein the subject has a bone fracture.

30. A method according to claim 29, wherein the bone fracture is a non-union fracture or an osteoporotic fracture.

31. A method according to any of claims 23 to 30, wherein the monobutyrin is from about 100μg/ml to 5mg/ml.

32. A method according to claim 31 , wherein the monobutyrin is from about 10μg/ml to 100μg/ml.

33. A method according to any of claims 23 to 32, wherein the composition further comprises a second agent capable of promoting musculoskeletal tissue repair, regeneration and/or formation.

34. A method according to claim 33, wherein the second agent is selected from the group consisting of bone morphogenetic factors, cartilage derived morphological protein, epidermal growth factor, fibroblast growth factor, platelet derived growth factor, parathyroid hormone, insulin-like growth factor, sodium fluoride, bisphosphonates, calcium carbonate, prostaglandins, vitamin D, vitamin K, oestrogen and mixtures thereof

35. A method according to any of claims 23 to 34, wherein the composition is administered locally.

36. A method according to any of claims 23 to 34, wherein the composition is administered systemically.

37. A composition comprising monobutyrin or derivatives, fragments and/or analogues thereof and a second agent capable of promoting musculo-skeletal tissue repair, regeneration and/or formation for use as a medicament.

38. A composition according to claim 37, wherein the musculo- skeletal tissue is bone, cartilage, synovium, muscle, tendon, ligament or meniscus.

39. A composition according to claim 37 or 38, wherein the second agent is selected from the group consisting of bone morphogenetic factors, cartilage derived morphological protein, epidermal growth factor, fibroblast growth factor, platelet derived growth factor, parathyroid hormone, insulin-like growth factor, sodium fluoride, bisphosphonates, calcium carbonate, prostaglandins, vitamin D, vitamin K, oestrogen and mixtures thereof.

40. A composition according to claim 37 or 39, wherein the monobutyrin is from about 100μg/ml to 5mg/ml.

41. A composition according to claim 40, wherein the monobutyrin is from about 10μg/ml to 100μg/ml.

42. The use of monobutyrin or derivatives, fragments and/or analogues thereof in the manufacture of a medicament for the treatment of diseases or clinical conditions that may be alleviated by the promotion of musculo-skeletal tissue repair, regeneration and/or formation.

43. The use of claim 43, wherein the tissue is bone, cartilage, synovium, muscle, tendon, ligament or meniscus.

44. The use of claim 42 or 43, wherein the disease or clinical condition is osteoporosis, arthritis, diabetes, cancer, Paget's disease, Schϋllers disease, renal bone dystrophy, brachypodism, Hunter-Thompson chondrodysplasia, a spinal deformation, bone dysplasia, scoliosis, peridonatal disease, osteomalacia or fibrous osteitis.

45. The use of claim 44, wherein the osteoporosis is steroid- induced osteoporosis, idiopathic juvenile osteoporosis, postmenopausal osteoporosis, post-traumatic osteoporosis, senile osteoporosis,

46. The use of claim 44, wherein the arthritis is osteoarthritis or rheumatoid arthritis

47. The use of claim 44, wherein the cancer is osteosarcoma or myeloma,

48. The use of claim 44, wherein the subject has a bone fracture.

49. The use of claim 48, wherein the bone fracture is a non-union fracture, a delayed union fracture, a mal-union fracture or an osteoporotic fracture.

50. The use of any of claims 42 to 49, wherein the monobutyrin is from about 100μg/ml to 5mg/ml.

51. The use of claim 50, wherein the monobutyrin is from about 10μg/ml to 100μg/ml.

52. A composition, method, use or device as substantially described herein with reference to the description and Examples.