WO2011033306A1 - Inhibition of tendon adhesions - Google Patents

Abstract

The invention provides composition having an osmolality of at least 1200m Osmol for use as a medicament for the inhibition of tendon adhesion formation. The compositions of the invention may comprises a sugar phosphate, such as mannose-6-phosphate (M6P) and/or glucose-6-phosphate (G6P). Composition comprising M6P are particularly favoured. The compositions may improve tendon repair by increasing the quality of replacement tendon formed after injury, and may be well suited to administration by injection.

Description

INHIBITION OF TENDON ADHESIONS

The present invention relates to medicaments for the inhibition of tendon adhesion formation, and to methods by which tendon adhesion formation may be inhibited. The invention also relates to relates to medicaments for improving tendon function, and to methods by which tendon function may be improved.

Tendons are the connective tissue that joins muscles to bones, and thus allows muscle contraction to be converted into bone movement. Tendons are primarily composed of collagen, which is produced by tenocyte cells within the tendon.

The collagen in tendons is highly organised, and is formed into structures in which the collagen is aligned in parallel. The smallest such structures are collagen fibrils, and these in turn make up collagen fibres. A collection of collagen fibrils makes up a primary fibre bundle (or subfascicle), a collection of primary fibre bundles makes up a secondary fibre bundle (or fascicle), and a collection of these secondary bundles makes up a tertiary fibre bundle. The tendon itself is composed of a number of tertiary fibre bundles. The primary, secondary and tertiary fibre bundles are each enclosed by a layer of endotenon, and the outer surface of the tendon is covered by the epitenon.

Tendons may be sheathed or unsheathed. In the case of sheathed tendons the tendon sheath surrounds the outside of the tendon and is made up of two layers - the synovial sheath and the fibrous tendon sheath.

Tendons may be subjected to a wide range of injuries, including sharp or blunt trauma. Typical tendon injuries include: traction/torsion injuries, crush injuries, lacerations, ruptures and surgical injuries, as well as degenerative conditions, such as repetitive strain injury.

Injuries to tendons are common. It has been reported that there are over 23 million patient visits to US hospitals per year following damage to their tendons or ligaments. The cause of this injury can usually be split by age group, where the younger patient has been involved in a sporting repetitive strain injury or a high impact trauma. The older patient usually has a degenerative tendon or ligament injury. Adhesions can form between the tendon and surrounding tissue resulting in a marked loss of function and pain following injury or surgery. A particularly active area for tendon repair is in the hand and wrist, where there are over half a million surgical procedures conducted per year in the US alone, with a similar number of procedures estimated to occur in Europe.

Approximately 30% of all patients undergoing primary surgical tendon repair have to submit for a second operation (tenolysis) to surgically remove adhesions (scarring) in order to restore function to the finger. There is therefore a need for medicaments or methods of treatment that are able to reduce the requirement for tenolysis surgery.

Unfortunately there is currently a paucity of products on the market indicated for use in the reduction of post-surgical adhesions, such as in patients undergoing hand/wrist surgery.

It has previously been suggested that incidences of adhesion formation in various tissues may be decreased through the use of hyperosmotic formulations. These hyperosmotic formulations may be provided to sites at which it is desired to decrease adhesion formation. Though there is no consensus regarding the properties required of such hyperosmotic formulations, or the methods by which they achieve their results, the formulations generally have an osmolality in the region of 300mOsmol to 500mOsmol.

Mannose-6-phosphate (M6P) is a sugar that is known to exert a number of different biological activities. In particular, M6P is known to reduce scarring that occurs as part of the healing response to injury. This activity of M6P has generally been ascribed to the sugar's ability to inhibit the activity of TGF-β, a growth factor that has three isoforms that are known to influence the body's wound healing and scarring response.

One of the therapeutic applications in which it has been suggested that M6P may be used is improvement of the range of motion that may be achieved after flexor tendon repair. In this context it has been reported that M6P is able to increase the range of motion, and also breaking strength, of repaired tendons, as compared to control treatments. M6P is shown to reduce TGF- -induced collagen I expression in vitro, and the beneficial effect on range of motion is believed to arise as a result of decreased adhesion formation.

An important finding associated with this prior art use of M6P in tendon healing is that the extent of the beneficial effects achieved varies inversely with the amount of the sugar used. Thus a low dose of M6P at a concentration of l mg/Ι ΟΟμΙ provides a greater beneficial effect than a higher dose, such as 2mg/100^l. This inverse relationship between dose and beneficial effect is noted in connection with all properties assessed: in vitro collagen I production; in vivo range of motion, and breaking strength. This appears to be consistent with the previous reports regarding optimal doses of M6P to be used in the neutralisation of TGF-β activity.

It is an aim of certain aspects or embodiments of the invention to provide new medicaments for the inhibition of tendon adhesion formation. It is an aim of certain aspects or embodiments of the invention to provide new methods for the inhibition of tendon adhesion formation. It is also an aim of certain aspects or embodiments of the invention to provide alternative medicaments for the inhibition of tendon adhesion formation, and an aim of certain aspects or embodiments of the invention to provide alternative methods for the inhibition of tendon adhesion formation. Furthermore, it is an aim of certain aspects or embodiments of the invention to provide improved medicaments for the inhibition of tendon adhesion formation, and of certain aspects or embodiments of the invention to provide improved methods for the inhibition of tendon adhesion formation.

In a first aspect of the invention there is provided a composition having an osmolality of at least 1200mOsmol for use as a medicament for the inhibition of tendon adhesion formation. It may be preferred that the composition comprises mannose-6- phosphate (M6P) at an osmolality of at least 1200mOsmol.

Compositions in accordance with the first aspects of the invention may be referred to as medicaments of the invention.

In a second aspect the invention provides a method of inhibiting tendon adhesion formation, the method comprising administering to patient in need of such inhibition a composition having an osmolality of at least 1200mOsmol. Methods of treatment in accordance with this second aspect of the invention may be referred to as methods of the invention. It will be appreciated that the medicaments of the invention will generally be suitable for use in practicing the methods of the invention, and represent preferred means for use in such methods.

The present invention is based upon the inventors' surprising finding that compositions, such as solutions of M6P, having very high osmolality as compared to the physiological conditions found in the body are highly effective in terms of their ability to inhibit tendon adhesion.

A medicament in accordance with the invention may preferably have an osmolality of between about 1200mOsmol and 5000mOsmol, more preferably between about 1300mOsmol and 3000mOsmol, still more preferably between about 1400mOsmol and 2500mOsmol, and most preferably has an osmolality of around 1500mOsmol (equivalent to 600mM).

The formation of adhesions after tendon injury is, as discussed above, one of the most frequent and troublesome complications associated with the tendon healing process. Inhibition of tendon repair using the medicaments or methods of the invention may be apparent through a reduction in the incidences of adhesion formation that occur after treatment; and/or a reduction in the extent of adhesion formation that occurs after treatment. The medicaments or methods of the invention are thus able reduce or avoid this detrimental effect of healing.

The medicaments or methods of the invention are able to decrease adhesion formation to a greater extent than treatments described in the prior art. This is illustrated by comparison within the same experimental model of the results achieved using prior art treatments and results achieved using the medicaments or methods of the invention. The inhibition of tendon adhesion formation also has beneficial effects on the clinical outcome achieved by patients treated with the medicaments or methods of the invention. These include an increase in tendon function and/or a decrease in the requirement for tenolysis surgery.

As mentioned above, the prior art had previously suggested that medicaments formulated to provide l mg/Ι ΟΟμΙ (approximately 38mM, and with an osmolality of about 368mOsmol) were optimally suited to improve the range of flexor tendon motion following repair of the injured tendon. This improvement in range of motion is believed to arise from a reduction in the formation of adhesions. The teaching of the prior art has previously suggested that M6P has a "bell-shaped" dose-response curve. This suggests that very low doses of M6P have no beneficial effect on scarring or adhesions, while increasing doses improve efficacy until an optimal dose is reached. Beyond this optimal dose increasing the quantity of M6P administered decreases the effectiveness of treatment, until eventually doses are reached that have detrimental effects on scar or adhesion formation. Generally it has been suggested that M6P be used at a concentration of between 10mM (318mOsmol) and 60mM (418 mOsmol), with optimum doses of M6P being reported at concentrations of around 20mM (338mOsmol) to 50mM (398mOsmol). Outside these doses effectiveness decreases as higher concentrations of M6P are used, until doses of 100mM (497mOsmol) or more have detrimental effects.

For reference it is worth noting that the osomolalities of the various concentrations of M6P discussed in the prior art are far lower than those utilised in the medicaments or methods of the invention. For example, when made up in a diluent of PBS a 10mM solution of M6P has an osmolality of 318mOsmol), a 20mM solution of M6P has an osmolality of 338mOsmol, a 50mM solution of M6P has an osmolality of 398mOsmol and a 60mM of M6P has an osmolality of 418 mOsmol.

The inventors have found that medicaments or methods of the invention are able to decrease the incidences of tendon adhesion that form after tendon injury to a greater extent than may be achieved using the optimal doses suggested by the prior art. In the experimental studies described elsewhere in the specification, treatment of tendon wounds with the medicaments or methods of the invention (represented, for experimental purposes, by use of a solution of M6P with an osmolality of 1500mOsmol) was able to reduce the incidence of tendon formation to a greater extent than did treatment of comparator wounds with a M6P solutions at concentrations described in the prior art as beneficial (exemplified in the Experimental Results section by a 50mM solution of M6P having an osmolality of 398mOsmol. It will be understood that the ability of the medicaments of the invention to inhibit tendon adhesion formation to a greater extent than prior art treatments indicates that the medicaments of the invention will be able to improve tendon function, most likely to a greater extent than prior art treatments.

The ability of the medicaments or methods of the invention to inhibit tendon adhesion formation means that even in the event that adhesion formation does occur after treatment using the medicaments or methods of the invention, the severity of adhesion formation will generally be reduced. This can be seen in the experimental data discussed in more detail below, where the medicaments or methods of the invention decrease the extent of adhesion formation more effectively than do the doses suggested in the prior art. It will be appreciated that lessening the extent or severity of adhesion formation will also represent a beneficial outcome for a patient seeking to inhibit tendon adhesion formation after injury.

Briefly, in an experimental model predisposed to tendon adhesion formation, the use of medicaments and methods of the invention (represented by the administration of a 1500mOsmol solution of M6P in PBS - as discussed above) is able to reduce the area of tendon adhesion formed in the experimental model to a greater extent than treatments representing the prior art (represented by use of a 50mM solution of M6P in PBS with an osmolality of 398mOsmol).

The beneficial therapeutic effects provided by the medicaments of the invention become particularly apparent over the course of the healing process. The extent to which tendon adhesions are inhibited (as compared to controls) increases from three to eight weeks after treatment. This is in contrast to the effects achieved using other, lower osmotic formulations. With lower osmotic formulations, an initial improvement in tendon repair (as shown by decreased adhesion area after three weeks) gradually decreases over time, until eight weeks from injury the benefits achieved (though still present) are not statistically significant as compared to controls.

While the medicaments and methods of the invention may preferably utilise compositions comprising M6P they are by no means limited to the use of this compound. The inventors believe that any compound suitable for administration to a patient may be used to provide the necessary osmolality. The major requirements are that the compound (or compounds) used in the manufacture of the medicament of the invention should not be provided in a form that harms a patient or the tendon healing process (e.g. the compounds should not elicit adverse cytotoxic reactions or induce an inflammatory response), and should be capable of dissolution at high enough concentrations to provide the required osmolality. While M6P or other sugar phosphates (such as those considered below) are compounds meeting these criteria there are many other such compounds that may be suitable for use in the medicaments or methods of the invention. In keeping with these considerations, the medicaments of the invention may comprise any organic or inorganic compound, or mixture thereof, capable of being dissolved to provide a solution of suitable osmotic strength, so long as the composition produced has an osmolality of at least 1200mOsmol.

In the event that the medicaments of the invention comprises an organic compound it is preferred that this compound is selected from the group consisting of: a hexose, a substituted hexose (e.g derivatives of sulfur, phosphorus, nitrogen, heavy metals), pentose, substituted pentose (e.g derivatives of sulfur, phosphorus, nitrogen, heavy metals), organic acids (e.g. citric acid), organic carboxylic acids, substituted carboxylic acids and derivatives such as acid anhydrides, esters, amides, monosaccharides, disaccharides, oligosaccharides, substituted monosaccharides, substituted disaccharides, substituted oligosaccharides, fatty acids, nucleic acids, nucleosides, nucleotides, boronic acids, poly-ethylene glycols, glycerine, glycerols, amino acids, substituted amino acids, organic primary amines, secondary amines, tertiary amines and quaternary amines, mixtures of the above, and salts derived from the above.

Sugar phosphates, such as M6P or glucose-6-phosphate (G6P) constitute preferred compounds for use in the medicaments of the invention. It may be preferred that the osmolality of a composition to be used as a medicament of the invention be entirely, or nearly entirely (for example at least 75%, 85% or 95%), ascribable to M6P or G6P present in the composition.

Sugar phosphonates represent another preferred group of compounds that may be used in the medicaments of the invention. Suitable examples of sugar phosphonates that may be used to produce compositions having an osmolality of at least 1200 mOsmol include those described in International Patent Application No. PCT/GB96/01840, published as WO 97/05883. The disclosure of this document, as it concerns the identity, manufacture and characterisation of suitable sugar phosphonates, is incorporated herein by reference. However, the inventors studies described in the present specification illustrate that, when used in the medicaments of the present invention, such sugar phosphonates achieve their therapeutic effect as a result of the osmotic activity, in contrast to the TGF^-inhibitory activity ascribed to such compounds in the prior art. An amount of a medicament of the invention to be provided per site of treatment may be determined with reference to the size of the site requiring treatment to inhibit adhesion formation. For example, a human digital flexor tendon may be treated by epitendonous injection in which approximately 150μΙ_ of a medicament of the invention is administered to each side of the site of injury (so that a total of 300μΙ_ is administered per site of injury). Optionally such treatment may be followed by bathing the site of injury with a larger volume of a medicament of the invention. Merely by way of guidance, the inventors have found that the site of an injury may be bathed with a volume approximately four times that originally administered. Thus in the case where approximately 150μΙ_ of a medicament of the invention is administered to each side of a site of injury, each side of the site of injury may optionally be bathed with a further volume of the medicament in the region of 600μΙ_.

As will be appreciated by those skilled in the art, tendons in different body sites may have larger or smaller cross-sectional areas than a digital flexor tendon, and even digital flexor tendon size may vary from individual to individual. The treatment of larger or smaller tendons to inhibit adhesion formation will generally require the use of proportionally larger or smaller volumes of a medicament of the invention.

The inventors have found that the use of medicaments or methods of the invention is able to provide a greater inhibition of tendon adhesion formation after injury than can be achieved by following the teaching of the prior art. The inventors have found that the medicaments or methods of the invention are also able to provide a prolonged inhibition of adhesion formation that persists for a greater length of time after healing than do the improvements provided by prior art treatments.

Without wishing to be bound by any hypothesis, the inventors believe that the osmolality of the compositions used as medicaments of the invention are so high that they give rise to "osmotic shock" effects in cells that may otherwise be involved in the production of tendon adhesions. They believe that the osmolality of the medicaments of the invention is sufficient to induce at least one pathway or response from the group consisting of: up-regulation of p38 phosphorylation; and/or disruption of the cytoskeleton; and/or a decrease in cell motility; and/or a decrease in the rate of cell proliferation; and/or a decrease in cell metabolic activity, in cells at the site to which the composition is administered. The medicaments of the invention may achieve their effects through exerting these activities on cell types (at the site where the composition is administered) selected from the group consisting of: cells of the tendon, cells of the tendon sheath, and cells of subcutaneous tissues in proximity to the site of injury. In particular, the cells to be affected by treatment at the site where the composition is administered may be selected from the group consisting of: epitenon fibroblasts; endotenon fibroblasts; and tendon sheath fibroblasts.

The medicaments of the invention are able to exert the requisite effects on cells at the treated site without causing cell death or other irreversible damage. This appears to arise as a result of the fact that the medicaments of the invention maintain residence at the site of treatment for a period that is sufficient to induce at least one of the pathways or responses outlined above, but that is not so long that the cells are permanently damaged. It may previously have been expected that solutions having the same osmolality as the medicaments of the invention may lead to cell damage or cell death were they not cleared before having the opportunity to do so.

It may be preferred that medicaments of the invention have a half-life at the site of administration of less than five hours, preferably less than four hours, yet more preferably less than three hours, and most preferably around two hours. Suitable protocols by which residency of medicaments at a site of administration can be assessed are described in the Experimental Results section.

The inventors' in vitro studies have shown that the osmotic shock effects of the medicaments of the invention lead to an efflux of water from cells, and transient cell crenation (a shrinking of cells that occurs as a result of water loss) which is associated with a transient reduction in cellular functions such as cell migration and proliferation. Without wishing to be bound by any hypothesis, the inventors believe that these osmotic shock effects, possibly in combination with other factors such as hydroflotation (discussed below), contribute to the inhibition of tendon adhesion formation observed in vivo.

Hydroflotation occurs when local hypertonic conditions established on administration of the medicaments of the invention induce intracellular and extracellular water to be drawn from surrounding tissues into the area surrounding a tendon where a medicament of the invention has been administered. The presence of this water gives rise to a physical barrier between the tendon and surrounding tissues (such as the tendon sheath) that disrupts the formation of structures that may otherwise contribute to adhesion formation.

As noted above, these osmotic effects appear to be responsible for the therapeutic effectiveness of the medicaments of the invention. Since osmotic pressure, and so osmolality, is a colligative property (i.e. a property that depends on solute concentration, but not on the identity of the solute in question), this provides further evidence that the therapeutic effects noted may be achieved by medicaments of the invention incorporating any suitable solute, so long as they achieve the requisite osmolality. This is supported by the finding that both mannose-6-phosphate and glucose-6-phosphate, sugar phosphates that have quite different biological activities in many contexts, are able inhibit the formation of tendon adhesions when utilised in medicaments of the invention.

Compelling evidence that the therapeutic activity of the medicaments of the invention is not mediated through a TGF^-based mechanism can be found in the results of the inventors' studies that demonstrate that the medicaments of the invention do not influence the cellular apparatus involved in TGF-β signalling. These results, which are described in more detail in the Experimental Results section, illustrate that the levels of cellular receptors for TGF-β and Smads (molecules that mediate TGF-β signalling intracellular^) are not substantially changed after administration of medicaments of the invention. If the medicaments of the invention did, in fact, achieve their effects through modulating TGF-β signalling, this would give rise to characteristic changes in the levels of the cellular receptor and intracellular signalling molecules.

Prior art therapies designed with a view to inhibiting TGF-β activity make use of much lower concentrations of compounds such as M6P than do the medicaments or methods of the invention. These lower concentrations are selected with a view to "quenching" the naturally occurring TGF-β activity associated with tendon injury, and utilise amounts of M6P that are sufficient to reduce TGF-β activity, but not sufficient to achieve the effects noted when the medicaments or methods of the present invention are employed.

Further to their ability to inhibit tendon adhesion formation, the inventors have observed that the medicaments of the invention are also able to bring about the formation of replacement tendon that more closely approximates the structure of unwounded tendon. Accordingly, in a preferred embodiment the medicaments of the invention may be used further to improve tendon repair by increasing the quality of replacement tendon formed after injury.

In addition to a surprisingly beneficial inhibition of tendon adhesion formation, the medicaments or methods of the invention are also able to improve the quality of the replacement tissue formed on healing of tendon wounds.

The organisation of collagen fibrils and fibres within tendon structures is of great importance in providing tendons with their requisite mechanical properties. Tendons in which the organisation of the collagen structures is disrupted are of lesser quality than those exhibiting a level of organisation that more closely approximates that found in undamaged tendon.

The inventors have found that the replacement tendon produced after treatment with the medicaments or methods is of greater quality than that produced after healing of control treated injuries or tendon wounds treated in accordance with the prior art. The increase in quality of replacement tendon is manifest in an increase in organisation of the constituent collagen. The use of the medicaments or methods of the invention to increase the quality of replacement tendon formed after injury, so that it more closely resembles unwounded tendon, represents an optional (but preferred) manner in which they may be used to improve tendon healing beyond the inhibition of tendon adhesion formation.

The medicaments or methods of the invention may be used to promote increased organisation of the replacement tendon produced after injury. The organisation of collagen structures in a tendon (whether treated with the medicaments or methods of the invention, or not) may be assessed with reference to the polarisation of the tendon, as discussed further in the Experimental Results section.

Analysis in this manner illustrates that the use of the medicaments or methods of the invention allows the production of replacement tendon having an organisation of collagen that more closely approximates that found in normal tendon that has not been wounded. This provides an indication that the function of the replacement tendon will also more closely approximate that of normal unwounded tendon. The medicaments or methods of the invention are able to promote increased polarity of the replacement tendon formed after injury. The extent of polarisation can be quantified experimentally, and this may be used as a method to quantify the increase in quality and organisation of replacement tendons produced after treatment using the medicaments or methods of the invention.

Improvement in the quality of replacement tendon formed after treatment with the medicaments or methods of the invention may be demonstrated with reference to increased strength of the replacement tendon formed after treatment, as compared to replacement tendon formed on healing of untreated or control treated tendons injuries. An increase in the strength of replacement tendon formed may be demonstrated by an increase in the breaking strength of the tendon.

Additionally or alternatively, an improvement in the quality of replacement tendon formed after treatment with the medicaments or methods of the invention may be demonstrated with reference to increased elasticity of the replacement tendon formed after treatment, as compared to replacement tendon formed on healing of untreated or control treated tendons injuries.

Mechanical properties indicative of an increased quality of replacement tendon, such as the breaking strength and/or elasticity of the replacement tendon, may be investigated using a tensiometer, or any other suitable device. Examples of protocols for use in the assessment of replacement tendon quality are discussed further in the Experimental Results section.

The medicaments of the invention may preferably be administered at a site of tendon injury. This may be a site where a tendon injury has already occurred, or may be a site where a tendon injury is to occur (such as an injury caused by a surgical procedure), in accordance with the prophylactic use of the medicaments or methods of the invention.

The use of the medicaments or methods of the invention to inhibit tendon adhesion formation may lead to an improvement in the function of tendon after injury. Such improvements may arise as a result of the reduction in the incidences of adhesion formation; and/or the reduction in the extent of adhesion formation; and may be further added to by the increase in the quality of replacement tendon formed. So important are these improvements in tendon function that they give rise to further aspects of the invention. In a third aspect there is provided a composition having an osmolality of at least 1200mOsmol for use as a medicament for improving tendon function after injury. The invention also provides a corresponding method of improving tendon function after injury. These methods may be practiced using medicaments in accordance with the third aspect of the invention.

Clinical end points allowing the assessment of improvement of tendon function are considered elsewhere in the specification.

The medicaments of the invention may be used to inhibit adhesion formation after tendon injury. In a fourth aspect the invention provides a composition having an osmolality of at least 1200mOsmol for use as a medicament for the inhibition of tendon adhesion formation after injury. The invention also provides a corresponding method of inhibiting tendon adhesion formation after injury. Preferably these methods may be practiced using medicaments in accordance with the fourth aspect of the invention.

The medicaments or methods of the invention may be used to reduce the requirement for subsequent tenolysis surgery. Since the medicaments or methods of the invention are able to inhibit tendon adhesion formation, and thereby decrease the incidences of adhesion formation, or to reduce the severity of any adhesions formed, they may be used as part of a care regime able to reduce the requirement for tenolysis surgery at a later date.

Except for where the context requires otherwise, the considerations set out in the specification and claims in respect of the formulations, uses, or the like, of medicaments or methods of the invention should be considered to be equally applicable to medicaments or methods in accordance with any of the aspects of the invention.

In order to aid in the understanding of the invention, various terms used in the specification and claims will be defined further below. Further preferred embodiments will also be described in more detail. "Inhibition of tendon adhesion"

The medicaments and methods of the invention may be used to inhibit tendon adhesion formation that may otherwise occur as a result of a tendon injury.

Inhibition of tendon adhesion may be assessed with reference to the area of tendon adhesion occurring in tendons receiving treatments using the medicaments or methods of the invention, as compared to the area of adhesion occurring in tendons receiving suitable control treatments. Experimental protocols by which such assessments may be made are described in more detail elsewhere in the specification.

Inhibition of tendon adhesion formation may be demonstrated by any decrease in the level of tendon adhesion formation occurring in tendons treated in accordance with the invention, as opposed to control-treated tendons. It may be preferred that the decrease is a statistically significant decrease. A suitable decrease in the level of tendon adhesion formation may be in the region of 5%, 10%, 20%, 50% or more.

"Injury"

The inventors believe that the medicaments or methods of the invention may be used to inhibit tendon adhesion formation that may occur after any sort of tendon injury caused by physical, chemical or thermal insult.

Injuries include, but are not limited to, physical insults in which a tendon is crushed or cut. Examples of cut injuries include those in which the tendon is partially cut, and those in which the tendon is fully cut (i.e. the tendon is severed). The medicaments or methods of the invention may be of use in inhibiting tendon adhesion formation after injuries including accidental injuries and intentional injuries, for example injuries occurring as a result of surgical procedures.

It may be preferred that the medicaments or methods of the invention are for use in inhibiting tendon adhesion formation after injury selected from the group consisting of: sharp trauma injury; blunt trauma injury; traction injury; torsion injury; crush injury; lacerations; ruptures; bicep tendon rupture; penetrating injuries; surgical injuries; sports injuries, such as mallet injury; and degenerative conditions, such as trigger thumb or repetitive strain injury. Sites of injuries

The inventors believe that the medicaments or methods of the invention may be used to inhibit tendon adhesion formation at any site within the body. The medicaments or methods of the invention may be used for inhibiting tendon adhesion formation in sheathed or unsheathed tendons. The medicaments or methods of the invention may be used to inhibit tendon adhesion formation in flexor tendons (including superficial flexor tendons and deep flexor tendons) and extensor tendons.

While the medicaments or methods of the invention have been shown to be effective in inhibiting tendon adhesion formation in the digital flexor tendons, it is anticipated that they will also be of benefit in inhibiting tendon adhesion formation at other sites, including (but not limited to): the hand; the fingers; the wrist; the foot; the limbs; the shoulder (including the supraspinatus tendon which may be associated with rotator cuff injury, frozen shoulder and painful arc); and the Achilles tendon.

The use of the medicaments or methods of the invention in inhibiting tendon adhesion formation after injuries to the hand, wrist or fingers represents a preferred embodiment of the invention.

In the hand there are two flexor tendons to each finger, one arising from the flexor digitorum profundus (FDP) muscle and one arising from the flexor digitorum superficialis (FDS) muscle. The function of these muscles is to provide flexion of the finger exerted through the insertions into the phalangeal bones of their respective tendons. One flexor tendon is also present in each thumb, arising from the flexor pollicis longus (FPS) muscle.

The palmar aspect of the hand is divided into five zones according to the anatomy of the flexor tendons to the fingers. Zone I extends from just distal to the insertion of the FDS tendon into the middle phalanx to the insertion of the FDP tendon into the distal phalanx. Zone II extends from the A1 pulley to just distal to the insertion of the FDS tendon. Zone III extends from the distal border of the carpal tunnel to the A1 pulley. Zone IV includes the carpal tunnel and zone V is proximal to the carpal tunnel and includes the origin of each tendon from its respective muscle belly. In zone II the digital flexor tendons are enclosed in a synovial membrane named the flexor tendon sheath. The flexor sheath of the thumb and little fingers commonly extends proximally to the wrist, termed the radial and ulnar bursae. The flexor tendons are also enclosed in a synovial membrane in zone IV. Flexor tendon injuries in the hand are common with an estimated half of these occurring in zone II. Injuries in zone II are frequently complicated by the formation of tendon adhesions. Accordingly, such injuries may gain particular benefit from treatment using the medicaments or methods of the invention.

In hand surgery, outcomes following the repair of severed digital flexor tendons are variable, and decreased post-operative range of motion in the hand may result from the formation of adhesions between the flexor tendon and its surrounding sheath. A study of complications following flexor tendon repair in the hand identified 49% of cases with peritendinous adhesions. The resulting disrupted biomechanical and structural properties of the healing tendon leads to a significant reduction in the active range of motion and subsequent function. This may necessitate further surgery, tenolysis, to improve the poor range of movement resulting from adhesions. There is also a high rate of failure in tendon repair surgery. Up to 10% of tenolysis surgeries fail due to re-rupture or fixation of the tendon as a result of adhesions. Current practice to optimise clinical outcome following tendon repair centres on both surgical technique and rigorous post-operative physiotherapy, however many patients still experience stiffness and decreased function.

In light of the above, it will be appreciated that injuries of the tendons of the hand may be preferred tendon injuries to be treated with medicaments or methods in accordance with any aspect of the invention. Details of the ways in which such treatment may be practiced are set out below.

The medicaments or methods of the invention are suitable for prophylactic use, for example administration prior to injury of a tendon as a result of a surgical procedure. In such cases it will be appreciated that the site of injury should be taken to encompass sites at which an injury to a tendon will occur. Routes of administration

The medicaments of the invention may be formulated for use in any appropriate method by which a composition having an osmolality of at least 1200mOsmol can be provided to a site where it can inhibit tendon adhesion formation.

It is preferred that the medicaments of the invention are administered into a tendon, at a tendon's surface, or adjacent to a tendon (routes of administration that may alternatively be termed intra-tendinous, epi-tendinous, or peri-tendinous, respectively). In the case of a sheathed tendon, administration to the tendon's surface or adjacent the tendon may be achieved by administration of the medicament within the tendon sheath.

The medicaments or methods of the invention may preferably make use of administration by injection. Injection into the sheath surrounding the tendon represents a preferred route of administration for use in accordance with the medicaments or methods of the invention. Without wishing to be bound by any hypothesis the inventors believe that the sheath may serve to maintain the medicaments in contact with the tendon, thereby improving the efficacy of the medicaments.

A suitable volume of a medicament to be administered may be determined with reference to the cross sectional area of the tendon, with larger or smaller tendons, or larger or smaller injuries, requiring the administration of larger or smaller volumes of a medicament. It will be appreciated that, in the case of administration into the sheath of sheathed tendons, the amount of a medicament of the invention to be administered may be influenced by the volume of medicament that can be retained within the sheath.

Without wishing to limit the uses of the medicaments or methods of the invention, the following provides a preferred protocol by which the medicaments or methods may preferably be used in a clinical context.

Medicaments of the invention may preferably be administered after any surgical procedure on a tendon to be treated has been completed. By way of example, when the medicaments of the invention are to be used in connection with surgery to repair the digital flexor tendon they may be administered to the flexor sheath at the level of the injury/tendon repair.

The following provides details of suitable routes of administration (as part of a suitable method of treatment) by which medicaments of the invention may be used in the treatment of injuries associated with tendons of the hand. The treatment of such injuries, and the particular suggestions regarding treatments set out below, represent preferred embodiments of the invention.

Surgical procedures involving the treatment of tendons, such as those of the hands, may be performed under sterile conditions using an operating microscope; or under loupe magnification. A bloodless surgical field may be maintained during the tendon repair procedure using a pneumatic tourniquet. After retrieval/preparation of the two ends of the severed tendon, repair may be performed using a 4 strand core suture and an epitendinous suture. Pulleys may be vented as necessary. Following tendon repair the tourniquet, if used, may be deflated and full haemostasis may be achieved prior to administration of a medicament of the invention. Once haemostasis has been achieved the medicament of the invention may be administered. The sheath may be left open following treatment.

In administering the medicaments of the invention, a cannula (preferably an 18G cannula) may be inserted 1 -3cm proximally into the flexor sheath canal. Care should be taken not to damage the tendons or repair site. A volume of 150μΙ_ of a medicament of the invention may be instilled slowly into the sheath as the cannula is withdrawn. The cannula may then be removed and inserted distally along the flexor sheath canal and a further volume of 150μΙ_ of a medicament of the invention may then be instilled in exactly the same way to give a total volume of 300μΙ_ of the medicament of the invention administered to the site of injury.

After the medicament of the invention has been administered, the digit may be moved through its full range of flexion and extension in order to facilitate spreading of the medicament within the sheath and ensure that all surfaces of the tendon are coated. The wounds may then be closed in accordance with the surgeon's usual practice.

Post-operatively the digital wound(s) may be dressed with an appropriate nonadherent dressing. The hand may be splinted with a Plaster of Paris dorsal extension blocking splint, with the MCP joint in flexion. A high arm sling (such as a Bradford sling) may be applied in the operating theatre. The plaster of Paris splint may subsequently be exchanged for a lightweight thermoplastic (or equivalent) dorsal extension blocking splint

A medicament of the invention may also be used to bathe the wound site following injection. This technique may be applied to wounds and treatments on sheathed or unsheathed tendons, and may result in improved efficacy of treatment.

Timing of administration

The medicaments of the invention may be of benefit if provided at any time before the healing process is completed. That said, it generally be preferred that the medicaments or methods of the invention be utilised within seven days of an injury occurring, more preferably within four days, yet more preferably within three days, and most preferably within two days of injury. These timings of administration are particularly suitable for use in the treatment of accidental injuries.

It will be appreciated that surgical intervention may give rise to intentional injuries, and that such intentional injuries may be formed some time after an earlier injury; for example in tenolysis surgery. In the case of surgical injuries of this sort it may be preferred that the medicaments or methods of the invention are utilised around the time of surgery, and preferably at the time of surgery (for example just before a surgical wound is closed). Prophylactic use of the medicaments or methods of the invention represents another preferred embodiment.

Formulation

The medicaments of the invention (or any other medicament for use in a method of the invention) may be formulated in any appropriate manner, selected with reference to the routes of administration, and capable of producing a medicament having an osmolality of at least 1200mM. Suitable formulations may be selected in accordance with known protocols, including the use of animal models and clinical trials. Methods of preparing suitable formulations will be well known to those skilled in the art and are set out in texts such as Remington's Pharmaceutical Sciences 18^th Edition (1990). Accordingly, the following information should be viewed as of relevance for guidance only, save for where it provides information that would not be apparent on consideration of the prior art.

A preferred example of a formulation of a medicament of the invention suitable for administration by injection into the tendon sheath comprises:

A 1500mOsmol solution of M6P (equivalent to 16.93mg/100^L) formulated and dissolved in phosphate buffered saline (PBS) at pH 6.5 to 7.5 to form a colourless solution.

The medicaments of the invention may be supplied in 2ml_, sterile glass vials (with a 1 ml_ fill volume). The medicaments of the invention may take the form of a slightly viscous (1 .7 - 2.2 mPa.s at 25^<€) solution.

Although they utilise solutes, such as M6P at a high osmolality, the medicaments of the invention may preferably have a relatively low viscosity. The inventors believe that this is of benefit in allowing capillary uptake of the medicaments of the invention around the site at which they are administered, thus improving availability of the medicament at the injured site.

Viscosity of a medicament of the invention may be assessed using a rheometer. Merely by way of example, a medicament of the invention utilising 1500mOsmol M6P may have a viscosity of between approximately 1 .5mPa.s and 3mPa.s, more preferably between 1 .6mPa.s and 2.6mPa.s, and most preferably between 1 .7mPa.s and 2.2mPa.s, as referred to above.

It may be preferred that M6P be used in the form of its disodium salt. The disodium salt has greater solubility in aqueous solvents than does the monosodium salt, and this is of benefit in obtaining the high concentrations of M6P used in the medicaments or methods of the invention.

The medicaments or methods of the present invention may preferably be used in the inhibition of tendon adhesion formation after injuries of human subjects. That said, it will be appreciated that non-human animals are also prone to tendon injury, and may also benefit from the medicaments or methods of the invention. The medicaments or methods of the invention may be of particular benefit in the treatment of non-human domestic or agricultural animals, or of animals having high monetary values (such as racehorses, or the like).

Clinical end points for assessment of tendon function

The extent of clinical function may be assessed on a regular basis, often associated with treatment such as physiotherapy. The final extent of hand tendon function, and optionally the need for tenolysis surgery in the case of poor functional outcomes, is conventionally assessed approximately six months after treatment.

Suitable clinical assessments of function may be selected with reference to the site of injury that has been treated. Generally, assessments will consider the flexion or extension achievable. In the case of injuries to the wrist, a suitable assessment may address the range of movement that can be achieved. In the case of injuries to the foot, a suitable assessment may address gait and the ability of the foot to bear weight. In the case of injury to the Achilles tendon a suitable assessment may address the patient's ability to stand on tip toes.

The following assessments are particularly useful in assessing function of a previously injured finger may, but may be adapted for use in assessing function after injuries at a range of body sites.

Flexion

Flexion may be assessed at the Proximal Interphalangeal Joint (PIPJ) and Distal Interphalangeal Joint (DIPJ) using a goniometer for both passive and active movement.

Extensor Lag

The combined extensor lag at the PIPJ and DIPJ may be measured using a goniometer for both active and passive movement.

Quality of Motion

An assessment may be made of the quality of tendon gliding, by examination and subject questioning about the stiffness of motion in the affected finger(s). Quality of motion will be categorized as below:

Excellent: normal gliding - no resistance

Good: mild resistance to gliding Fair: moderate resistance to gliding

Poor: marked resistance to gliding or "triggering"

Performance analysis

An analysis of performance may be made for the active Range of Motion at week 26 after treatment. Summary statistics of further performance data may be calculated to facilitate the design of future studies.

Flexion and extensor lag (degrees) for the PIPJ and DIPJ joints for both passive and active movement may be collected. These measurements may then be used to calculate the active Range of Motion (ROM) using the following formula:

(PIP Flexion + DIP Flexion) - (Extension Deficit of DIP and PIP) = Range of Motion

Active Range of Motion at week 26 may be analysed using a two-group t-test. This along with active Range of Motion data from other time points will be summarised using descriptive statistics (mean, standard deviation, median, range and interquartile range). Passive Range of Motion may also be calculated and summarised in a similar manner.

The flexion and extension deficit measurements may be used to calculate Total Active Motion (TAM) percentage using the following formula:

(PIPFlexwn + DIP Flexion}- (Extension Deficit <tf DlP ndPIP) , .

:— ¹— _: - ^{: 1} ¾ 100 = % of .Normal motion

These data may be summarised using descriptive statistics. Total Passive Motion may be calculated and summarised similarly.

The ROM and TAM measures may be calculated appropriately based on such studies. The active and passive flexion and extensor lag measurements may also be summarised using descriptive statistics.

Recovery of movement may also be assessed by categorising TAM percentage using Strickland's Original classification as follows: Grade Percentage range

• Excellent 85 - 100%

• Good 70 - 84%

• Fair 50 - 69%

• Poor 0 - 49%

This may be summarised by determining the number and percentage of subjects with each severity grade by visit.

Quality of motion may be assessed by classifying the tendon gliding quality into one of the following categories.

Quality of Motion Description

• Excellent: Normal gliding - no resistance

• Good: Mild resistance to gliding

• Fair: Moderate resistance to gliding

• Poor: Marked resistance to gliding or "triggering"

This may be summarised by determining the number and percentage of subjects with each Quality of motion category by visit.

The invention will now be further described with reference to the accompanying Experimental Results and Figures in which:

Figure 1 is a graph comparing the percentage reduction in adhesion area achieved on treatment with a medicament of the invention (600mM, 1500 mOsmol) or with lower doses of M6P such as those taught by the prior art (50mM, 398mOsmol) and or other doses of M6P not encompassed by the medicaments of the invention (200mM, 697 mOsmol);

Figure 2 is a graph comparing the percentage improvement in tendon polarisation achieved on treatment with a medicament of the invention 600mM, 1500 mOsmol) or with lower doses of M6P such as those taught by the prior art (50mM, 398mOsmol) and or other doses of M6P not encompassed by the medicaments of the invention (200mM, 697 mOsmol); Figure 3 is a micrograph comparing histological sections (stained with haematoxylin and eosin) of tendons experimentally treated with a medicament of the invention (left hand panel) or a control (right hand panel) and illustrating that medicaments of the invention are able to improve tendon repair by decreasing tendon adhesion formation;

Figure 4 is a micrograph comparing polarised images of histological sections (stained with haematoxylin and eosin) of tendons experimentally treated with a medicament of the invention (left hand panel) or a control (right hand panel) and illustrating that medicaments of the invention are able to improve the quality of replacement tendon formed after injury by increasing the organisation of collagen;

Figure 5, in a first panel, shows immunoblots comparing the level of phosphorylation of p38 over time in cells exposed to medicaments of the invention (comprising either M6P or G6P) or to controls;

Figure 5, in a second panel, shows micrographs of cells cultured in the presence of medicaments of the invention (comprising M6P or G6P) or controls, where the cells have been exposed to FITC phalloidin, which binds to filamentous actin thereby allowing their visualisation;

Figure 6 shows graphical representations of data comparing chemokinesis and chemotaxis of cells exposed to medicaments of the invention (comprising M6P or G6P) or appropriate controls;

Figure 7 shows graphs illustrating cell proliferation over time when exposed to medicaments of the invention (comprising M6P or G6P) for varying periods compared to appropriate controls;

Figure 8 shows micrographs of explant cultures in which explants are exposed to medicaments of the invention compared to appropriate controls;

Figure 9 shows micrographs of optical sections through various whole wounded or unwounded tendons subjected to treatment with the medicaments of the invention or control treatments, in which live and dead cells are differentially labelled; Figure 10 shows micrographs of tissue sections taken from unwounded tendons (C panels), tendons treated with a medicament of the invention (A panels), and tendons treated with a PBS control (B panels), in which immunhistochemistry allows visualisation of distribution of the M6P receptor (CI-M6PR), the TGF-β receptor 1 (TGF-βΡιΙ ), Smad 2 and Smad 3;

Figure 1 1 shows haematoxylin and eosin staining of histological sections, comparing structures in surgically lacerated tendons after no treatment (panel A) and treatment with a medicament of the invention (panel B);

Figure 12 shows micrographs illustrating morphology of cells over time after treatment with medicaments of the invention comprising either M6P or G6P, non- treated control cells are also shown for comparison;

Figure 13 is a graph showing force-extension curves generated using a tensiometer , and comparing results achieved in tendons treated with a medicament of the invention (solid black line), tendons receiving no treatment (solid grey line) and tendons treated with placebo (dotted line); and

Figure 14 is a graph illustrating tensiometry data generated from tendons treated with a medicament of the invention, tendons receiving no treatment, and tendons treated with placebo, and comparing maximum force required to achieve tendon breakage in Newtons ("N"), maximum force required to achieve tendon breakage normalised to the size of the tendon ("N/mm2") and Young's modulus ("Youngs") indicative of the tendon's elastic properties.

Experimental Results

Methods:

• Study investigated the effect of M6P on adhesion formation in mice tendon healing.

• Male C57 Black-6 mice (aged 10-12 weeks) were used, 5 animals allocated to each of the drug groups

- M6P; 50mM (in PBS)

- M6P; 200mM (in PBS)

- M6P; 600mM (in PBS)

• Mouse tendon wounding was performed on the deep digital flexor tendons of both hind paws. Surgery involved exposure of the deep digital flexor tendon and partial laceration of the tendon (involving approximately 50% of the tendon fibres). The tendon injury of one hindpaw was treated once, just before closure of the skin wound, with either 2μΙ_ of Mannose 6 phosphate at 50mM, 200mM or 600mM (which was infiltrated into the sheath using a glass micropipette) and the contra-lateral hindpaw tendon injury was treated once with 2μΙ_ of 0.9% (w/v) saline as a control treatment.

• Following administration of treatment the skin was closed over the tendon wound with a single 10/0 polyamide suture and the hind paw cleaned with 0.9% saline. Following tendon injury, the hind paws were immobilised (to promote tendon adhesion) by a second incision distal to the ankle joint wherein the common deep and superficial flexor tendons were completely divided, with the proximal end buried to avoid the tendon reuniting.

• At eight weeks following injury, animals were euthanized and both hind paws harvested and processed for histology (at 8 weeks the adhesion is mature with collagen synthesis and cellular proliferation back to baseline levels). To calculate the area of adhesion and the volume of the deep digital flexor tendon, a total of five histological sections were analysed per tendon covering a depth of 280 μηι.

• For each animal, the adhesion area of the medicament-treated and control saline-treated tendons was tabulated. The medicament-treated adhesion area values per animal were then expressed as either a difference from, or a percentage of, the contra-lateral saline-treated tendon adhesion area. Statistical significance was calculated using a paired Student's t-test.

• Remodelling of the tendon architecture was also measured by layering histological images onto polarised images. Briefly, images captured of haematoxylin and eosin stained histology with bright field microscopy where captured in precisely the same position with the polarising filter (Leica Microsystems, Germany.), sited at 45° to the tendon which caused the tendon aligned to give maximum polarisation. Images were analysed on Image Pro Plus version 4.5 (Media Cybernetics, USA) and the area of tendon was mapped using the outlining function on haematoxylin and eosin stained images. The latter image was layered onto the polarised image to generate a precise outline on the polarised image.

Culture of Tendon Fibroblasts

Flexor tendons from rat hindpaws were dissected out and placed into L15 air- buffered medium. Excised flexor tendons were then minced into 5mm tissue pieces using a sterile scalpel blade and the tendon tissue seeded into a Petri dish. Growth medium (DMEM/10% FBS (v/v)) was slowly added to the tissue which was then incubated for 3 days to allow fibroblast outgrowth. After 3 days the growth medium was replaced and cells were cultured until 80% confluent. Cells were subcultured by incubation in Trypsin/EDTA for 5mins at 37^<C/5%C0₂ and centrifuged at 2000rpm for 5mins. Pelleted cells were resuspended and seeded in new culture vessels at a density of 6.2x10³ cells/cm².

Cell Proliferation

Tendon fibroblasts were plated out at 10⁴ cells per well in a 96 well plate, and incubated overnight at 37^°C to allow the cells to attach to the culture vessel. Cells were exposed to M6P or G6P for 1 , 5, 10, 15, 30 and 45 min. Following exposure, M6P or G6P were removed and all wells washed twice with PBS, and wells were treated with growth medium (DMEM + 10% FBS) to allow recovery and cell growth. A positive control was included which was cells without medicament treatment. Cell proliferation was determined using a cell counting kit (CCK-8, Fluka) at 16h, 40h, 64h, 160h, 184h and 208 h post treatment. The percentage cell proliferation relative to the positive control (DMEM/10% FBS with no treatment) was determined.

Cell Migration Assay (Chemotaxis)

Tendon fibroblasts were seeded onto a 96-well transwell filter (pore size 8μηι), at 5x10⁴ cells/filter, in 50μΙ_ DMEM/10%FBS. DMEM/10%FBS was added into the lower chamber at a volume of 150μΙ_. Cells were incubated overnight to allow cell adhesion to the filter. Following incubation, medium was aspirated from upper and lower chambers and filters washed with sterile PBS. Cells were incubated in M6P or G6P, added to both upper and lower chambers of the transwell, for various lengths of time. Following exposure to treatment, chambers were washed in PBS as described above. To initiate cell migration, DMEM/10%FBS was added to the lower chamber of the transwell, and serum-free DMEM was added to the upper chamber. Cells were incubated overnight. Following incubation, medium from the upper chamber was aspirated off and the well inserts were placed into a receiver plate containing accutase. Transwell inserts were incubated in accutase for 5mins to remove migrated cells from the underside of the filter into the receiver plate. Cell Titre Glo/10%FBS was added to the accutase containing migrated cells. The receiver plates were shaken for 2 mins and then incubated at room temperature for 10mins, following which migrated cell numbers were determined using a SpectraMax plate reader.

Cell Migration Assay (Chemokinesis)

Tendon fibroblasts in DMEM/10%FBS were seeded into a 24-well plate at 2.1 x10⁴ cells/well and incubated overnight. Medium was removed and cells washed with sterile PBS. Cells were treated with M6P or G6P. Following treatment, cells were washed in sterile PBS and incubated in DMEM/10%FBS and the movement of fibroblasts assessed using timelapse video microscopy. Cells undergoing no treatment were exposed to DMEM/10% FBS (positive control) or DMEM alone (-ve control). Images were taken every 15mins, over a 20h duration, at x7.5 magnification. Resultant timelapse images were imported into ImageJ software and the motility cells in each treatment recorded by use of the Manual Tracking plugin. Motility data was then imported into the Chemotaxis and Migration Tool plugin (Ibidi, Germany) to generate plots of migration paths and distances from a normalised point of origin. Further comparisons between treatment groups were made determining the % of cells migrating pre-determined distances.

Analysis of p38 Phosphorylation

Rat tendon fibroblasts were seeded at 300,000 cells per well of a 6 well plate in DMEM supplemented with 10% FBS, 0.1 mM non essential amino acids, 2mM glutamax and 100 U penicillin/100μ9 streptomycin. After one day the cells were serum starved in DMEM supplemented with 0.1 mM non essential amino acids, 2mM glutamax and 100U penicillin/100μ9 streptomycin. The cells were then treated with M6P or G6P for 5, 10, 15, 30 and 60 minutes with untreated cells as a control. After treatment the cells were lysed in lysis buffer. Samples were then subjected to gel electrophoresis, transferred onto nitrocellulose and immunblotted with an antibody to phosphor-p38 and visualised.

Ex Vivo Cell Migration and Proliferation

Flexor tendons from rat hindpaws were dissected out and placed into L15 air- buffered medium. Tendons were oriented longitudinally in collagen-l matrix and incubated for 1 h to allow the collagen to polymerize. DMEM/10%FBS was added and the tendons were incubated for 48h. Following incubation the dissected tendon was photographed using a digital camera attached to a light microscope. Medium was then removed from tendon explants and replaced with M6P or G6P for 1 h. After the 1 h incubation the treatment was removed and replaced with DMEM/10%FBS. Tendons exposed to DMEM/10% FBS alone acted as positive controls. Tendons were photographed every 24h to assess and compare the outgrowth of fibroblasts from tendons.

In Vivo Cytotoxicity Assay

The middle digit, flexor digitorum profundus (FDP) tendons of C57/BLJ mice were either unwounded, wounded + no treatment, wounded and treated with PBS or wounded and treated with M6P. Animals were then harvested at 30min, 24h and 72h post surgery and treatment. Using an operating microscope at 10-40 times magnification, the FDP tendon was dissected free and immediately placed in sterile C0₂ independent media containing Live/Dead assay kit solution containing calcein AM and ethidium homodimer and incubated at 37^<€, 5% C0₂ for one hour prior to inspection by fluorescent microscopy. Using this method live cells appear green and dead cells appear red.

Results:

• At 8 weeks post-surgery, the data showed a significant reduction in tendon adhesion formation compared to saline control for the 600mM M6P treatment group with a mean reduction in adhesion area of 85669 μηι2 and a percentage reduction in adhesion area of 47 % (p = 0.02). In addition, the data showed a dose-responsive percentage reduction in adhesion area: 28 % reduction for the 50mM M6P treatment group, 25 % for the 200mM M6P treatment group and 47.5% reduction for the 600mM M6P treatment group (Figure 1 )

• Histologically, notable differences in the amount of adhesion that formed were evident in 600mM treated tendons when comparing paired samples (Figure 3)

• Restoration of normal tendon architecture was measured by the degree of polarisation. The treatment of tendon wounds with 50mM and 200mM did not significantly improve polarisation. Of all the treatments only 600mM of M6P was found to significantly improve the polarisation, and therefore the collagen organisation of the tendon (p = 0.047). Comparing data from M6P treated tendon wounds with non wounded tendon, indicated that the polarisation of the M6P treated tendon was 87.7% compared with 100% in unwounded tendon. (Figures 2 and 4)

• Analysis of the level of phosphorylation of p38 over time in cells exposed to medicaments of the invention (comprising either M6P or G6P) or to controls showed that the medicaments of the invention were able to rapidly induce p38 phosphorylation (Figure 5).

• Cells cultured in the presence of medicaments of the invention (comprising either M6P or G6P) underwent cytoskeletal reorganisation, with disruption of the structures found in cells exposed to control treatment (Figure 5).

• Chemokinesis and chemotaxis were both decreased in cells exposed to medicaments of the invention as compared to the levels in controls (Figure 6).

• Cell proliferation was also transiently decreased in cells exposed to medicaments of the invention (comprising M6P or G6P). The extent and length of the decrease observed increased as the length of time that the cells were exposed to the medicaments increased (Figure 7).

• Tendon explants cultured in the presence of medicaments of the invention demonstrated decreased cellular outgrowth as compared to controls (Figure 8).

• Analysis of the levels of live or dead cells in tissues treated with the medicaments of the invention or controls indicated that medicaments of the invention did not significantly increase cell death compared to control treatment, and thus were not cytotoxic (Figure 9).

Medicaments of the invention achieve their therapeutic effects independent of TGF-β activity

The possible influence of the medicaments and methods of the invention on TGF-β signalling at sites where the medicaments were administered was investigated as follows.

Methods

Flexor tendons in the hind paws of C57 black 6 mice were experimentally injured by partial surgical laceration. The lacerated tendon was then treated with either a medicament of the invention (a 1500mOsmol solution of M6P/M6P at a concentration of 600mM), or isotonic PBS (which served as a control).

At days 1 -3 after injury the experimental animals were euthanized and the tendons treated with medicaments of the invention or control solution recovered and processed for immunohistochemistry.

Suitable specific antibodies were used to visualise the distribution of the M6P receptor (CI-M6PR), and the downstream signalling components TGF-β receptor 1 (TGF-βΡιΙ ), Smad 2 and Smad 3. The distribution of these targets in the tendons treated with medicaments of the invention or control solutions was compared with that found in unwounded tissues.

Photomicrographs comparing immunohistochemistry in these treated, control treated, and unwounded tissues are shown in Figure 10.

Results

The results of the immunohistochemical analysis illustrated that injured tendon lacked the CI-M6PR, providing a clear indication that this receptor, which interacts with the TGF-β signalling pathway is not involved in generating the therapeutic effects observed. This was further confirmed by the injured tendon's lack of molecules such as TGF-βΡιΙ , Smad 2 and Smad 3 that are necessary for propagation of signals associated with the TGF-β pathway. In contrast, these markers were found in uninjured regions of the tendon (where their presence served as a positive control).

These findings clearly illustrate that medicaments of the invention achieve their activity in a manner that is independent of the TGF-β signalling pathway. This is consistent with the fact that agents such as M6P incorporated in medicaments of the invention are used at far higher concentrations than those that have previously been shown to beneficially influence the TGF-β signalling pathway.

Medicaments of the invention exert an influence at the tissue level via hydroflotation

Figure 1 1 shows histological sections comparing the structures found at sites of tendon injury treated with a medicament of the invention (panel B), with those found in tendons where an equivalent injury is not provided with treatment (panel A).

As can be seen in panel A, when no treatment is provided then the tendon and sheath are found in close proximity to one another. This creates conditions that predispose towards adhesion formation (typically associated with an ingrowth of cells from the tendon sheath towards the tendon).

In contrast, panel B illustrates that treatment with the hyperosmotic medicaments of the invention causes an osmotic influx of intracellular and extracellular water to the site where the medicament has been provided. This water causes hydroflotation that separates the tendon and tendon sheath, and creates a barrier that discourages ingrowth of cells. Hydroflotation induced by the medicaments of the invention thus contributes to an inhibition of tendon adhesion formation.

Medicaments of the invention exert an influence at the cellular level via cell crenation

Cells subject to osmotic stress undergo crenation, in which the cell size is rapidly reduced as a result of water loss. Crenation, and other processes associated with osmotic shock, give rise to a transient reduction in cell functions associated with cell migration and proliferation. Methods

Rat flexor tendons were grown in DMEM containing 10% fetal bovine serum (FBS) and then transferred to media without FBS. After transfer cultured cells were treated with a medicament of the invention comprising either M6P or G6P, and maintained in the presence of this medicament for at least 60 minutes. Control cell populations were maintained in serum-free DMEM for the same period. Micrographs showing cell morphology were taken at various times during this period.

Results

Figure 12 shows micrographs illustrating that medicaments of the invention (comprising either M6P or G6P) give rise to cell crenation in cultured rat flexor tendon cells over a period of 60 minutes after administration. This effect is observed in medicaments based on either M6P or G6P, but is not found in cultured cells that are not exposed to medicaments of the invention.

These results illustrate not only that the medicaments of the invention are able to cause crenation in tendon cells, but also show clearly that the medicaments are able to achieve activity independent of the solute used to achieve the requisite osmolality.

Tendon Residency Experiments

The following study, utilising a rabbit model of flexor tendon injury, was undertaken to determine residency of the medicaments of the invention at the site of administration. The ability of medicaments of the invention to achieve a beneficial effect during only a relatively short period of residency would provide a further indication that the medicaments of the invention achieve their activity through osmotic shock, rather than via cellular receptors.

Method

New Zealand White rabbits were pre-medicated with atropine and ketamine prior to induction of anaesthesia with isofluorane in oxygen. The forepaws of the rabbits were shaved and cleaned before exposing the digital flexor tendons. These were then wounded with a 50% partial laceration followed by administration of 80μΙ_ of a medicament of the invention (comprising M6P at 1500mOsmol) to the tendon sheath space. Liquid samples were collected from the intra-synovial junction of the treated tendon sheath at regular intervals and diluted in water to a suitable range for high performance anion exchange with pulsed ampourimetric detection (HPAE-PAD) quantification (i.e. 1 :100 followed by a 1 :25 dilution in PBS).

20μΙ_ of the diluted sample was injected on a strong anion exchange column designed for selective carbohydrate separations. M6P is eluted using a gradient of 47.5 mM sodium hydroxide and 500 mM sodium acetate at 1 mL/min over 20 min, and detected using a Four-Potential Waveform.

Results

Analysis indicated that the medicaments of the invention had a half-life at the site of administration of less than 2 hours when administered into the tendon space of an injured (severed) tendon in rabbit in vivo. This short half-life is consistent with the suggestion that the medicaments of the invention achieve their activity via osmotic shock of the cells at the site of injury, rather than by a pharmacological effect (e.g. through antagonism of cellular receptors).

Investigation of lubricity of the medicaments of the invention

The following in vitro study was taken to determine the lubrication properties of a medicament of the invention comprising a 1500mOsmol solution of M6P.

Methods

In a friction test using a AR2000 research rheometer, a fixed downward force (10N) was applied to the upper plate and no gap was defined. The upper plate was then rotated at a known rate and rubs against the lower plate, lubricated by a very thin film of a sample. The torque required to maintain the defined rotational rate was recorded throughout the test. Samples used include the medicament of the invention and phosphate buffered saline (PBS). These were tested in comparison to a number of controls (de-ionised water, a light general-use grease and a highly lubricious cellulose gel "Ultraslime"). Tests were performed in triplicate with the first run being discounted to an experimental artefact.

Results

The medicament of the invention demonstrated an increase in lubrication properties (lubricity) compared to an isotonic solution of PBS (as exemplified in rotating plate friction testing), indicating that treatment with the medicaments of the invention in vivo may contribute to maintaining the gliding properties of the flexor tendon. It has previously been shown that lubrication of tendons (using treatments other than those in accordance with the present invention) results in improved functional outcomes following flexor tendon injury and repair. Without wishing to be bound by any hypothesis, the inventors believe that the lubricating properties of the medicaments of the invention may contribute to the beneficial effects observed.

Investigation of replacement tendon quality using tensiometry

A study was conducted to investigate the ability of the medicaments and methods of the invention to improve the mechanical properties of treated tendons, as opposed to untreated tendons, as assessed by tensiometry. Improved mechanical properties of treated tendons may be taken as indicative of improved quality of replacement tendon tissue formed on healing of wounds treated with the medicaments or methods of the invention.

The study was undertaken using 15 female New Zealand White rabbits. Each rabbit underwent unilateral complete transection of the deep flexor digitorum tendon. The transacted tendon was then repaired immediately with modified Kessler suture (a surgical technique well known in the field of tendon repair).

Methods

N=5 experimental animals were assigned to each of the following groups:

• Placebo treatment (consisting of PBS)

• Treatment with a medicament of the invention (comprising 600mM M6P) N=2 experimental animals were assigned to a group receiving no treatment (shown as "6 wk untreated" in the accompanying Figures).

Briefly the surgical procedure involved a longitudinal incision in the volar surface of the middle digit of the right forepaw. The incision extended between the metacarpophalangeal and proximal interphalangeal joints. Overlying tissues were carefully dissected to expose the flexor digitorum pfofundus tendon. The tendon was transacted between the A3 and A4 pulleys with the wrist and digit extended. The tendon was then immediately repaired using Prolene 5-0, with a modified Kessler suture. The in the case of those groups receiving placebo treatment or treatment with a medicament of the invention, 100μΙ_ of the appropriate solution was then applied to the tendon repair site and surrounding tissues and tendon sheath placed back in position. The subcutaneous tissue was closed using Monocryl 4-0- sutures and the skin was closed using Prolene 4-0.

After surgery and treatment the relevant limb was held in flexed position with cast for 7 weeks.

At 7 weeks post surgery the experimental animals were euthanized, their casts removed, and the injured/repaired tendon separated by micro-dissection (after removal of the superficial tendon).

Mechanical properties of the tendons were assessed by tensiometry. The microdissected tendons were mounted into the pneumatic grips of the tensiometer and stretched at the rate of 20 mm/min. Tensile strength test was terminated once failure was observed.

Results

Individual force-extension curves generated using a tensiometer are shown in Figure 13. This Figure compares tensiometry measurements from tendons receiving no treatment (solid grey line) as well as those treated with placebo (dotted line), and a medicament of the invention (solid black line). It can be seen from this Figure that the greatest ability to withstand applied force was exhibited by tendons treated using the medicaments of the invention.

Figure 14 shows mean tensiometry data obtained from wounded/repaired FDP tendon receiving no treatment as well as those treated with placebo, and a medicament of the invention. In this Figure, "N" represents the maximum force required to achieve failure of the tendon (i.e. breakage) in Newtons; "N/mm2" represents maximum force required to achieve failure of the tendon normalised to the size of the tendon; and "Youngs" represents the Young's modulus indicative of the tendon's elastic properties. An increased Young's modulus value indicates that the injured/repaired tendon has improved mechanical properties. It can be seen that tendons treated with the medicaments of the invention exhibit increased breaking strength and increased Young's modulus as compared to untreated tendons or tendons treated with placebo.

Conclusions

The tensiometry study illustrated that treatment of injured tendons with medicaments of the invention increased breaking strength of the FDP tendon.

While the sample group investigated was too small to allow significantly significant results to be generated the trend shown by the results is that treatment with the medicaments of the invention increases maximum load at 7 weeks post-surgery - compared to both placebo treated and untreated controls.

A similar trend is shown with regard to the elastic properties at of tendons at seven weeks post-surgery - where tendons treated with the medicaments of the invention exhibit improved elasticity as compared to both placebo treated and untreated controls.

All of these results indicate that not only are the medicaments of the invention able to inhibit incidences of adhesion formation, but they are also able to increase the quality of replacement tendon formed after injury.

Claims

Claims

1 . A composition having an osmolality of at least 1200mOsmol for use as a medicament for the inhibition of tendon adhesion formation.

2. A composition according to claim 1 , wherein the osmolality of the composition is between 1200mOsmol and 5000mOsmol.

3. A composition according to claim 1 or claim 2, wherein the osmolality of the composition is between 1400mOsmol and 3000mOsmol.

4. A composition according to any preceding claim, wherein the osmolality of the composition is approximately 1500mOsmol.

5. A composition according to any preceding claim, wherein the composition comprises an organic compound selected from the group consisting of: a hexose, substituted hexose (e.g derivatives of sulfur, phosphorus, nitrogen, heavy metals), derivitised hexose, pentose, substituted pentose (e.g derivatives of sulfur, phosphorus, nitrogen, heavy metals), derivitised pentose, organic acids (e.g. citric acid), organic carboxylic acids, substituted carboxylic acids and derivatives such as acid anhydrides, esters, amides, monosaccharides, disaccharides, oligosaccharides, substituted monosaccharides, derivitised monosaccharides, substituted disaccharides, derivitised disaccharides, substituted oligosaccharides, derivitised oligosaccharides, fatty acids, nucleic acids, nucleosides, nucleotides, boronic acids, poly-ethylene glycols, glycerine, glycerols, amino acids, substituted amino acids, organic primary amines, secondary amines, tertiary amines and quaternary amines, mixtures of the above, and salts derived from the above.

6. A composition according to any preceding claim, wherein the composition comprises a sugar phosphate.

7. A composition according to any preceding claim, wherein the composition comprises mannose-6-phosphate (M6P) and/or glucose-6-phosphate (G6P).

8. A composition according to claim 7, wherein the composition comprises M6P.

9. A composition according to any preceding claim, wherein the composition is resident at the wound site for a period of time suitable to inhibit tendon adhesion formation, without eliciting adverse effects on tendon healing.

10. A composition according to any preceding claim, wherein the osmolality of the composition is sufficient to induce at least one pathway or response from the group consisting of:

i) up-regulation of p38 phosphorylation; and/or

ii) disruption of the cytoskeleton; and/or

iii) a decrease in cell motility; and/or

iv) a decrease in the rate of cell proliferation; and/or

v) a decrease in cell metabolic activity

in cells at the site to which the composition is administered.

1 1 . A composition according to claim 10, wherein the cell types to be affected by treatment at the site where the composition is administered are selected from the group consisting of: cells of the tendon, cells of the tendon sheath, and cells of subcutaneous tissues in proximity to the site of injury.

12. A composition according to claim 1 1 , wherein the cells to be affected by treatment at the site where the composition is administered are selected from the group consisting of: epitenon fibroblasts; endotenon fibroblasts; and tendon sheath fibroblasts.

13. A composition according to any preceding claim, wherein the medicament further improves tendon repair by increasing the quality of replacement tendon formed after injury.

14. A composition according to any preceding claim, for use in the treatment of tendon injuries in body sites selected from the group consisting: of limbs; extremities; the hand; the forearm; the shoulder; and Achilles tendons.

15. A composition according to any preceding claim, for peri-tendinous administration.

16. A composition according to any preceding claim, for epi-tendinous administration.

17. A composition according to any preceding claim, for intra-tendinous administration.

18. A composition according to any preceding claim for administration prior to injury.

19. A composition according to any preceding claim for administration to an existing injury.

20. A composition according to any preceding claim for use to reduce the requirement for subsequent tenolysis surgery.

21 . A composition having an osmolality of at least 1200mOsmol for use as a medicament for improving tendon function after injury.

22. A composition having an osmolality of at least 1200mOsmol for use as a medicament for the inhibition of tendon adhesion formation after injury.

23. A composition according to claim 21 or claim 22 comprising M6P.