FIELD OF THE INVENTION

This invention relates to the treatment of musculoskeletal fibroproliferative disorders such as fibromatosis and, in particular, Dupuytren's disease. In particular it relates to a composition or therapeutic agent or to a combination of such compositions or therapeutic agents for the treatment, prophylaxis or prevention of progression of musculoskeletal fibroproliferative disorders, especially Dupuytren's disease, to the use of such

composition/therapeutic agent or combination of compositions/therapeutic agents for the treatment, prophylaxis or prevention of progression of musculoskeletal fibroproliferative disorders, especially Dupuytren's disease and to a method of treating musculoskeletal fibroproliferative disorders, especially Dupuytren's disease.

BACKGROUND OF THE INVENTION

Dupuytren's disease, which is alternatively known as palmar fibromatosis (or in its established disease state Dupuytren's contracture), is a disease associated with the build up of extracellular matrix materials including collagen on the connective tissue of the hand (the palmar fascia) causing it to thicken and shorten with the physical effect of causing the fingers to curl, most commonly the ring finger and little finger.

Dupuytren's disease affects approximately 5% of the white Caucasian population. The commonest manifestation is progressive flexion contracture of the digits of the hand, resulting in significantly compromised function. It affects both males and females, but the incidence is higher in males.

The causes of Dupuytren's disease are not well understood and underlying disease is not currently curable.

Treatment of Dupuytren's disease has traditionally been invasive surgical techniques. Primarily, the treatment has involved surgical excision of the offending tissue. In severe or recurrent disease, the surgical excision may be combined with excision of the overlying palmar skin and resurfacing of the cutaneous defect with full-thickness skin graft. Surgery is typically followed by prolonged rehabilitation, usually lasting 3 months and complications have been reported in up to 20% of cases. Such surgical correction is the mainstay treatment of later stage disease when secondary changes to tendons and joints have developed. A less invasive surgical intervention is needle fasciotomy in which the fibrous bands (contractures) in connective tissue are divided using the bevel of a needle.

Enzymatic cleavage of the affected tissue has been the focus of development to reduce invasiveness associated with surgery and improve recovery time. This approach has led to trials of collagenase. A bacterial collagenase, Clostridial collagenase, has been granted FDA approval as Xiaflex™ to Pfizer and Auxilium. USRE39941, US5589171 and US6086872 describe the use of bacterial collagenase for the enzymatic cleavage of connective tissue in the treatment of Dupuytren's disease. Bacterial collagenases suffer from certain disadvantages: for example lack non-selective cleaving of various collagen materials including collagen type IV associated with blood vessels; and, in the case of Xiaflex™, possible allergic reactions and potential immunogenicity; and administration may cause haemorrhage whilst the prolonged activity of collagenase limits the dose that can be administered locally due to risk of side effects as the drug disperses.

WO 2010/102202 describes a novel temperature sensitive recombinant collagenase in which the activity is observed at significantly below body temperature, but which is comparatively inactive at body temperature. Thus Dupuytren's syndrome can be treated by administering such recombinant collagenase at lower temperatures, which it is claimed restricts the duration of activity, increases the possible local dose and reduces collagenase-related side effects.

To date collagenase therapies have appeared relatively effective in treatment of contracture of the metacarpophalangel joint, whilst the correction of proximal interphalangeal joints has been much less satisfactory. Furthermore, as with surgical interventions, recurrence can be expected, but in the case of early collagenase trials, which involve enzymatically cutting the cord, recurrence is high, especially for disease affecting the proximal interphalangeal joint.

Other non-surgical treatments that have been proposed include steroid injections for early disease and to reduce recurrence, application of vitamin E cream applied as topical therapy, ultrasonic therapy and low-dose radiation therapy (for slowing the progression of early stage disease), such as X-rays and electron beam therapy.

Most research for treatments of Dupuytren's disease has focused on detecting pre-disposition to Dupuytren's (e.g. US-A-2004/0161761) and on the extracellular matrices produced, which has resulted in the collagenase-based treatments. There has been very little conclusive insight into potential treatments gained from studies into the biochemical pathway of Dupuytren's disease.

There remains a need for novel therapeutic intervention in the treatment and/or prevention of (e.g. progression of) Dupuytren's disease and other musculoskeletal fibroproliferative disorders.

PROBLEM TO BE SOLVED BY THE INVENTION

There remains a need for improvements in the treatment of Dupuytren's disease and other musculoskeletal fibroproliferative disorders, particularly fibromatosis and like diseases including and preferably selected from plantar fibromatosis (or Ledderhose's disease), adhesive capsulitis (frozen shoulder) and Peyronie's disease (fibromatosis of the penis).

It is an object of this invention to provide a composition and method for the treatment or prophylaxis (e.g. prevention of progression or recurrence) of one or more of Dupuytren's disease, plantar fibromatosis, adhesive capsulitis and Peyronie's disease.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the invention, there is provided a composition for use in the treatment or prophylaxis of a musculoskeletal fibroproliferative disorder, the composition comprising a cell-cell junction (or intercellular junction) inhibitor or modulator.

In a second aspect of the invention, there is provided a cell-cell junction (or intercellular junction) inhibitor or modulator for use in the treatment of a musculoskeletal fibroproliferative disorder.

In a third aspect of the invention, there is provided the use of a cell- cell junction (or intercellular junction) inhibitor or modulator in the manufacture of a medicament for the treatment of a musculoskeletal fibroproliferative disorder.

In a fourth aspect of the invention, there is provided a method for the treatment of a musculoskeletal fibroproliferative disorder, the method comprising administering to a patient in need thereof an effective amount of a cell- cell junction (or intercellular junction) inhibitor or modulator.

In a fifth aspect of the invention, there is provided a method or composition for reduction or prevention of recurrence of Dupuytren's disease post- surgical fasciectomy, post-needle fasciotomy or post-enzyme-mediated

extracellular matrix degradation, the method comprising locally administering to a patient a cell-cell junction (or intercellular junction) inhibitor or modulator.

In a sixth aspect of the invention, there is provided a composition comprising a combination of at least two of an adherens junction inhibitor or modulator, a gap junction inhibitor or modulator and a mechanosensitive ion channel inhibitor or modulator.

In a seventh aspect of the invention, there is provided a method for the treatment of a musculoskeletal fibroproliferative disorder comprising administration to a patient in need thereof a combined effective amount of at least two of an adherens junction inhibitor or modulator, a gap junction inhibitor or modulator and a mechanosensitive ion channel inhibitor or modulator.

ADVANTAGES OF THE INVENTION

The compositions and methods of the present invention enable musculoskeletal fibroproliferative disorders, such as Dupuytren's (and other fibromatosis and like disease characterized by contracture) to be slowed, halted or reversed.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a photograph of a clinical presentation of a patient with Dupuytren's disease.

Figure 2 shows images of nodules and cord in an intraoperative view;

Figure 3 is a chart showing a distribution of a-SMA rich cells in tissue excised from different parts of diseased Dupuytren's tissue;

Figures 4A-4D are charts illustrating the effect on contraction and a-SMA and COL1 expression in myofibroblasts and non-palmar dermal fibroblasts of gadolinium;

Figures 5A-5F are charts illustrating the effect on contraction and a-SMA expression in myofibroblasts and non-palmar dermal fibroblasts of carbenoxolone; Fig 5B showing distribution of gap junctions in myofibroblasts and non-palmar dermal fibroblasts;

Figures 6A-6B illustrate the expression of a-SMA and COL1 and expression of OB cadherin and N-cadherin in myofibroblasts and non-palmar dermal fibroblasts; Figure 6C showing the distribution of adherens junctions in myofibroblasts and non-palmar dermal fibroblasts; and

Figure 7 is a chart illustrating the rate of contraction of Dupuytren's nodular cells untreated and treated with N-cadherin antibody and E-cadherin antibody.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides for an improved treatment of a musculoskeletal fibroproliferative disorder, especially Dupuytren's disease (or other fibromatosis and like disease such as plantar fibromatosis, adhesive capsulitis and Peyronie's disease), which comprises administration to a patient in need thereof, especially a patient showing signs of early disease state, a therapeutic, prophylactic or progression-inhibitive amount of a cell-cell junction inhibitor or modulator. Further, the invention provides, by administration of a cell-cell junction inhibitor or modulator to a patient having or showing signs of developing

Dupuytren's disease, prevention of disease manifestation and/or progression, optionally as an adjunctive (or concomitant) therapy to a primary surgical intervention (e.g. a fasciotomy or fasciectomy) or primary therapeutic treatment (e.g. an extracellular matrix degradation, depletion or cleaving agent, such as a matrix metalloproteinase or collagenase). Still further, the invention provides, by administration of a cell-cell junction inhibitor or modulator to a patient, prevention of recurrence of disease as an adjunctive therapy to primary surgical intervention or therapeutic treatment of established disease.

Whenever the term 'cell-cell junction' is used herein, the term 'intercellular junction' may be used in the alternative.

Musculoskeletal fibroproliferative disorders are characterized by excessive or uncontrolled production of extracellular matrix in association with a musculoskeletal structure, often associated with contraction in later stage disease. As mentioned above, musculoskeletal fibroproliferative disorders include fibromatosis disorders. (The terms 'musculoskeletal fibroproliferative disorders' and 'fibromatosis disease' may be used interchangeably herein, where the context allows). The present invention is concerned with the treatment and, in particular, the inhibition of progression and recurrence (e.g. after primary treatment by surgery or therapy) of such diseases. In particular, the present invention is concerned with diseases selected from Dupuytren's disease, plantar fibromatosis, adhesive capsulitis and Peyronie's disease, especially Dupuytren's disease. The remainder of this document will discuss compositions and methods for treatment of musculoskeletal fibroproliferative disorders generally, with specific reference to Dupuytren's disease. Where the context allows, it should be understood that the disclosure may be read also with the generality or other specified diseases in place of Dupuytren's disease.

By cell-cell junction inhibitor or modulator, it is meant an agent that interferes, inhibits or modulates cell-cell junctions or mechanosensitive channels. Without being bound by theory, it is believed that cell-cell junctions play a critical role in the development of Dupuytren's disease and other musculoskeletal fibroproliferative disorders by facilitating the contraction of and between myofibroblast cells whereby extracellular matrix may be contracted and remodeled to form a cord or other adhesion, such as in frozen shoulder. By inhibiting those cell-cell junction interactions in myofibroblast cells, it is believed that contraction may be inhibited.

Preferably, the cell-cell junction inhibitor or modulator is an inihibitor or modulator of one or more of adherens junctions, gap junctions or mechanosensitive ion channels. Whilst mechanosensitive channels are not, strictly speaking inter-cellular channels, they are implicated in cell-cell junction activity since it is believed that they are associated with adherens junctions insofar as when the cell feels contraction, mechanosensitive channels open to allow Ca ²⁺ ion into the cell, which is necessary for effecting cell contraction via a-smooth muscle actin (a-SMA). Tension may be exerted on the cell via the adherens junction by a neighbouring cell.

Optionally, the treatment comprises administering to a patient a combination of inihibitors or modulators of adherens junctions and/or inhibitors or modulators of gap junctions and/or inhibitors or modulators of mechanosensitive ion channels.

Preferably, the administration according to the treatment of the present invention is local administration (e.g. by injection into or adjacent to the affected tissue). Most preferably, e.g. for the treatment or prevention of

Dupuytren's disease, the delivery is by injection into a, the or each nodule site (where, typically, there is an accumulation of myofibroblasts).

Any suitable dose may be delivered that is effective in the treatment or prophylaxis or progression inhibition of the musculoskeletal fibroproliferative disease according to the present invention.

According to one embodiment, the composition comprises a mechanosensitive ion channel inhibitor or modulator. By mechanosensitive ion channel inhibitor, it is meant an agent that blocks or inhibits mechanosensitive ion channel inhibitor formation, maintenance or function by blocking a component necessary for the formation or function of a mechanosensitive ion channel inhibitor.

Any suitable mechanosensitive ion channel inhibitor may be used in accordance with the present invention.

Optionally, the mechanosensitive ion channel inhibitor may be selected from calcium channel blockers, a low affinity selective compound isolate from tarantula venom, GsMTx-4, aminoglycosides, amiloride or gadolinium.

Preferably the mechanosensitive ion channel inhibitor is gadolinium, optionally administered in a complex such as diethylenetriamine pentaacetic acid

bismethylamide complex.

The mechanosensitive ion channel inhibitor may be administered in any suitable dose. Preferably, where using gadolinium, each administration (e.g. injection to each nodule site), may comprise a dose in the range 0.01 to 100 μιηοΐεβ, preferably, 0.05 to 50 μιηοΐεβ, more preferably, 0.1 to 10 μιηοΐεβ and optionally 0.5 to 5 μιηοΐεβ or from 1 to 2 μιηοΐεβ.

Typically, each site may be injected with 0.05 to 0.5 ml, more preferably 0.1 to 0.3 ml of agent formulation.

According to one embodiment, the composition comprises an adherens junction inhibitor or modulator, preferably an adherens junction antagonist. By an adherens junction antagonist it is meant an agent that blocks or inhibits adherens junction formation, maintenance or function by blocking a component necessary for the formation or function of adherens junctions.

Preferably, the adherens junction inhibitor or modulator comprises one or more anti-cadherin or, preferably one or more cadherin antagonist. An anti-cadherin agent is one that inhibits or blocks the formation or function of a cadherin. The anti-cadherin or cadherin antagonist may be effective against any cadherin which may be implicated in myofibroblast contraction, e.g. it may be one or more of N- cadherin, cadherin 12 type 2, P-cadherin, Tl -cadherin, T2-cadherin, VE-cadherin, R-cadherin, K-cadherin, cadherin 7 type 2, cadherin 8, type 2, OB-cadherin, T- cadherin, H-cadherin, M-cadherin, KSP-cadherin, Ll-cadherin, cadherin 18 type 2, cadherin 19 type 2 cadherin 20 type 2 and cadherin 23. Optionally, the anti- cadherin has broad spectrum anti-cadherin activity susch as β-catenin antibody, a- catenin antibody or vinculin antibody.

The anti-cadherin may be broad spectrum or specific cadherin antibodies (e.g. specific to any of the cadherins referred to above), such as an OB- cadherin antibody or an N-cadherin antibody such as those (or producible by the method) described in WO-A-2011/119888 or WO-A-2010/054377, the disclosures of which antibodies and antibody production methods are incorporated herein by reference.

Preferably, the anti-cadherin or cadherin antagonist is effective against N-cadherin and/or OB-cadherin and more preferably the anti-cadherin is an N-cadherin antagonist or an OB-cadherin antagonist. Still more preferably the anti-cadherin is an N-cadherin antagonist or inhibitor. Preferably, the anti-cadherin or cadherin antagonist, especially the N-cadherin antagonist, is a cadherin antibody, preferably an N-cadherin antibody. An example of an N-cadherin antibody is the commercially available monoclonal anti-N-cadherin antibody, clone GC-4, available from Sigma- Aldrich ^® (and having the product number 3865 and MDL no: MFC D00164511). The inventors have surprisingly found that N-cadherin antagonism is effective in reducing contraction of myofibroblasts from Dupuytren's patients. Previous studies (e.g. Hinz et al, Mol. Biol. Cell, 2004 15, 4310-20) have suggested that only an OB-cadherin antagonist was effective in inhibiting myofibroblast contraction, whilst an N-cadherin antagonist was not.

The anti-cadherin, preferably the anti-N-cadherin may be administered in any suitable dose. For example, when administering an anti- cadherin antibody, a suitable amount may be determined by methods as known in the art, but may comprise, for example, administering an amount of from 1 to 500 μg, optionally 5 to 250 μg, 10 to 100 μg or 50 to 75 μg.

According to a second embodiment, the composition comprises a Gap junction inhibitor or modulator. By Gap junction inhibitor, it is meant an agent that blocks or inhibits Gap junction formation, maintenance or function by blocking a component necessary for the formation or function of a Gap junction. Optionally, the Gap junction inhibitor may be selected from one or a combination of a glycerrhetinic acid, a fatty alcohol such as 2-octanol, antimalarials such as mefloquine or primaquine, thapsigargin, halothane and amiloride.

Preferably, Gap junction inhibitors useful in accordance with the present invention include anti-connexin agents such as connexin inhibitors, in particular connexin 43 inhibitors. Optionally, the anti-connexin is an antibody to a connexin, preferably an antibody to connexin 43. Preferably, the connexin inhibitors are selected from glycerrhetinic acid and derivatives thereof and more preferably the connexin inhibitor is carbenoxolone (18-a-glycyrrhetinic acid).

The Gap junction inhibitor may be administered in any suitable dose. Preferably, where using a connexin 43 inhibitor such as carbenoxolone, each administration (e.g. injection to each nodule site) may comprise a dose in the range of 0.005 to 100 μιηοΐεβ, preferably, 0.01 to 50 μιηοΐεβ, more preferably, 0.05 to 10 μιηοΐεβ and optionally 2 to 8 μιηοΐεβ.

Where the agent defined according to any of the above embodiments is an antibody, it may be a monoclonal antibody or fragment thereof; a chimeric monoclonal antibody (such as a human-murine chimeric monoclonal antibody); a fully human monoclonal antibody; a recombinant human monoclonal antibody; or a humanized antibody fragment, such as a Fab, F(ab')2 or Fv fragment. Optionally, the fragment may pegylated or encapsulated (e.g. for stability and/or sustained release).

Optionally, the treatment comprises administering to a patient a combination of inihibitors or modulators of N-cadherin and inhibitors or modulators of mechanosensitive ion channels, such as calcium channel blockers or gadolinium.

It is believed that the effectiveness of a cell-cell junction inhibitor or modulator in the treatments of the present invention is due to the crucial role played by cell-cell junctions in contractile effect of differentiated myofibroblast cells, which are understood also to be the main culprits in induction of uncontrolled matrix generation in Dupuytren's disease (and other fibromatosis diseases). The inventors have demonstrated that certain cell-cell interactions affect contracture and can be inhibited resulting in reduced rates of contracture in Dupuytren's nodular cells.

The consensus in the literature is currently that clinical nodules are the precursor to established Dupuytren's contractures. Dupuytren's disease occurs in people with genetic predisposition and further risk factors to manifestation of Dupuytren's disease include local trauma, poor lifestyle (e.g. smoking and drinking alcohol and poor diet), liver disease and diabetes. Established disease presents as flexion contracture which may typically be presented as contracture of the metacarpophalangeal joints (MCPJ) alone, less frequently contracture of the proximal interphalangeal joints (PIP J) alone, and often both. A phase III clinical trial of enzymatic fasciotomy using bacterial collagenase reported (Hurst et al, N. Engl J. Med, 2009, 361, 968-979) that 77% of MCPJ contractures were effectively treated (to within 5° of full extension) compared with 40% of PIP J contractures. An earlier stage trial (Badalamente et al, J Hand Surg Am, 2007, 32, 767-774) showed recurrence rates of 57% in patients with PIP J contractures at 2 years follow-up.

Numerous studies have shown that the presence of myofibroblasts is concomitant with early and active disease and that such cells are implicated in proliferative extra-cellular matrix (ECM) generation or deposition and, in particular, collagen deposition. TGF- ?1 leads to the development of the myofibroblast phenotype. Myofibroblasts are also believed to be responsible for contractile behavior. Myofibroblasts characteristically express a-smooth muscle actin (a-SMA), which is the actin isoform typical of vascular smooth muscle cells. a-SMA is believed to be the protein responsible for the contractility of

myofibroblasts and is the most reliable marker for myofibroblasts.

As mentioned above, the present invention preferably comprises a composition and method for treating, and more preferably inhibiting or halting the progression or recurrence of, musculoskeletal fibroproliferative disorders, such as fibromatosis disease, especially Dupuytren's disease, by administering to a patient a therapeutic, prophylactic or progression-inhibiting amount of a cell-cell junction inhibitor or modulator (preferably an N-cadherin antagonist such as a anti-N- cadherin antibody and/or a mechanosensitive ion channel inhibitor such as gadolinium). Preferably, the administration is local administration (e.g. by injection into or adjacent to the affected tissue).

There are two main embodiments of this invention.

A first main embodiment of the invention comprises a composition and method for treating early disease state musculoskeletal fibroproliferative disorders, especially early disease state Dupuytren's disease, by administering to a patient presenting early state disease, e.g. prior to the presence of palpable cord, an effective amount of a cell-cell junction inhibitor or modulator (preferably an N- cadherin antagonist such as a anti-N-cadherin antibody and/or a mechanosensitive ion channel inhibitor such as gadolinium).

According to the first embodiment, a composition comprising a cell- cell junction inhibitor or modulator (preferably an N-cadherin antagonist such as a anti-N-cadherin antibody and/or a mechanosensitive ion channel inhibitor such as gadolinium) may be administered to a patient for preventing disease progression (to established disease state) and resultant flexion contracture. Preferably, the method comprise local administration (e.g. by injection) directly into the clinical nodule(s). Optionally, the method further comprises administering to the patient, preferably locally (and more preferably directly to the clinical nodule(s) identified), an extracellular matrix degradation, depletion or cleavage agent, which is preferably a collagen degradation, depletion or cleavage agent and may be, for example a matrix metalloproteinase (MMP) and/or a collagenase (but may be, for example, a MMP or collagenase up-regulating or inducing agent). It is believed that the matrix metalloproteinase or collagenase may disrupt collagen and extra- cellular matrix local to the clinical nodule(s) thereby enhancing access of administered a cell-cell junction inhibitor or modulator to the proliferative fibrotic foci and thus enhance efficacy of treatment. It is believed that administration of the cell-cell junction inhibitor or modulator in this manner may be considered prophylactic or progression halting or inhibiting treatment. According to this embodiment, the primary treatment is a cell-cell junction inhibitor or modulator to which the extracellular matrix degradation or cleavage agent is preferably adjunctive.

In one particular embodiment which involves the combined treatment of a patient presenting early disease state musculoskeletal

fibroproliferative disorders, especially Dupuytren's disease, with a cell-cell junction inhibitor or modulator (preferably an N-cadherin antagonist such as a anti-N- cadherin antibody and/or a mechanosensitive ion channel inhibitor such as gadolinium) and an extracellular matrix degradation, depletion or cleavage agent (e.g. matrix metalloproteinase and/or coUagenase), the cell-cell junction inhibitor or modulator and the extracellular matrix degradation, depletion or cleavage agent (e.g. coUagenase) may be administered simultaneously or sequentially, together or separately. Preferably, both a cell-cell junction inhibitor or modulator (preferably an N-cadherin antagonist such as a anti-N-cadherin antibody and/or a

mechanosensitive ion channel inhibitor such as gadolinium) and the extracellular matrix degradation, depletion or cleavage agent (e.g. coUagenase) are administered locally, for example by injection. Optionally, they may be administered

simultaneously, e.g. administering a composition comprising both a cell-cell junction inhibitor or modulator and coUagenase (e.g. by injectable solution) or by applying two separate compositions at the same time. Alternatively, the cell-cell junction inhibitor or modulator and the extracellular matrix degradation, depletion or cleavage agent (e.g. coUagenase) are administered separately. When

administered separately, they may be administered in any order a suitable time apart. Preferably, when administered separately the extracellular matrix degradation, depletion or cleavage agent (e.g. coUagenase) is administered first followed by the cell-cell junction inhibitor or modulator (preferably an N-cadherin antagonist such as a anti-N-cadherin antibody and/or a mechanosensitive ion channel inhibitor such as gadolinium), which may be administered a suitable time after the extracellular matrix degradation agent, e.g. after no less than 5 minutes, and preferably within 48 hours, more preferably within 24 to 48 hours, still more preferably within 6 hours and most preferably within 15 minutes to 3 hours. Optionally, a cell-cell junction inhibitor or modulator (preferably an N-cadherin antagonist such as an anti-N-cadherin antibody and/or a

mechanosensitive ion channel inhibitor such as gadolinium) and the extracellular matrix degradation, depletion or cleavage agent are administered simultaneously for the treatment of early disease state musculoskeletal fibroproliferative disorders. Optionally, a composition is provided for local administration (e.g. injectable solution, sustained release composition or implant) for treating early disease state musculoskeletal fibroproliferative disorders, preferably Dupuytren's disease, which composition comprises an effective amount of a cell-cell junction inhibitor or modulator (or configured to release an effective amount of a cell-cell junction inhibitor or modulator if, for example, the composition is a sustained release composition) optionally in combination with an extracellular matrix degradation, depletion or cleavage agent (preferably a matrix metalloproteinase and/or collagenase) preferably in an adjunctive amount and a pharmaceutically acceptable carrier.

Preferably, according to this embodiment, the cell-cell junction inhibitor or modulator (preferably an N-cadherin antagonist such as an anti-N- cadherin antibody and/or a mechanosensitive ion channel inhibitor such as gadolinium) is provided in an amount effective to inhibit disease progression without inducing systemic complications. Optionally, therefore, the cell-cell junction inhibitor or modulator is provided in an amount to reduce progression or recurrence by at least 30%, preferably at least 50% and more preferably at least 70%.

Preferably, an effective amount of a cell-cell junction inhibitor or modulator is that which will result in a reduction in clinical nodule size (e.g. at least a 20%), or even at least a 50%>, reduction in size, as measured by degree of protrusion or lateral or longitudinal extent) in up to two weeks post administration. Efficacy of a cell-cell junction inhibitor or modulator treatment preferably is observable by an overall reduction in the progression of disease. Without being bound by theory, it is believed that myofibroblasts require tension to develop and persist and the cell-cell junction inhibitors used in the present invention can reduce local tension thereby reducing the tendency of new myofibroblasts to develop.

Preferably, according to this embodiment, an extracellular matrix degradation, depletion or cleavage agent, e.g. a matrix metalloproteinase and/or coUagenase, is provided in a cell-cell junction inhibitor or modulator adjunctive amount, by which it is meant an amount effective to enhance the efficacy of the a cell-cell junction inhibitor or modulator. In any case, it is preferred that the extracellular matrix degradation, depletion or cleavage agent (e.g. matrix metalloproteinase or coUagenase) is provided in an amount of up to 1-2 mg.

Preferably, the extracellular matrix degradation, depletion or cleavage agent (e.g. matrix metalloproteinase or coUagenase) is administered in an amount significantly below (e.g. 0.01 to 0.5 times) the extracellular matrix degradation, depletion or cleavage agent (e.g. matrix metalloproteinase or coUagenase) dose that would be required to achieve an enzymatic fasciotomy in established disease state fibromatosis. Preferably, the extracellular matrix degradation, depletion or cleavage agent (e.g. matrix metalloproteinase or coUagenase) is provided in an amount of 0.01 to 2 mg, more preferably 0.05 to 1 mg and still more preferably 0.1 to 0.8 mg.

The extracellular matrix degradation, depletion or cleavage agent, e.g. matrix metalloproteinase or coUagenase, may assist the a cell-cell junction inhibitor or modulator (preferably an N-cadherin antagonist such as a anti-N- cadherin antibody and/or a mechanosensitive ion channel inhibitor such as gadolinium) in accessing the cell mass, as well as assisting in disaggregating of the extracellular matrix of the clinical nodule.

A second main embodiment of the invention comprises a composition and method for treating established disease state Dupuytren's (or other musculoskeletal fibroproliferative) disease by administering to a patient an effective amount of a cell-cell junction inhibitor or modulator (preferably an N- cadherin antagonist such as a anti-N-cadherin antibody and/or a mechanosensitive ion channel inhibitor such as gadolinium), preferably in combination with, simultaneous to, sequentially with, in association with, concomitantly with, in combined administration with or adjunctive to surgical fasciectomy, a fasciotomy and/or a extracellular matrix degradation, depletion or cleavage agent (e.g. a matrix metalloproteinase or collagenase) treatment, preferably a collagenase treatment. Preferably, the method comprises surgical fasciectomy, needle fasciotomy or extracellular matrix degradation, depletion or cleavage agent (e.g. a matrix metalloproteinase or a collagenase) administration, which provides improvement (i.e. enabling greater extension of the affected digits), more preferably correction (i.e. to within 5° of full extension) and most preferably full correction (complete extension) of the established disease. Most preferably, the method comprises administration of extracellular matrix degradation, depletion or cleavage agent (e.g. a matrix metalloproteinase or a collagenase) to sites local to the disease site.

Where the treatment comprises extracellular matrix degradation, depletion or cleavage agent (e.g. a matrix metalloproteinase or a collagenase), a cell-cell junction inhibitor or modulator may be provided for combined treatment by simultaneous, sequential or separate administration, e.g. for combined, concomitant or adjunctive therapy. Preferably, in this embodiment, a cell-cell junction inhibitor or modulator is adjunctive to the extracellular matrix degradation, depletion or cleavage agent (e.g. a matrix metalloproteinase or a collagenase) treatment.

According to this embodiment, a cell-cell junction inhibitor or modulator

(preferably an N-cadherin antagonist such as a anti-N-cadherin antibody and/or a mechanosensitive ion channel inhibitor such as gadolinium) may be administered separately, before or after, administration of the extracellular matrix degradation, depletion or cleavage agent (e.g. a matrix metalloproteinase or a collagenase), e.g. up to 4 to 6 weeks before or after administration of the extracellular matrix degradation, depletion or cleavage agent (e.g. a matrix metalloproteinase or a collagenase), preferably up to 14 days before or after administration of the extracellular matrix degradation, depletion or cleavage agent (e.g. a matrix metalloproteinase or a collagenase), still more preferably at least 30 minutes before or after administration of the extracellular matrix degradation, depletion or cleavage agent (e.g. a matrix metalloproteinase or a collagenase) and more preferably after the extracellular matrix degradation, depletion or cleavage agent (e.g. a matrix metalloproteinase or a collagenase), e.g. in the period 4 hours to 7 days after the extracellular matrix degradation, depletion or cleavage agent (e.g. a matrix metalloproteinase or a collagenase), whereby administration of a cell-cell junction inhibitor or modulator may gain better access to the disease site but be administered at a point when myofibroblasts can be optimally inhibited.

Preferably, according to this embodiment, a cell-cell junction inhibitor or modulator is provided in an amount effective to inhibit disease recurrence without inducing systemic complications.

Preferably, an effective amount of a cell-cell junction inhibitor or modulator is that which will prevent a palpable or as measured (e.g. by ultrasound scan) increase in clinical nodule presentation and/or in clinical nodule size (e.g. a 25% increase in size, as measured by degree of protrusion or lateral or longitudinal extent) in up to two to twelve weeks post administration. Efficacy of a cell-cell junction inhibitor or modulator treatment preferably is observable by an overall prevention in the recurrence of disease (e.g. post surgery or enzymatic treatment). Preferably, by administering an effective amount of a cell-cell junction inhibitor or modulator, re-establishment of established state disease manifested by flexion contracture can be managed or prevented, e.g. flexion contracture maintained to 10° or less, more preferably 5° or less further contraction compared with post- correction treatment extent, within a period after administration of the a cell-cell junction inhibitor or modulator, e.g. up to 6 weeks, preferably up to 6 months. Optionally, repeat administrations may be provided in order to achieve this (e.g. two to four weekly).

Preferably, according to this main embodiment of the invention, the treatment comprises administration local to disease site of an extracellular matrix degradation, depletion or cleavage agent (e.g. a matrix metalloproteinase or a collagenase). The extracellular matrix degradation, depletion or cleavage agent (e.g. a matrix metalloproteinase or a collagenase) should be administered in an amount sufficient to enable improvement and/or correction of disease-associated contraction (e.g. to 5° or less of full extent in the case of Dupuytren's) within 24 or 48 hours of administration). Preferably, an extracellular matrix degradation, depletion or cleavage agent, such as a coUagenase (e.g. Clostridium coUagenase), is provided for local administration in an amount of up to 10 mg administered in one or more locations along each contracture, preferably from 0.1 to 5 mg per administration and more preferably from 0.15 to 2 mg and most preferably 0.5 to 1 mg.

In one embodiment in which extracellular matrix degradation, depletion or cleavage agent (e.g. a matrix metalloproteinase or a coUagenase) is administered for contracture improvement and a cell-cell junction inhibitor or modulator administered for recurrence inhibition, there may be provided a single combined dose of the extracellular matrix degradation, depletion or cleavage agent (e.g. a matrix metalloproteinase or a coUagenase) and a cell-cell junction inhibitor or modulator.

In one embodiment, an extracellular matrix degradation, depletion or cleavage agent may be administered (e.g. injected) into diseased cord tissue in an effective amount, whilst a cell-cell junction inhibitor or modulator may be administered (e.g. injected) into clinical nodule(s) and/or cord tissue in a recurrence-inhibitory amount.

The extracellular matrix (ECM) degradation, depletion or cleavage agent may be any suitable agent capable of degrading, cleaving or causing or inducing degradation or cleavage of extracellular matrix, including collagen. For example, the ECM degradation or cleavage agent may be an ECM degradation enzyme or an ECM degradation enzyme expression up-regulator (e.g. relaxin). Preferably the ECM degradation or cleavage agent is a matrix metalloproteinase or a coUagenase, more preferably a coUagenase, such as a bacterial coUagenase (e.g. clostridial coUagenase), human or humanised co agenase or mutant or recombinant coUagenase or recombinant matrix metalloproteinase (e.g. recombinant matrix metalloproteinase I, preferably human recombinant matrix metalloproteinase I). Preferably, the co agenase is time or temperature dependent or is

photo dynamically activated or deactivated, to allow higher local doses to be administered without systemic or long-lasting side-effects. Optionally, it is a

Cathepsin-L or a mutant or recombinant thereof. Examples of suitable coUagenase for use in the present invention include those described in: GB-A-2323530, US 5589171, USRE39941, US6086272 & WO-A-2010/102262 (and for established disease optionally in the amounts described therein, the disclosure of which collagenases and amounts and modes of administration are incorporated herein by reference).

By early disease state it is meant that indications of disease are present, e.g. histological markers or more particularly clinical nodules in tissue, but in the absence of, for example, palpable cord or significant contracture. By early disease state Dupuytren's disease, it is meant that indications of Dupuytren's disease are present, e.g. histological markers or more particularly clinical nodules in palmar and/or digital tissue, but in the absence of significant (e.g. at least 5°) flexion contracture (or, for example, palpable cord).

By established disease state, it is meant that clinical nodules are present, palpable cord is present and contracture is evident. By established disease state Dupuytren's disease, it is meant that clinical nodules are present on the palm and digits of the hand and flexion contracture is evident (e.g. at least 5°).

Varying histological stages of Dupuytren's disease have been categorised in the literature, most succinctly by Rombouts (J Hand Surg Am, 14, 644-652, 1989) and later authors, into three distinct stages: 1) a proliferative stage with high cellularity and the presence of mitotic figures; 2) a fibrocellular stage charactised by high cellularity but no mitotic figures and the presence of reticulin network; and 3) a fibrous stage with few cells separated by broad bundles of collagen fibres. Stage 1) disease is believed to correlate with early disease state as discussed above (i.e. presence of nodules but no contracture) and Dupuytren's stages 2) and 3) is believed to correlate with our Established Disease State (characterized by digital contracture). The present inventors have found that during early established disease state, active myofibroblasts are collected in the established nodules and cords, especially in relation to the MCP and PIP joints and these drive the progression of flexion contractures of the digit. By clinical nodule, it is meant a palmar or digital nodule evident as a palpable subcutaneous lump.

By histological (or histopathological) nodule, it is meant a collection of cells (mainly myofibroblast cells with some inflammatory cells such as macrophages and mast cells) which may range from tiny foci of cells to larger collections of cells, but not clinically palpable.

Without being bound by theory, it is believed that the initial clinically palpable nodule(s) is the focus of proliferating fibroblasts in disease progression, but that numerous histological nodules will form at various locations in the palm and/or digits which will ultimately contribute to cord formation, contraction and flexion contracture.

Where 'nodule' is used herein it may be clinical or histological nodules (or either) as will be apparent from the context.

According to two alternative embodiments specific to Dupuytren's, a first embodiment may relate to a composition and method for treating

Dupuytren's disease characterized by joint contractures of less than 20° and a second embodiment may relate to a composition and method for treating

Dupuytren's disease characterized by joint contractures of at least 20°. The contracture of 20° is identified as a transition phase, since at less than 20° contracture, many patients may choose to stop progression of the disease without wishing to undergo surgery since their mobility and operative use of the hand is still largely adequate, whilst at greater than 20°, many patients will find surgery or other collagen depleting therapy (such enzymatic fasciotomy) essential to restore full function to the hand.

The inventors' investigations reveal that cell-cell junctions are an optimal therapeutic target for early Dupuyten's disease (i.e. early disease state). In established disease state Dupuytren's disease, an ideal combination is a matrix metalloproteinase such as collagenase with a cell-cell junction inhibitor reduce the impact of any recurrence, which is typically associated with enzymatic fasciotomy.

In each of the embodiments, the cell-cell junction inhibitor or modulator may be provided in a multiple administrations over an extended (or continuous) term in order to prevent or inhibit disease progression or recurrence. Where recurrence is to be avoided, intermittent treatment may be provided by, e.g. low-dose fortnightly, monthly or six-monthly administration. Alternatively, continuous treatment may be provided by low-dose releasing sustained or delayed intermittent release implant or patch. Alternatively, repeat doses may be initiated by signs of disease progression in the early disease state and may optionally comprise a combined extracellular matrix degradation or cleavage agent (e.g. a matrix metalloproteinase or a coUagenase) and cell-cell junction inhibitor or modulator treatment (e.g. consistent with the first embodiment described above).

In one embodiment of the invention, the progression of early disease state disease (e.g. Dupuytren's disease) to established disease state can be prevented, inhibited or halted by the local administration of a cell-cell junction inhibitor or modulator.

Preferably, the cell-cell junction inhibitor or modulator may be administered separately or simultaneously in combination with or adjunctively to a coUagenase and/or matrix metalloproteinase. A coUagenase, especially a photo- responsive or temperature dependent coUagenase, may be administered for local effect to enhance the cell-cell junction inhibitor or modulator disease progression inhibition effect by enhancing access to treatment sites by cleaving early stage extracellular matrix formation. A temperature dependent coUagenase is one which (typically a recombinant or mutant coUagenase) has coUagenase activity dependent upon temperature and typically is active at below body temperature, e.g. at 25°C and below, thereby allowing extremely high doses of coUagenase to act very locally (e.g. by injecting at the disease site at say 20°C without having any systemic action or other side effects associated with longevity of action).

The composition and method of the present invention may utilise any suitable means of administration, which is preferably local. In particular, the cell-cell junction inhibitor or modulator should be administered locally, e.g. by applying directly into a surgical incision during surgery, by injection (preferably directly into the clinical nodule(s) and/or cord tissue), by release from a sustained and/or delayed release particles or device that may be implanted into or close to the disease site or a sustained and/or delayed release patch formulation, by topical application or any other suitable route. A composition is preferably suitably formulated and typically comprises the required dose of cell-cell junction inhibitor or modulator along with a pharmaceutical acceptable carrier or excipient.

Formulations for parenteral administration may typically comprise a sterile aqueous preparation of the active ingredient, which is preferably isotonic with the blood of the recipient. Formulations for intra-articular administration may be in the form of a sterile aqueous preparation of the active ingredient.

Formulations suitable for topical administration may include liquid and semi liquid preparations such as liniments, lotions and applications; oil-in- water and water-in- oil emulsions such as creams, ointments and pastes; and solutions and suspensions.

In a further aspect, there is provided a formulation for frequent, e.g. daily, periodic or occasional (preferably daily), topical application to the musculoskeletal fibroproliferative disorder area (e.g. the hands, and in particular palms and digits, in the case of Dupuytren's disease) for use, for example, by early disease state or post-operative patients for the inhibition of disease progression or recurrence, the formulation comprising a cell-cell junction inhibitor or modulator suitable for topical administration (e.g. selected from such cell-cell junction inhibitor or modulator defined above) and a suitable excipient. The formulation may be provided as a cream or lotion, a patch or a medicated glove (in which the glove is impregnated for release of the active component from the internal surface). Preferably, the formulation comprises cell-cell junction inhibitor or modulator in a concentration for administration by topical application of a low dose, such as 0.001 to 0.05, preferably 0.001 to 0.01, of the dose of the selected cell-cell junction inhibitor or modulator that would yield a systemic response.

Optionally, the compositions and methods of the present invention may further comprise further active ingredients that may be effective in the treatment or progression-inhibition of musculoskeletal fibroproliferative disorders such as Dupuytren's disease. For example, combination therapy or concomitant or adjunctive co-administration of a cell-cell junction inhibitor or modulator and an agent of the vascular endothelial growth factor family, such as VEGF-A, VEGF-B, VEGF-C or VEGF-D or an agent encoding said VEGF or a functional fragment thereof (such as described in WO-A-2004/082705), which combination is preferably a development retarding combination (or composition) for use in association with surgery, or needle or enzyme fasciotomy. Additionally or alternatively such method or composition as described herein may further comprise an activator of PPARy (such as pioglitazone) for reducing myofibroblast populations local to the disease site.

In another aspect, there is provided a method of treatment and composition comprising the cell-cell junction inhibitor or modulator in one or more compositions and/or formulations as described above for the treatment of keloid and/or hypertrophic scars. Such methods and composition may be administered in a similar manner to that described above.

The invention will now be described and illustrated in more detail, without limitation, with reference to the following Examples.

EXAMPLES

The following Examples illustrate the effect on contractility of Dupuytren's nodular cells (herein referred to as myofibroblast cells) and non- palmar human dermal fibroblast cells (herein referred to as HDF cells) by treatment with certain agents.

Figure 1 illustrates a clinical presentation of Dupuytren's disease in a patient's little finger, with contracture of the metacarpophalangeal and proximal interphalangeal joints. The clinical presentation is also clearly illustrated in Figure 1 of Rehman et al. Arthritis Research & Therapy 2011, 13:238.

From data from greater than 100 Dupuytren's cords, myofibroblasts from the majority of patients are concentrated in nodules, located on the palmar aspect of the hand and at the level of the affected joints (see Figure 2). According to Figure 2, nodules rich in myofibroblasts are located in the vicinity of the finger joints. Figure 2 shows: A: intraoperative view of Dupuytren's cord, with location of proximal interphalangeal joint (PIP J; 1) marked; B: Low magnification photomicrograph of histological section stained for a- smooth muscle actin. A collection of a-SMA rich cells in a nodule is located in the vicinity of the PIP J; C: High magnification view of nodular area, showing a -SMA positive cells

(myofibroblasts).

Previous work (Verjee et al; Verjee LS, Midwood K, Davidson D,

Essex D, Sandison A and Nanchahal J (2009), 'Myofibroblast distribution in

Dupuytren's cords: correlation with digital contracture', J Hand Surg Am 34:1785- 1794) has shown that cells from and close to the nodules in Dupuytren's patients have a significantly higher percentage of a-SMA positive cells than non-nodular cells (e.g. cord cells) implying such nodular cells have a higher myofibroblast concentration. A chart of total cell count for each of several different cell types and the percentage of a-SMA positive cells within that population is shown in Figure 3.

Cells isolated from nodules excised from patients with Dupuytren's disease were cultured up to a maximum of passage 2 in a three dimensional collagen gel under isometric conditions in a culture force monitor, following the methodology of Verjee et al (Verjee LS, Midwood K, Davidson D, Eastwood M and Nanchahal J (2010), ost-transcriptional regulation of alpha-smooth muscle actin determines the contractile phenotype of Dupuytren's nodular cells', J Cell Physiol. 224: 681-690). A culture force monitor used in the present experiments is such as illustrated in Figure 1 (page 683) of Verjee et al, J Cell Physiol. (2010) 224: 681- 690.

Example 1

The effect of mechanosensitive ion channel inhibitors on

Dupuytren's nodular cell contraction as compared with contraction of non-palm dermal fibroblast cells was explored further. Gadolinium was used as the mechanosensitive ion channel blocker. It is believed that gadolinium blocks cationic mechanosensitive channels possibly through binding to anionic phospholipids. An isometric force contraction experiment was conducted using the method set out in Methods below.

Addition of the mechanosensitive channel blocker gadolinium led to dose-dependent reduction in contractility of myofibroblasts (MF) (Figure 4A) but not of non-palmar human dermal fibroblasts (HDF) (Figure 4B). The reduced contractility of myofibroblasts was accompanied by a reduction of a-SMA and COLl message expression (Figure 4C) as well as a-SMA protein (Figure 4D) in myfibroblasts but not in HDF. Cell viability was unaffected by gadolinium concentrations up to 600 μΜ.

Figure 4 illustrates that blocking mechanosensitive junctions downregulates myofibroblast activity. In Figures 4A and 4B, gadolinium (300μΜ) inhibited the contractility of myofibroblasts (Figure 4A) in a dose-dependent manner, but not HDF (Figure 4B) [data shown are from n>3 patients (each in triplicate) and are expressed as mean ± S.E.M. ** P < 0.01, *** P < 0.001].

Gadolinium (300 μΜ) reduced a-SMA and COLl mRNA (Figure 4C) and a-SMA protein (Figure 4D) in myofibroblasts compared to the respective untreated cells, whereas no significant difference was observed in HDF. Gene expression was assessed by quantitative RT-PCR and data are presented as fold change compared to GAPDH, and normalized to expression in untreated myofibroblasts or HDF respectively. Protein expression was assessed by western blotting using vimentin as a loading control. Gels shown are representative of gels from 3 patients. Data are shown as the mean ± S.E.M from n>3 patients (each assay was performed in triplicate). ** P < 0.01.

As can be seen, the mechanosensitive ion channel inhibitor gadolinium inhibited contraction of collagen gel populated by Dupuytren's nodular cells (myofibroblast cells) whilst having no impact on contraction rates of non- palmar human dermal fibroblast cell populations from the same patients.

Furthermore, the inhibitory effect of the mecahnosensitive ion channel inhibition by gadolinium on Dupuytren's myofibroblast (nodule) cell contraction is dose dependent. Thus, gadolinium and mechanosensitive junction inhibitors more generally may be effective in reducing the contraction and thus useful in treating or inhibiting the progression of Dupuytren's disease and other musculoskeletal fibroproliferative disorders.

Example 2

The effect of gap junction inhibitors on Dupuytren's nodular cell

(myofibroblast cell, MF) contraction as compared with contraction of non-palmar human dermal fibroblast (HDF) cells was explored. Carbenoxolone was used as the gap junction inhibitor. Carbenoxolone is a saponin that inhibits gap junction communication by disassembling gap junction plaques composed of connexin 43. Connexin 43 in the plaques is phosphorylated and, without being bound by theory, carbenoxolone is believed to act by dephosphorylating and not by inhibiting phosphorylation.

An isometric force contraction experiment was conducted using the method set out in Methods below.

The expression and effect of blockade of gap junctions in myofibroblasts and human non-palmar dermal fibroblasts (HDF) were compared. In the basal state, myofibroblasts demonstrated lower levels of Cx43 both at message and protein levels (Figure 5 A) and as visualized by immunofluorescence (Figure 5B) compared to HDF. Selective blockade of gap junctions by

carbenoxolone led to a dose dependent decrease in isometric contraction of myofibroblasts (Figure 5C) but not of HDF (Figure 5D). Impaired cell viability was not seen after 24h following addition of carbenoxolone up to a dose of 200 μΜ. In addition to the reduced contractility, carbenoxolone resulted in a reduction in expression of COL1 and a-SMA mR A in myofibroblasts together with a reduction in a-SMA at mRNA and protein levels in HDF (Figure 5E). Combined treatment of myofibroblasts with neutralizing antibody to N-cadherin and carbenoxolone did not result in reduction of contractility above that seen with carbenoxolone alone (Figure 5F). However, contractility was completely abolished by addition of cytochalasin D (Figure 5F), as would be expected.

In more detail, with reference to Figures 5A-F, in Figure 5 A, it can be seen that baseline levels of connexin mRNA and protein were significantly higher in HDF compared to myofibroblasts. Gene expression was assessed by quantitative RT-PCR and data are presented as fold change compared to GAPDH, and normalized to expression in HDF. Data are shown as the mean ± S.E.M from n>3 patients (each assay was performed in triplicate). *** P < 0.001. Protein expression was assessed by western blotting using vimentin as a loading control. Gels shown are representative of gels from 3 patients. In Figure 5B,

immunofluoresence staining of myofibroblasts and HDF cultured in monolayer show connexin (small dots) and nuclei stained with DAPI (large blobs).

[Representative images from 5 patients]. Figures 5 C and D: Carbenoxolone inhibited the contractility of myofibroblasts (Figure 5C), but not HDF (Figure 5D). Data are shown as the mean ± S.E.M from n>3 patients (each assay was performed in triplicate). ** P < 0.01, *** P < 0.001. Figure 10E: Carbenoxolone (ΙΟΟμΜ) reduced a-SMA and COL1 mRNA and a-SMA protein in myofibroblasts compared to HDF. Carbenoxolone also reduced a-SMA mRNA and protein expression in HDF. Gene expression was assessed by quantitative RT-PCR and data are presented as fold change compared to GAPDH, and normalized to expression in untreated MF or HDF respectively. Protein expression was assessed by western blotting using vimentin as a loading control. Gels shown are representative of gels from 3 patients. Data are shown as the mean ± S.E.M from n>3 patients (each assay was performed in triplicate). *P < 0.05, ** P < 0.01, *** P < 0.001. Figure 5F: The addition of anti-N cadherin antibody (10μg/ml) in combination with carbenoxolone (ΙΟΟμΜ) did not result in a further decrease in myofibroblast contractility when compared to carbenoxolone (ΙΟΟμΜ) alone. Contractility of MF was abolished following the addition of cytochalasin D

(20μg/ml). Data are shown as the mean +/- SEM for n>3 patients (each performed in triplicate). *P < 0.05, *** P < 0.001.

As can be seen, carbenoxolone (a gap junction inhibitor) inhibited contraction of collagen gel populated by Dupuytren's myofibroblast cells (nodular cells) whilst having no impact on contraction rates of non-palmar dermal fibroblast cell (HDF) populations from the same patients. Furthermore, the inhibitory effect of the gap junction inhibition by carbenoxolone on Dupuytren's myofibroblast (nodule) cell contraction is dose dependent. It may be concluded that inhibition of contracture in Dupuytren's myofibroblast (nodule) cells may be achieved by a gap junction inhibitor (such as carbenoxolone).

Example 3

The role, expression and effect of blocking of OB-cadherins and N- cadherins were explored, using a culture force monitor experiment as described in Methods below.

First examined were the adherens junctions in myofibroblasts (MF) and in non-palmar human dermal fibroblasts (HDF) and the expression of OB- and N-cadherins compared. MF expressed higher levels of COL1 and a-SMA mRNA and higher levels of a-SMA protein than HDF (Figure 6A). MF also expressed more OB-cadherin and less N-cadherin at both message and protein levels compared to HDF (Figure 6B). The intercellular adherens junctions between MF, visualized by immunofluoresecent staining for β-catenin, were better defined and were more numerous than between HDF (Figure 6C).

In more detail, with reference to Figure 6: Figure 6 A shows baseline levels of a-SMA and COL1 mRNA, and a-SMA protein, were significantly higher in Dupuytren's myofibroblasts (MF) compared to human dermal fibroblasts (HDF). Figure 6B: Baseline levels of OB cadherin mRNA and protein were also higher in MF compared to HDF, whereas N cadherin expression was significantly higher in HDF compared to MF. Gene expression was assessed by quantitative RT-PCR and data are presented as fold change compared to GAPDH, and normalized to expression in HDF. Protein expression was assessed by Western blotting using vimentin as a loading control. Gels shown are representative of gels from 3 patients. Data are shown as the mean ± S.E.M from n>3 patients (each assay was performed in triplicate). ** P <0.01. (C) Immunofluoresence staining of MF and HDF cultured in monolayer showing β-catenin (small dots) and nuclei stained with DAPI (large blobs). [Images shown are representative of cells from 5 patients]. The effect of N-cadherin blocking was investigated using a commercially available N-cadherin antibody, the monoclonal anti-N-cadherin antibody, clone GC-4, available from Sigma- Aldrich ^® (and having the product number 3865 and MDL no: MFC D00164511). This resulted in reduced MF contractility in a dose dependent manner, compared to isotype controls or antibodies to the epithelial specific E-cadherin (obtained from Invitrogen) (Figure V).

As can be seen in Figure 7, the addition of anti-N cadherin, but not anti-E cadherin antibodies (5-2(^g/ml), inhibited MF contractility. Data are shown as the mean ± S.E.M from n>3 patients (each assay was performed in triplicate). *** P < 0.001.

Consistent with previous reports for myofibroblasts derived following treatment of fibroblasts with TGF-βΙ, it was found that myofibroblasts from Dupuytren's tissue expressed more OB-cadherin at both message and protein levels whilst HDF expressed more N-cadherin. However, the above examples illustrate that blocking N -cadherin was surprisingly effective in inhibiting isometric contraction of 3D collagen matrices by primary myofibroblasts from Dupuytren's nodules. This is in contrast to previous publications (e.g. Hinz et al, Mol Biol Cell, 15, 4310-20, 2004) that showed that only OB cadherin blockade inhibited myofibroblast contraction whilst N-cadherin blockade only inhibited fibroblast contraction. This surprising contrast with earlier publications may be due to the present inventors' use of cells and a system that more properly represent the in vivo environment cells in Dupuytren's disease. The present examples used early passage primary human cells from patients with Dupuytren's disease whereas in earlier publications subcutaneous rat fibroblasts up to passage 7 were compared to myofibroblasts generated by exposure to TGF-βΙ over 4 days in one case, whilst embryonic rat subcutaneous and lung fibroblasts and myofibroblasts generated by exposure to TGF-βΙ over 5 days were studied in another. Alternatively, or additionally, it may be that intercellular adherence between myofibroblasts is crucial to their function such that blockade of even the relatively few N cadherin- containing adherens junctions had a profound effect on contractility. Either way, this illustrates the surprising reduction in contractility of myofibroblasts by an N-cadherein inihibitor illustrates that N-cadherin inhibitors (and antagonists and antibodies) may be effective in treating or inhibiting the progression of Dupuytren's disease and other musculoskeletal fibroproliferative disorders.

Methods

Unless otherwise stated, the following methods were employed in the Examples above.

Patient samples

Tissue samples were obtained following informed consent (REC 07/H0706/81). Dupuytren's nodular tissue and matched full-thickness skin (harvested from the groin or medial aspect of arm) were obtained from patients with Dupuytren's disease undergoing dermofasciectomy.

Cell culture

Human dermal fibroblasts (HDF) were isolated from non-palmar skin and

Dupuytren's myofibroblasts (MF) were isolated from a-SMA-rich nodules (Verjee et al (2010) J Cell Physiol 224:681-90). Tissue samples were dissected into small pieces and digested in Dulbecco's modified Eagle's medium (DMEM) (Lonza) with 1% penicillin-streptomycin (PAA) and 5% fetal bovine serum (FBS) (Gibco) with type I collagenase (Worthington Biochemical Corporation) + DNase I (Roche Diagnostics) for up to 2h at 37°C. Cells were cultured in DMEM with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO ₂ . Cells up to passage 2 were used for experiments.

Culture Force Monitor (CFM)

Measurement of the isometric contractile forces generated by cells within 3D collagen matrices was performed as previously described (Verjee et al, 2010 - referenced above). Briefly, 2xl0 ⁶ cells were seeded in 2.5 ml of type I collagen (FirstLink), and the resulting 3D matrices are suspended between two flotation bars and held stationary at one end whilst the other is attached to a force transducer. Fibroblast populated collagen matrix generated tensional forces were continuously measured and data logged every minute (dynes: lxlO ^"5 N). Cell populated matrices were cultured in DMEM with 10% FBS and 1% penicillin- streptomycin at 37°C in a humidified incubator for 24h with 5% CO ₂ and treated with anti-N-cadherin antibody (Sigma), anti-E-cadherin antibody (Invitrogen), IgG isotype control, gadalolinium or carbenoxolone (Sigma). Cell-mediated force generation was abolished by addition of a saturating dose of 2(^g/ml cytochalasin D (Sigma) to fibroblast populated collagen matrices (Townley et al. (2009) J Hand Surg Eur Vol 34:783-7). Experiments using each patient sample were performed in triplicate.

Quantitative RT-PCR

Cells were cultured in monolayer and treated with gadalolinium or carbenoxolone for 24h and total RNA was extracted from each sample using the QIAamp RNeasy Mini Kit (Qiagen) according to manufacturer's instructions. Isolated RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop

Technologies). For real-time quantitative reverse transcription PCR, Inventoried TaqMan® Gene expression Assays were used for a-SMA (Hs00426835-gl), COL1 (Hs00164004-ml), N-cadherin (Hs00362037-ml), OB-cadherin

(Hs00901475-ml) and Cx43 (Hs00748445-sl) (Applied Biosystems) with Reverse Transcriptase qPCR™ Mastermix No ROX (Eurogentec). Samples were run on the ABI 7900HT Fast Real-Time PCR System (Applied Biosystems). Expression was normalized to GAPDH (Hs02758991-gl) (Applied Biosystems) and compared to the level of gene expression in either baseline respective cell types or to the level of gene expression in HDF, which were assigned the value of 1 using delta CT analysis performed with SDS software (Applied Biosystems). Western blots Cells were cultured in monolayer and treated with gadalolinium or carbenoxolone for 24h prior to protein extraction. Cell lysates were prepared in lysis buffer (25 mM HEPES (pH 7.0), 150 mM NaCl, and 1% Nonidet P-40), containing protease inhibitor cocktail (Roche Biochemicals) and then electrophoresed on 10% SDS polyacrylamide gels (Life Technologies), followed by electrotransfer of proteins onto PVDF transfer membranes (Perkin Elmer Life Sciences). Membranes were blocked in 5% BS A/TBS + 0.05% Tween and incubated overnight at 4°C with primary antibodies against a-SMA primary antibody, anti-N-cadherin antibody (Sigma), connexin and vimentin (Abeam). Horseradish peroxidase-conjugated anti- mouse IgG (Dako) or anti-rabbit IgG (Amersham Biosciences) were used as secondary antibodies. Bound antibody was detected using the enhanced chemiluminescence kit and visualized using Hyperfilm MP (Amersham

Biosciences). Immunofluoresence

Cells were cultured in monolayer with DMEM, 10% FBS and 1% penicillin- streptomycin for 24h then fixed for 10 min with 3% paraformaldehyde in PBS and permeabilized with 0.2% Triton X-100 (Sigma). Cells were stained with either a rabbit monoclonal anti-β catenin or a rabbit polyclonal anti-connexin (Abeam) followed by Alexa Fluor 568-conjugated goat anti-rabbit antibody (Invitrogen), Alexa Fluor 488 Phalloidin (Invitrogen) and DNA with DAPI (Sigma). Secondary antibody alone was used as an immuno labelling control. Images were acquired using confocal microscopy oil immersion objectives (60x) and the signal was analyzed by Ultraview confocal microscopy (PerkinElmer). Cell viability was assessed using a Live/Dead Viability/Cytotoxicity Kit (Invitrogen).

Statistics

The rate of FPCL contraction (dynes/h) was calculated by measuring the average gradient of the curve between 6 and 24 h. Analysis of single variance was used for comparing all conditions. All statistical analyses were performed using software (GraphPad Software version 5.0c). Significance was achieved if p<0.05. The invention has been described with reference to a preferred embodiment. However, it will be appreciated that variations and modifications can be effected by a person of ordinary skill in the art without departing from the scope of the invention.