WO2010063994A1

WO2010063994A1 - Non-surgical improvement of scars

Info

Publication number: WO2010063994A1
Application number: PCT/GB2009/002790
Authority: WO
Inventors: Mark William James Ferguson; Sharon O'kane; Mark Cooper; John Hutchinson
Original assignee: Renovo Ltd
Current assignee: Renovo Ltd
Priority date: 2008-12-01
Filing date: 2009-12-01
Publication date: 2010-06-10
Anticipated expiration: 2011-06-01
Also published as: TW201023878A; AR074441A1; GB0821941D0

Abstract

The present invention provides TGF-β3 for use as a medicament for provision to a scar to bring about the non-surgical improvement thereof. The medicaments may preferably be injectable medicaments. The invention also provides corresponding methods of treatment. A suitable amount of TGF-β3 to be provided may be between about 100 ng and 10,000 of TGF-β3 per centimetre of scar to be non-surgically improved, and is preferably about 500 ng or 1000 ng of TGF-β3 per centimetre of scar to be non-surgically improved. The scar to be improved may be a scar of the skin. Scars to be improved may be between 2 and 6 months post-wounding. Improvement using the medicaments or methods of the invention may reduce the redness and/or contrast of the scar.

Description

Non-surgical improvement of scars

The present invention relates to medicaments and methods for use in the non-surgical improvement of scars that have resulted from injury.

Many different processes are at work during the scarring response, and much research has been conducted into discovering what mediates these processes, and how they interact with each other to produce the final outcome. Many of the factors that influence the quality of the scar that is eventually formed have been found to be related to events occurring early in the wound healing process.

In the case of a scar that results from healing of a wound, the scar may be defined as the structure produced as a result of the reparative response. The body's reparative response to wounding has arisen as the evolutionary solution to the biological imperative to prevent the death of an injured animal. In order to overcome the risk of mortality due to infection or blood loss, the body reacts rapidly to repair the damaged area, rather than attempt to regenerate the damaged tissue. Since the damaged tissue is not regenerated to attain the same tissue architecture present before wounding, a scar may be identified by virtue of its abnormal morphology as compared to unwounded tissue.

The scarring response is common throughout all adult mammals. It involves a number of inter-related phases, which follow one another (with some degree of overlap) from the first moments of the injury, and ultimately result in the formation of a replacement tissue termed a "scar". The various phases influence one another, and it is this cumulative influence that determines the properties of the scar produced.

The first phase of the wound healing process is the inflammatory response. This is triggered by damage to blood vessels, which then leads to the clotting cascade. Degranulation of platelets, occurring as part of this cascade, causes the release of numerous soluble signalling factors (including TGF-βs) that recruit inflammatory cells to the site of injury. These inflammatory cells, predominantly of the monocyte/macrophage or neutrophil lineages, begin to remove debris left from the injury, deposit a provisional extracellular matrix (ECM) in the wound void, and release their own signalling molecules that cause recruitment of other cells types crucial to wound healing. These cells, the provisional matrix and further inwardly migrating cells are vital to the second phase of wound healing; granulation tissue formation.

Granulation tissue, so called because of its "granular" macroscopic appearance, is a mixture of inflammatory cells, fibroblasts, new extracellular matrix and new blood vessels that develops to fill the wound defect. Granulation tissue formation "peaks" at three to seven days post-wounding, depending on the species involved. Cells migrate into the wound area from the surrounding tissue and, once in situ, deposit new matrix components. The matrix deposited during this process replaces the provisional matrix laid down during inflammation, and is often deposited on top of the provisional matrix, which acts as a scaffold.

Extracellular matrix deposition continues for approximately two to four weeks after wounding, and it is the extracellular matrix laid down during this time that forms the basis for the scar left after the healing process is completed.

Wound contraction and re-epithelialisation are responsible for closure of the wound, as well as re-establishment of a functional covering. During wound contraction contractile elements in myofibroblast cells reduce the volume of the wounded area. At the same time, keratinocytes from the surrounding unwounded tissue (and, if present, from epidermal appendages remaining in the wounded area) migrate across the surface of the granulation tissue, thereby re-forming the epidermal barrier.

In the final phase of wound healing, scar remodelling, enzymes released by cells remaining in the scar slowly re-shape the matrix, thus contributing to maturation of the scar. Scar remodelling and maturation have been shown to occur in a number of stages during the twelve months following injury.

While the scarring response is well suited to serve its primary purpose of replacing lost tissue and preventing further damage or death through blood loss or infection, there are a number of ill effects associated with scarring. These tend to arise due to differences between the properties of the scar tissue produced and the original undamaged tissue that this replaces. Scar tissue may interfere with growth, cause deformities, impair function and prove aesthetically unattractive. Like the unwounded connective tissue they replace, scars are composed mainly of extracellular matrix. This material is deposited by cells present during the healing process to replace that which has been damaged or lost as a result of injury. The biological imperative appears to be to produce tissue that can fill the void at an injured site, rather than to produce an accurate replacement of that which preceded wounding. Often a scar will comprise connective tissue that has an abnormal organisation compared to unwounded tissue, as is frequently observed in scars of the skin. Alternatively or additionally, a scar may comprise connective tissue that is present in an abnormally increased amount. Most scars consist of both abnormally organised and excess connective tissue, as described further below.

In normal skin the extracellular matrix molecules form fibres which, when viewed microscopically, have a characteristic random arrangement that is commonly referred to as "basket-weave". This basket-weave arrangement is disrupted in scars. Fibres in scars exhibit a marked degree of alignment with each other as compared to the random arrangement of fibres in normal skin. In general the fibres observed within scars are also of smaller diameter than those seen in normal skin. Both the size and arrangement of ECM may contribute to the scars altered mechanical properties, most notably increased stiffness, when compared with normal skin.

Viewed macroscopically, scars may be depressed below the surface of the surrounding tissue, or elevated above the surface of their undamaged surroundings. Scars may be relatively darker coloured than normal tissue (hyperpigmentation) or may have a paler colour (hypopigmentation) compared to their surroundings. In the case of scars of the skin, either hyperpigmented or hypopigmented scars constitute a readily apparent cosmetic defect. It is also known that scars of the skin may be redder than unwounded skin, causing them to be noticeable and cosmetically unacceptable. It has been shown that the cosmetic appearance of a scar is one of the major factors contributing to the psychological impact of scars upon the sufferer, and that these effects can remain long after the cause of the scar has passed.

In addition to their psychological effects, scars may also have deleterious physical effects upon the sufferer. These effects typically arise as a result of the mechanical differences between scars and normal tissue. The abnormal structure and composition of scars mean that they are typically less flexible than their normal tissue counterpart. As a result scars may be responsible for impairment of normal function (such as in the case of scars covering joints which may restrict the possible range of movement) and may retard normal growth if present from an early age.

Scars occur at many body sites, and the effects of scarring at these sites will generally be related to loss or disruption of function in the scarred area. Some of the disadvantages associated with scarring of the skin have been discussed above. Scarring of the internal organs may lead to the formation of strictures and adhesions that significantly or totally impair function of the organ in question. Scarring of tendons and ligaments may cause lasting damage to these organs, and thereby reduce the motility or function of associated joints. Scarring associated with blood vessels, and particularly the valves of the heart, may occur after injury or surgery. Scarring of blood vessels may lead to restenosis, which causes a narrowing of the blood vessel and thus reduces the flow of blood through the scarred area. Scarring in the central and peripheral nervous system may prevent transmission along the nerve and may prevent or reduce reconnection of damaged nerve tissue, and/or functional neuronal transmission.

Given that scarring is a commonly occurring problem, and the number of damaging effects associated with scarring, there is a need to develop medicines and strategies that can be used to avoid or reduce scarring. Currently the majority of therapies intended to improve scarring focus either on reducing the extent of scar formation in response to injury, or on physical manipulation of scars that have been formed.

Perhaps the most commonly used approach to reduce the impact of scars is surgical excision. In this approach, the scar is excised, and steps taken to reduce the level of scarring that may be expected on healing of the new wound formed by this excision. Such steps include the use of plastic surgery techniques to reduce scar formation, realignment of the newly formed scar (to avoid crossing lines of tension, which may lead to increased scarring, or to camouflage the new scar in existing skin folds) and application of the best possible care post-surgery.

Various other techniques for surgical revision of scars are known to those skilled in the art. For example, elevated scars may be treated by means such as dermabrasion, in which the surface of the scarred area is precisely abraded away by mechanical treatment; lasers (CO₂, ErYag, Pulsed Dye) may also be used to "de-bulk" raised scars, and to more closely approximate the margins of scars with the height of the surrounding area. Cryotherapy, in which scars are subjected to extreme low temperature in an attempt to reduce scar bulk and colour differences, is a further technique by which scars may be surgically revised.

Another form of scar revision treatment uses Nd:Yag lasers to attempt to reduce redness that contributes to the visual impact of scars. Treatment of this sort is intended to destroy blood vessels in the scar, thus reducing the total blood vessel number and alleviating scar redness (also referred to as rubor).

Surgical revision techniques are currently widely used to beneficial effect, however they are not universally applicable. In particular patients who have already been subject to the trauma of scarring are frequently highly averse to undergoing additional surgical procedures that will, necessarily, be associated with further scarring. Furthermore, some of those most at need of scar revision are individuals with a predisposition to bad or pathological scarring. It may be unadvisable to subject such patients to further wounding lest the resultant scars prove worse than those already present.

A number of non-surgical methods of scar revision have been developed to^' act as an alternative to, or adjunct to, surgical scar revision. These non-surgical techniques tend to be popular with patients having an aversion to surgery. They may also be clinically useful in cases where a patient is predisposed to poor scarring results. Some of the most common non-surgical scar revision treatments include compression therapies (e.g. silicon sheets/gels, pressure garments, ear clips, hydrating creams/ointments); corticosteroid therapy; radiotherapy; anti-neoplasties (e.g. 5-fluorouracil); retinoic acid; Verapamil and immunomodulators (such as Imoqimod).

Perhaps the simplest method suggested to avoid the generation of wide scars is the application of adhesive tape over a wound site. It has been suggested that this treatment may be of use in maintaining the margins of the wound in contact with one another, and thus avoiding "spreading" of the wound that may otherwise produce a broader scar.

Long term pressure, often applied by compression bandages or other such dressings, may be used in an attempt to soften stiff scars, and to reduce the height of scars that stand proud above the surrounding skin. Another treatment used in a similar vein is the use of high-pressure water. This is commonly applied to sites of scarring via hosepipes, at approximately body temperature.

lntralesional steroid injections have also been used in attempts to reduce scarring, but these treatments are often associated with thinning of the skin covering the scar, a result which may itself be aesthetically unacceptable.

In the case of scars that are depressed below the surface of the unwounded surrounding tissue, administration of temporary or permanent filler compounds has been suggested as a method by which the height discrepancy may be overcome, thus reducing the aesthetic impact of the scar.

Despite the range of non-surgical scar revision techniques that have been developed, none of these are considered universally applicable. Furthermore, many of the techniques identified to date are subject to low or questionable efficacy, require high levels of patient compliance and/or multiple clinical visits, and are associated with high scar recurrence rates. Accordingly, there remains a need for new medicaments and methods, alternative medicaments and methods, and improved medicaments and methods for non-surgical scar improvement.

One of the targets that has been investigated for use in the inhibition of scarring is Transforming Growth Factor Beta 3 (TGF-β3). The TGF-βs are a family of growth factors that exist in three mammalian isoforms, TGF-β1 , TGF-β2 and TGF-β3. The biological effects of these isoforms are generally comparable in many respects, but, in the context of wound healing and scarring, they are exert very different effects.

All three mammalian TGF-β isoforms play important roles in the formation of scars. TGF- β1 and TGF-β2 both promote biological activities associated with scar formation (such as accumulation of extracellular matrix components), while TGF-β3 acts to reduce scar formation following a wound.

TGF-β3 has been shown experimentally to inhibit the formation of scars that may otherwise occur on healing of wounds. This has been demonstrated in a number of scientific studies in which TGF-β3 is administered locally to the site where scarring is to be inhibited at the time the wound is formed. Wounds treated with TGF-β3 heal to produce scars that are less pronounced than those formed on healing of control or untreated wounds. This is apparent both macroscopically, where the scar formed is less noticeable compared to the skin around it, and microscopically, where the architecture established in the repaired area more closely approximates that of the unwounded skin.

The anti-scarring effect achieved by TGF-β3 is believed to arise as a consequence of changes that this growth factor brings about during the key early stages of the wound healing response that directly influence the quality of the subsequent scar. Studies (Shah et a/., 1995) have shown that administration of TGF-β3 to wounds reduces the number of monocyte and macrophage cells that are present over the first seven days of healing. It is thought that the reduction in the number of these cells present helps to lessen the inflammatory response at the wounded site. This response is otherwise normally amplified by soluble factors released by these cells which recruit further inflammatory cells to the wounded area. The reduced inflammatory response ensures that fewer cells are present to produce TGF-β1 and TGF-β2 that would otherwise contribute to scar formation.

The presence of TGF-β3, and reduced levels of pro-scarring TGF-β isoforms, in treated wounds establishes local conditions that favour a more normal organisation and deposition of extracellular matrix components. In particular, during the early phases of healing (between 1 and 7 days after wounding) wounds treated with TGF-β3 contain decreased levels of fibronectin, collagen I and collagen III when compared to untreated controls.

Fibronectin is one of the key components of the provisional matrix laid down early in the wound healing process. The major source of fibronectin is macrophages and fibroblasts within the wound area. The reduced number of these cells in TGF-β3-treated wounds may contribute to the decreased amount of fibronectin present. This decrease in fibronectin is most pronounced in the deep dermis. Fibronectin, along with other provisional matrix components such as fibrin, forms a scaffold for further ECM components that are deposited later in the wound healing process, and so the influence of this early stage of healing on the more permanent reparatory structures produced, may continue long after the initial phases have been completed. Collagen I and collagen III constitute the major components of the permanent scar structure that develops to fill the wounded area. Both of these molecules are found in reduced amounts during the first seven days of healing of wounds treated with TGF-β3. The reduction of collagen I in TGF-β3-treated wounds is most notable in the deep dermis (similar to fibronectin, and possibly as a consequence of the reduced provisional matrix scaffold), while the reduction in collagen III is most notable in the region just below the epidermis.

Interestingly, when the amount of collagen I and collagen III present in scars (70 days after wounding) formed on healing of TGF-β3-treated wounds is compared with the amount found in various control groups (scars formed on healing of untreated wounds, or wounds provided with the pro-scarring isoforms TGF-β1 or TGF-β2) it has been found that all contain a similar amount of these ECM components, despite the fact that the macroscopic appearance of the scars is very different (those treated with TGF-β3 being notably better than those from other experimental or control groups). This finding suggests that many of the processes involved in the later stages of the wound healing, in which collagen is deposited and the ECM matures, are shared in common between TGF- β3 treated and non-treated wounds. This further indicates that it is the ability of TGF-β3 to change processes occurring at the early stages of wound healing that give rise to the beneficial results noted. The effects of these changes are apparent long after the events that have been influenced are completed.

It is an object of certain aspects and embodiments of the invention to provide new medicaments for the non-surgical improvement of scars. It is an object of certain aspects and embodiments of the invention to provide new methods for the non-surgical improvement of scars. This improvement is intended for the treatment of scars in a subject which exist on account of a previous injury or trauma of the subject. The scar could be caused by a previous elective procedure which has undergone and completed the normal natural healing process and which at the end of that process has left some scar tissue.

It is also an object of the present invention is to reduce the redness of scars by treatment with a suitable therapeutically active ingredient in place of surgical procedures. The invention also aims to reduce the contrast of scars. In many patients treatment of a scar intended to improve its elasticity and vascularisation will not necessarily reduce the redness and/or contrast of the scar. This is because the process of increasing vascularisation actually results in an increase in the size and or number of blood vessels in the area of the former wound. Consequently, whilst the elasticity of a scar may be improved, its visual impact in terms of its redness or contrast may not improve. A further object of the invention thus resides in treating this category of patients in whom scar redness can be treated by therapeutic means. It is one intention that such treatment may or may not be independent of improvements in elasticity and/or vascularisation of the scar.

In a first aspect of the invention there is provided TGF-β3 for use as a medicament for provision to a scar for the non-surgical improvement thereof. This aspect of the invention also provides TGF-β3 for use in the manufacture of a medicament for provision to a scar for the non-surgical improvement thereof. The scar may be a scar formed as a result of a wound inflicted at least two to six months ago, for example two, three, four, five or six months prior to administration of the medicament. Administration of TGF-β3 to a scar may begin at least two to six months after closure of a wound, the healing of which has resulted in a scar (for example, two, three, four, five or six months after wound closure). Medicaments in accordance with the first aspect of the invention may preferably be formulated for local injection.

In a second aspect, the invention also provides a method of non-surgical improvement of a scar, the method comprising providing a therapeutically effective amount of TGF-β3 to a scar of a subject in need of such non-surgical scar improvement. Methods in accordance with the second aspect of the invention may comprise administration of a therapeutically effective amount of TGF-β3 to a scar to be non-surgically improved. Methods of the invention may comprise two or more incidences of treatment in which TGF-β3 is provided to the subject.

The present invention is based on the inventors surprising finding that TGF-β3, when provided to an existing scar, is capable of bringing about advantageous changes in the properties of the scar. These advantageous changes allow administration of TGF-β3 to bring about effective scar improvement without the need for surgical intervention. The properties of the scar subject to change may include both macroscopic and microscopic properties. Properties that may be subject to improvement using the medicaments and methods of the invention include, but are not limited to, those independently selected from the group consisting of: scar width; scar height; scar pigmentation; scar redness; scar volume and scar texture.

The finding that TGF-β3 may be used to improve scarring by non-surgical treatment of existing scars is highly surprising because the skilled person, on considering the way in which TGF-β3 affects the early stages of wound healing to inhibit scarring when applied at around the time of wounding, would not have expected that there would be any benefit in providing TGF-β3 at this much later stage of the scarring process, after the scar has already formed.

As described above, it has previously been shown that TGF-β3 is able to influence the early stages of the wound healing process to create a localised wound environment in which the inflammatory response is reduced, and extracellular matrix components are deposited in a manner that more closely approximates that found in the unwounded skin. This appears to occur as a result of TGF-β3 "dampening" the body's natural response, in which excessive abnormally organised matrix materials are deposited to fill the void left by injury.

The wound healing process is a dynamic one, in which actions taking place early in the healing process are built upon by those that follow, and are thus able to influence later events. Accordingly, the fact that TGF-β3 establishes conditions of controlled and orderly repair during the first days of the wound healing response serves to entrain the later stages of the healing and scarring process in a beneficial manner. Since the early days of the healing process are crucial in characterising the nature of the response and the replacement tissue formed, relatively small changes at this early stage may lead to changes of much greater magnitude in the later stages of the process.

The skilled person, being aware of all these points, would not expect that intervention using TGF-β3 to modify the properties of a scar that had already formed would have any beneficial effects. It would be thought that the ability of this growth factor to produce a local milieu in which inflammatory activity and matrix deposition is reduced would be being exerted at much too late a stage to have any effect. In existing scars, of the type to which TGF-β3 is to be provided in order to bring about a non-surgically improvement in accordance with the invention, the inflammatory response has effectively run its course. Furthermore, the extracellular matrix components of the scar have been deposited long ago and, once these are in place, it would not be expected that TGF-β3 would beneficially influence them in any way.

Accordingly, it can be seen that there is nothing in the prior art that would have led the skilled person to consider that TGF-β3 may be useful provided to scars in medicaments or methods by which scars may be non-surgically improved. TGF-β3 was perceived to have a relatively narrow therapeutic time window (around the time of scarring) in which it could beneficially influence the course of the healing process to reduce scarring, and the present proposed uses lie well outside that window.

Furthermore, there would be certain particular aspects of the reported influences of TGF- β3 on the wound healing response that would lead the skilled person to consider it to be quite unsuitable for use in the manner suggested in the present disclosure. For example, TGF-β3 has been shown to increase the number of blood vessels in wounds to which it is provided (Shah et.al. Journal of Cell Science 108, 985-1002 (1995). The skilled person would thus consider TGF-β3 to be quite unsuitable for use in applications where it was wished to reduce scar redness, since it would be expected to have the opposite effect.

In one embodiment, the TGF-β3 is for reducing redness of the scar. The scar is, for example, one which is between 2-6 months (or more) after healing of the wound from which the scar was formed. In one embodiment, the TGF-β3 does not restore elasticity to the scar but instead reduces the redness of the scar. It is considered that the TGF-β3 may reduce the contrast in colour, and in particular redness, of a scar in comparison to neighbouring "normal" i.e. unscarred skin. The reduction in redness and/or contrast is visually measurable.

In one embodiment, the scar is not a hardened scar and the TGF-β3 is not for restoring elasticity to the scar. In one embodiment, the TGF-β3 is not for reducing the hardness of a hypertrophic scar or a keloid scar.

It has previously been reported that the addition of pro-fibrotic forms of TGF-β (i.e. TGF- β1 or TGF-β2) is not sufficient to cause scarring in the absence of an initial wounding insult. By the same token, it would not generally be believed by those skilled in the art that scars that have already formed may improved by the application of TGF-β3.

Furthermore, it had previously been found that agents (such as antibodies) capable of neutralising TGF-β1 and TGF-β2 need to be added during the early phases of wound healing if they are to beneficially influence the extent of scar formation. This further indicates that early intervention in the activity of TGF-βs is believed by those of ordinary skill in the art to be important when seeking to reduce scarring.

Without wishing to be bound by any hypothesis, the inventors believe that the beneficial effects of TGF-β3 that make it suitable for use in non-surgical scar improvement arise as a result of the growth factor's ability to promote and hasten maturation of the scars to which it is provided. Maturation involves a number of processes, including the rearrangement of ECM constituents within the scar and maturation/pruning of the vascular network.

For the purposes of the present disclosure, TGF-β3 may be taken to comprise a protein comprising the amino acid sequence shown in Sequence ID No. 1. TGF-β3 having the sequence shown in Sequence ID No. 1 represents a preferred form of TGF-β3 for use in the medicaments or methods of the invention. However, that the advantages of nonsurgical reduction of scars achieved using this form of TGF-β3 may also be provided by therapeutically effective fragments or derivatives of TGF-β3. Accordingly, except for where the context requires otherwise, references to TGF-β3 in the various aspects or embodiments of the invention should be taken to also encompass therapeutically effective fragments or derivatives of TGF-β3. These are discussed in more detail below.

In the context of the present invention, non-surgical improvement of scars may be taken to refer to any non-surgical procedure or method of treatment in which a scar, formed on completion of the healing process, is treated in order to reduce the appearance of a scar or the functional impairment caused by a scar. The medicaments or methods of the invention may be used as the only mode of non-surgical scar improvement, or may be used in combination with other treatments able to improve a scar. Merely by way of example, the medicaments or methods of the invention may be used as part of a combination treatment for non-surgical improvement of a scar also comprising one, or more, of: compression therapies; corticosteroid therapy; radiotherapy; silicon sheets; anti-neoplasties (e.g. 5-fluorouracil); retinoic acid; Verapamil and immunomodulators (such as Imoqimod). TGF-β3 may be provided separately to scars receiving other nonsurgical treatments (such as those set out above). Alternatively, suitable medicaments in accordance with this embodiment of the invention may comprise active agents, such as corticosteroids; anti-neoplastic agents; retinoic acid, Verapamil; or immunomodulators in combination with TGF-β3.

The term "non-surgical improvement of scars" as used herein should be taken to specifically exclude methods of scar revision in which a scar is excised, incised, realigned, or otherwise surgically manipulated. The medicaments or methods of the invention may be suitable for use to further non-surgically improve scars produced as a result of such surgical revisions (or other surgeries), but only after wounds associated with the surgical aspect of the treatment have healed.

The time at which non-surgical improvement of scars using the medicaments or methods of the invention is initiated may be calculated with reference to the time that has elapsed since the formation of the wound that gave rise to the scar. However, this latter form of calculation will not generally be suitable in incidences where the healing process is unusually protracted (e.g. due to wound healing complications). In this case it may be preferred that the time at which non-surgical improvement of scars is initiated is calculated with reference to the time that has elapsed after healing of the wound causing the scar has been completed. A wound may be considered to have healed, and thus to have yielded a scar, once a complete epithelial coverage has been re-established over the wounded site. This can be determined by macroscopic assessment, for instance by a clinician responsible for a patient's treatment.

Non-surgical improvement of scars using the medicaments or methods of the invention may preferably be initiated within twelve months of wounding, more preferably within ten months of wounding, still more preferably within eight months of wounding, yet more preferably within six months of wounding, and most preferably within two months of wounding.

Non-surgical improvement of scars using the medicaments or methods of the invention may be initiated at least two months after wounding, or may be initiated at least four months after wounding, at least six months after wounding, at least eight months after wounding, at least ten months after wounding, or even twelve or more months after wounding.

The non-surgical improvement of scars may be initiated within twelve months of healing, within ten months of healing, within eight months of healing, within six months of healing, or even within two months of healing. That is to say that the TGFβ-3 may be provided to the scar within twelve months of healing, for example within ten months of healing, or within eight months of healing, possibly within six months of healing, or, for example, within two months of healing

In order to bring about effective non-surgical improvement of scars in accordance with the invention, treatment may be initiated at least two months after healing of the injury leading to formation of the scar, at least four months after healing, at least six months after healing, at least eight months after healing, at least ten months after healing, or even twelve or more months after healing.

Methods of non-surgical scar improvement in accordance with the invention may preferably comprise two or more incidences of treatment. The use of two or more incidences of treatment will generally have a greater beneficial effect on non-surgical scar improvement than will a single incidence of treatment. However, limiting the number of incidences of treatment e.g. to two, is likely to avoid the risk of reduced compliance by patients (who may be averse to larger numbers of repeated injections, or may find it difficult to schedule larger numbers of visits to receive treatments) and hence reduced completion of treatment regimes. More than two incidences of treatment may be desirable to treat scars that are relatively resistant to treatment, such as pathological scars. That said, the therapeutic benefits of non-surgical scar improvement in accordance with the invention may be achieved with only a single administration of TGF- β3 to a scar. This embodiment may be particularly suitable in the case that TGF-β3 is administered in a form that allows sustained release of the growth factor to the scar that is to be non-surgically improved.

In the event that two, or more, incidences of treatment are to be used in non-surgical scar improvement employing the medicaments or methods of the invention, it may be preferred to control the time that elapses between individual incidences. Preferred times between incidences of treatment may be from approximately 6 hours to approximately 48 hours. More preferably the timing between incidences of treatment may be from approximately 16 hours to 36 hours, still more preferably from approximately 20 to 30 hours and most preferably may be around 24 hours.

It may be preferred that the medicaments and methods of the invention be used in nonsurgical improvement of scars that have not previously been subject to scar revision or anti-scarring therapies. In particular, it may be preferred that the medicaments and methods of the invention are used on scars other than those that have previously been provided with TGF-β3 as part of an anti-scarring treatment.

Subject to any provisos set out elsewhere in the present disclosure, the medicaments and methods of the invention may be of benefit in the non-surgical improvement of any scar requiring such treatment. The medicaments and methods of the invention may be used in the non-surgical improvement of "normal" scars, or in the non-surgical improvement of "pathological scars."

For the purposes of the present invention, a "normal" scar may be considered to be any scar formed by the normal wound healing response {i.e., a wound healing response that is not perturbed such that it produces pathological scarring).

"Pathological scars" may be considered to include those scars which are formed as a result of abnormal wound healing response, such as hypertrophic scars, keloid scars or pterygium (a form of pathological scarring in the eye). Hypertrophic or keloid scars are both unusually severe forms of scarring that give rise to scars that are markedly elevated compared to the surrounding tissue, and which can often be subject to colour differences compared to their surroundings. Keloid scars characteristically overgrow the boundaries of the initial injury that gave rise to the scar, while hypertrophic scars are frequently subject to damaging contraction. Treatment of pathological scars using the medicaments or methods of the invention may be able to improve the appearance, and thus lessen the visual impact, of the treated scar.

Both keloid and hypertrophic scars may also be associated with pain or pruritus. Nonsurgical revision in accordance with the invention may also reduce discomfort, such as pain or pruritus, associated with these scars. It may be particularly preferred that the medicaments or methods of the invention are used for the non-surgical improvement of scars subject to "rubor perseverans." (Bond etal. Plast Reconstr.Surg. Feb;121(2):487-96, 2008) This persistent redness of scars leads to scars that are more noticeable compared to their surroundings than scars subject to normal maturation and resolution. It has been found that scars subject to "rubor perseverans" are not, as might previously have been thought, subject to a prolonged inflammatory response, and inflammation does not appear to be a major contributor to the redness observed. The inventors believe that non-surgical scar improvement in accordance with the present invention can be used to dramatically improve the appearance of scars with "rubor perseverans," causing treated scars to much more closely approximate the colour and appearance of unwounded skin than untreated scars at the same time post-wounding or post-healing.

It may be preferred that non-surgical scar improvement in accordance with the present invention is undertaken in scars undergoing remodelling. The inventors own publications have recently confirmed that remodelling occurs for at least a year after healing of human skin wounds (Bond etal. Plast Reconstr. Surg. May; 121 (5): 1650-8, 2008). It is a preferred embodiment of the invention that TGF-β3 be used as a medicament for nonsurgical improvement of scars undergoing remodelling, and/or that TGF-β3 be used in methods of non-surgical improvement of scars undergoing remodelling.

Remodelling of scars may continue for at least a year after closure of the wound from which the scar has formed. During the remodelling process ECM molecules within the scar undergo a characteristic process of maturation that contributes to the increased mechanical strength of the scar, as compared to the initial wound.

It is surprising that TGF-β3 is effective in treating the redness or contrast of existing scars since it has previously only been implicated in the early stages of the healing process, and the biological processes involved in early wound healing and in scar remodelling are considered to be quite distinct from one another.

Various terms used in the present disclosure will now be described further for the avoidance of doubt. It will be appreciated that, for the sake of brevity, some of these terms may be described with reference to only certain aspects of the invention. However, except for where the context requires otherwise, the following descriptions of these terms will be applicable to all aspects of the invention.

Medicaments or methods of the invention

For the purposes of the present disclosure, references to "medicaments of the invention" should be taken as encompassing TGF-β3-containing medicaments manufactured in accordance with the various aspects or embodiments of the invention. Furthermore, medicaments of the invention should be taken to comprise TGF-β3 (or therapeutically effective fragments or derivatives thereof) when used in accordance with the first aspect of the invention, or any suitable embodiment of this aspect, except if the context requires otherwise. Medicaments of the invention are able to provide TGF-β3 to a scar to be non- surgically improved. It may be preferred that medicaments of the invention are for administration to a scar to be non-surgically improved.

Methods of the invention should be taken to encompass any method of non-surgical scar improvement, or other corresponding method of treatment, in accordance with the various aspects or embodiments of the invention set out herein. It will be appreciated that medicaments of the invention will generally represent preferred compositions with which the methods of the invention may be put into practice.

Therapeutically effective amounts

A "therapeutically effective amount" of TGF-β3 in context of the present invention will be any amount of this growth factor that is capable of effecting the non-surgical improvement of a scar to which it is provided. Therapeutically effective amounts of TGF- β3 in accordance with the present invention will be those that give rise to an improvement in the appearance of treated scars (assessed microscopically and/or macroscopically) as compared to the appearance of untreated or control treated scars.

The inventors have, for the first time, identified such therapeutically effective amounts, and have further found that particular therapeutically effective amounts are associated with properties or characteristics that mean that their uses in the medicaments or methods of the invention may be of particular benefit. These preferred therapeutically effective amounts are considered in greater detail elsewhere in the specification. Generally however, a total therapeutically effective amount of TGF-β3 able to non- surgically improve a one centimetre length of a dermal scar may be in the region of 100 to 10,000 ng, more preferably in the region of 200 to 5,000 ng, yet more preferably in the region of 500 to 2,500 ng, and even more preferably in the region of 1000 to 2,000 ng. It is generally preferred that these therapeutically effective amounts of TGF-β3, and amounts of TGF-β3 referred to elsewhere in the present disclosure, are determined by quantitative Enzyme-Linked Immunosorbent Assay (ELISA) calibrated with the United Kingdom National Institute for Biological Standards and Control (NIBSC) Transforming Growth Factor Beta-3 (Human rDNA derived) Reference Reagent code 98/608, as described in more detail below.

It will be appreciated that these therapeutically effective amounts may be administered over the course of one or more incidences of treatment, and that this is a preferred embodiment of the invention. In the case that multiple incidences of treatment are to be employed, the amount of TGF-β3 to be provided in each incidence may be altered accordingly such that a known proportion of a therapeutically effective amount is provided over the course of the multiple incidences of treatment (e.g., in the case where a therapeutically effective amount of TGF-β3 is to be provided over two incidences of treatment, each incidence may involve the provision of half of the selected therapeutically effective amount). For instance when it is desired to provide between approximately 100 and 10,000 ng of TGF-β3 to a centimetre of scar over the course of a treatment regime comprising two instances of administration, a suitable medicament to be used will be one able to provide between approximately 50 ng and 5,000 ng of TGF-β3 per centimetre of scar to be non-surgically improved. Thus, in further examples, suitable medicaments of the invention may be for the administration of approximately 100 to 2500ng, approximately 250 to 1250 ng, or approximately 500 to IOOOng of TGF-β3 per centimetre of scar to be non-surgically improved.

500 ng or 1000 ng of TGF-β3 per centimetre of scar to be improved represent particularly preferred amounts of TGF-β3 to be administered per incident of treatment when providing therapeutically effective amounts of TGF-β3 via the medicaments or methods of the invention. These amounts may be administered by means of two incidents of treatment, thus respectively providing a total therapeutic amount of either 1 ,000 ng or 2,000 ng of TGF-β3 per centimetre of scar requiring non-surgical improvement. The inventors believe that non-surgical scar improvement, using a therapeutically effective amount of TGF-β3 in accordance with the present invention, may give rise to an appreciable reduction in scarring within twelve months after the final incidence of treatment, within eight months of the final incidence of treatment, within six months of the final incidence of treatment, or within four months, two months, or even one month of the final incidence of treatment.

Preferably non-surgical scar improvement using the medicaments or methods of the invention may give rise to a decrease in scarring of at least 10% compared to a suitable control (for example, the same scar prior to treatment, an un-treated portion of the same scar, a control treated scar or an untreated scar). Preferably non-surgical scar improvement in accordance with the invention may give rise to a decrease in scarring of at least 20%, more preferably at least 50%, even more preferably at least 75% and yet more preferably by at least 90% compared to a suitable control. Indeed, it is most preferred that non-surgical scar improvement in accordance with the invention may give rise to a decrease in scarring of 100% as compared to a suitable control.

Centimetre of scar

Whether in connection with the medicaments or methods of the invention, a therapeutically effective amount of TGF-β3 to be provided to a scar to be non-surgically improved may be defined with reference to the length or area of a scar to be treated. The relevant length or area may be determined in terms of the number of "centimetres of scar" that it is wished to non-surgically improve. For the purposes of the present disclosure, a "centimetre of scar" represents a unit by which the size of site at which scarring is to be non-surgically improved may be measured.

A centimetre of scar may be taken to comprise any square centimetre of a body surface that is subject to scarring, either in whole or in part. For example, a scar of two centimetres length and one centimetre width (i.e. with a total surface area of two square centimetres) will be considered to constitute "two scar centimetres". By the same token, a linear scar of two centimetres length, but of negligible width (i.e. with negligible surface area), will, for the purposes of the present invention, be considered to constitute "two scar centimetres", if it passes through two square centimetres of the body surface. Similarly, a scar having a length of two centimetres and a width of two centimetres (i.e. a total surface area of four square centimetres) will constitute four scar centimetres.

The size of a site in scar centimetres should generally be assessed prior to treatment (since non-surgical improvement using the medicaments or methods of the invention may be able to reduce the size of a scar). Measurements should generally be taken when the scar is in its relaxed state (i.e. when the part of the body bearing the site to be measured is in the position adopted when the body is at rest). For scars of the skin, the relevant size should be assessed when the skin is not subject to external tension.

The inventors believe that it may be preferred that the chosen therapeutically effective amount of TGF-β3 be provided to a scar to be non-surgically improved by means of at least two (and more preferably two) incidences of treatment. In keeping with this aim it will be recognised that it may be preferred that medicaments of the invention be formulated such that a therapeutically effective amount of TGF-β3 is provided by the amount of a medicament that is provided to a centimetre of scar (and by extension to a patient) over the course of at least two (and more preferably two) incidences of treatment.

Therapeutically effective fragments or derivatives of TGF-β3

Except for where the context requires otherwise, all references to TGF-β3 in accordance with medicaments or methods of the invention should also be taken to encompass therapeutically effective fragments or derivatives of TGF-β3. Suitable fragments or derivatives of TGF-β3 may comprise at least 10 amino acid residues, preferably at least 40 amino acid residues, more preferably at least 70 amino acid residues, and most preferably at least 100 amino acid residues of Sequence ID No. 1.

Without limitation, suitable examples of suitable forms of derivatives may be selected from the group consisting of: therapeutically effective peptide derivatives of TGF-β3 (or fragments thereof); therapeutically effective fragments or derivatives comprising or based on the pharmacophore of TGF-β3 of the invention; therapeutically effective peptoid derivatives of TGF-β3 (or fragments thereof); therapeutically effective D-amino acid derivatives of TGF-β3 (or fragments thereof); therapeutically effective peptidomimetics based on TGF-β3 (or fragments thereof); therapeutically effective peptide analogues of TGF-β3 (or fragments thereof); therapeutically effective pseudopeptides based on TGF- β3 (or fragments thereof); therapeutically effective retrp-inverso peptides based on TGF- β3 (or fragments thereof); therapeutically effective depsipeptide derivatives based on TGF-β3 (or fragments thereof); therapeutically effective β-peptide derivatives based on TGF-β3 (or fragments thereof); and therapeutically effective retropeptoid derivatives based on TGF-β3 (or fragments thereof).

The terms "peptide analogue", "peptide derivative" and "peptidomimetic" as used herein are intended to include molecules which mimic the chemical structure of a peptide and retain the functional properties of the peptide. Approaches to designing peptide analogs are known in the art. For example, see Farmer, P. S. in Drug Design (E. J. Ariens, ed.) Academic Press, New York, 1980, vol. 10, pp. 119-143; Ball. J. B. and Alewood, P. F. (1990) J. MoI. Recognition 3:55; Morgan, B. A. and Gainor, J. A. (1989) Ann. Rep. Med. Chem. 24:243; and Freidinger, R. M. (1989) Trends Pharmacol. Sci. 10:270. Examples of peptide analogues, derivatives and peptidomimetics include peptides substituted with one or more benzodiazepine molecules (see e.g., James, G. L. et al. (1993) Science 260:1937-1942), peptides with methylated amide linkages and "retro-inverso" peptides (see U.S. Pat. No. 4,522,752 by Sisto).

It will be appreciated that, for the purposes of the present invention TGF-β3 may be taken to encompass either the monomeric and dimeric forms of the protein. It has surprisingly been found that, although TGF-β3 typically acts as a dimer, TGF-β3 is able to exert its biological effects in both monomeric and dimeric form, and so either of these forms may be used in the medicaments or methods of the invention to bring about nonsurgical scar improvement. It will be appreciated that the molecular weights of monomeric TGF-β3 will be lower than the corresponding dimer, and so therapeutically effective amounts or doses of monomeric TGF-β3 should be calculated accordingly.

Preferably therapeutically effective fragments or derivatives of TGF-β3 suitable for use in the medicaments or methods of the invention may, when provided to a scar, give rise to a decrease in scarring of at least 10% compared to a suitable control (for example, the same scar prior to treatment, an un-treated portion of the same scar, a control treated scar or an untreated scar). Preferably therapeutically effective fragments or derivatives of TGF-β3 suitable for use in accordance with the invention may, when provided to a scar, give rise to a decrease in scarring of at least 20%, more preferably at least 50%, even more preferably at least 75% and yet more preferably by at least 90% compared to a suitable control. Indeed, it is most preferred that therapeutically effective fragments or derivatives of TGF-β3 may, when provided to a scar, be able to bring about a decrease in scarring of 100% as compared to a suitable control.

Methods of administration, formulations and compositions

The inventors believe that non-surgical scar improvement may be achieved in accordance with the present disclosure using any suitable mode of administration by which a therapeutically effective amount of TGF-β3 may be provided to a site of scarring requiring such improvement. However, it may generally be preferred that TGF-β3 is directly administered to a scar requiring treatment. In particular, localised injection of TGF-β3 represents a preferred mode of administration in the methods of the invention, and formulations suitable for local injection represent preferred forms of TGF-β3 for use as medicaments of the invention.

The ability of local injections to directly administer TGF-β3 into a scar to be non-surgically improved provides a number of advantages. Without wishing to be bound by any hypothesis, it is believed that injection helps to obviate problems that may otherwise be associated with the requirement for TGF-β3 to cross the epidermal barrier. It is also believed that the administration of TGF-β3 via injectable medicaments into the scar helps to create particularly beneficial local conditions for non-surgical scar improvement.

It may generally be preferred that administration of TGF-β3 in accordance with the invention by injection is practiced using an intradermal injection needle and syringe. However, an alternative route by which medicaments in accordance with the invention may be administered is by propulsion through the skin, as either liquid droplets or lyophilised powder. Such propulsion may be supersonic propulsion via explosive release of a compressed gas. This mode of administration is often referred to as "needle-less injection," and a number of suitable devices are commercially available (e.g., those sold under the names "powderject," "Jetpeel," Airgent," and the like).

It will be appreciated that therapeutically effective amounts of TGF-β3 may be provided to a scar by means other than injection (e.g. by topical medicaments as considered elsewhere in the specification), and may still give rise to the required non-surgical improvement. Indeed, in certain cases topical medicaments may be preferred since injections are frequently a cause for distress among patients, and a requirement for multiple injections may contribute to poor completion of courses of treatment. In the case of topical treatments it will be necessary to provide a method for delivering the TGFβ3 protein across the intact epidermis e.g. by encapsulating within a liposome or transfersome (Paul et.al. Vaccine. Jan-Feb; 16(2-3): 188-95 1998).

Treatment regimes, such as the methods of the invention, may utilise both administration by injection and other means. For example, non-surgical scar improvement may be achieved by a first incidence of treatment involving administration of TGF-β3 in the form of an injection, followed by a second (and optionally subsequent) incidence of treatment involving administration of TGF-β3 by a topical medicament. This may be preferred in the event that a clinician, or the like, administers the first incidence of treatment (for instance at a hospital or clinic), and the patient administers the second, and optionally subsequent, incidences of treatment themselves (for instance in their own home).

In the case that TGF-β3 is provided to a patient by means of injection, a preferred mode of administration may involve administration of a series of injections spaced approximately one centimetre apart from one another throughout the area to be treated. Thus in the case of a substantially linear scar, the scar may be marked (for example using a surgical marker) with a series of reference points approximately one centimetre apart from one another. In a preferred embodiment, the reference points at either end of the scar should extend at least half a centimetre from end of the scar to be treated. This arrangement is shown schematically in Figure 2. For administration of TGF-β3, a hypodermic needle will be inserted intradermal^ at Site B and advanced to Site A, 100μl_ of dosing solution will then be injected into the 1cm length of the dermis as the needle is withdrawn, ensuring that the composition incorporating the TGF-β3 is evenly distributed between Sites A and B. A "bleb" with blanching will be seen where the solution has been injected into the dermis. The needle will then be inserted at Site C in the direction of Site B and the dosing process repeated until dosing has been achieved at all parts of the scar to be non-surgically revised. This manner of administration may be thought of as producing a line of injections along the scar. A similar administration may be achieved by using syringes (pre-filled or filled at time of use) and needles especially designed for intradermal administration (Laurent et.al. Vaccine, 8833-8842, 2007) e.g. Becton Dickinson Soluvia™ system. It may be preferred that, when TGF-β3 administered to a scar, it is administered substantially to the centre of the scar to be improved. In the case of elongate scars, this may involve administration along a line extending substantially along the longitudinal centre of the scar. Administration in this manner may be particularly effective in the case of scars having a width of approximately 1 cm or less. In the case of scars having a width of greater than 1 cm, it may be preferred that TGF-β3 is administered by means of a series of approximately parallel lines extending longitudinally along the scar, such that a therapeutically effective amount of TGF-β3 is provided to each scar centimetre.

The inventors have found that formulations in which a therapeutically effective amount of TGF-β3 is provided in a volume of approximately 100μl of a medicament are particularly beneficial, and it is further preferred that these embodiments of the invention should involve administration of approximately 100μl of a medicament comprising TGF-β3 in each injection. Preferred formulations may be devised such that a therapeutically effective amount of TGF-β3 (or required proportion of such an amount) is administered in a volume of approximately 100μl. The inventors have found that volumes of approximately 100μl of a medicament comprising TGF-β3 are well tolerated by patients. In particular, in the case of injection of medicaments comprising TGF-β3, 100μl volumes of such medicaments may generally be administered per centimetre of scar to be treated without inducing further damage, or the like at the treated site. Furthermore, 100μl volumes of medicaments comprising TGF-β3 may be well absorbed by scars to which they are administered, and this is beneficial in controlling the accurate administration of therapeutically effective amounts of TGF-β3.

Injectable formulations of TGF-β3 in accordance with the present invention may preferably be suitable for intradermal injection.

While it is possible to use TGF-β3 provided by the present invention for therapy in any form, it may be preferable to administer it in a pharmaceutical formulation, e.g., in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Accordingly, in one aspect, the present invention provides a pharmaceutical composition or formulation comprising at least one active composition, or a pharmaceutically acceptable derivative thereof, in association with a pharmaceutically acceptable excipient, diluent and/or carrier. The excipient, diluent and/or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The compositions of the invention can be formulated for administration in any convenient way for use in human or veterinary medicine. The invention therefore includes within its scope pharmaceutical compositions comprising a product of the present invention that is adapted for use in human or veterinary medicine.

Acceptable excipients, diluents, and carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (A. R. Gennaro edit. 2005). The choice of pharmaceutical excipient, diluent, and carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice.

The inventors have found that sugars, such as maltose or trehalose, represent preferred excipients for use in the medicaments of the invention. Sugars, such as maltose, may provide a number of advantages to the compositions in which they are incorporated, including increasing the recovery of biologically active sugars from within the composition. Sugars such as maltose may also serve to reduce injection pain. Examples of suitable formulations are described in the inventor's co-pending patent application, published as WO2007007095.

It may be preferred that TGF-β3 is administered in combination with an anaesthetic agent such as lignocaine, or the like. Accordingly, it may be a preferred embodiment of the medicaments of the invention that they are for administration with an anaesthetic, and even that the compositions further comprise an anaesthetic agent such as lignocaine. Similarly, it may be a preferred embodiment of methods of treatment of the invention that an anaesthetic agent, such as lignocaine, be administered to the patient either prior to, or at the same time as, administration of the TGF-β3.

Further details of preferred formulations suitable for use in the medicaments of the invention, and preferred routes of administration of such medicaments, are provided elsewhere in the present specification. Pharmaceutically acceptable

As used herein, the phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe", e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the US Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeias for use in animals, and more particularly in humans.

Calculation of TGF-β3 content, potency and amounts administered

Doses of TGF-β3, and the protein content of medicaments containing TGF-β3 (and in particular recombinant human TGF-β3, which is a preferred form of TGF-β3 to be used in accordance with the invention), may preferably be determined by quantitative Enzyme- Linked Immunosorbent Assay (ELISA) calibrated with the United Kingdom National Institute for Biological Standards and Control (NIBSC) Transforming Growth Factor Beta- 3 (Human rDNA derived) Reference Reagent code 98/608. Determination of protein content in this manner allows the protein content of TGF-β3-containing medicaments, and thus the amount of TGF-β3 that will be provided to a centimetre of a body site by a given amount of such a medicament, to be calculated by the skilled person.

It is preferred that doses and therapeutically effective amounts of TGF-β3 referred to in the present disclosure be calculated in accordance with this NIBSC Reference Reagent. References in this application to particular doses or amounts of TGF-β3 should be interpreted accordingly. This protocol has been used in determining the protein content of solutions used in the Experimental Results section.

In the event that a skilled person is not able to obtain a reference sample of NIBSC Reference Reagent code 98/608, the inventors have found that ELISAs using their own TGF-β3 product (Lonza Bulk Drug Substance Lot 205-0505-005) as a standard give rise to values that are approximately 52% of those obtained with NIBSC Reference Reagent code 98/608. In the event that it is wished to use this alternative standard, instead of NIBSC Reference Reagent code 98/608, required amounts of TGF-β3 should be determined accordingly (e.g., if it is desired to use a therapeutically effective amount of TGF-β3 corresponding to 1000 ng as assessed with reference to the NIBSC Reference Reagent code 98/608, then this will equate to 520 ng of TGF-β3 as assessed with reference to Lonza Bulk Drug Substance Lot 205-0505-005).

The biological activity (i.e., potency) of TGF-β3 to be used in accordance with the present invention may be determined by the inhibition of proliferation of Mink Lung Epithelial Cell line (MLEC); American Type Culture Collection (ATCC) Cat No. CCL-64. In a preferred embodiment, biological activity may be quantified by means of an assay calibrated using the United Kingdom National Institute for Biological Standards and Control Reference Reagent code 98/608, referred to above. Reference Reagent code 98/608 is considered to have a specific biological activity of 10,000 Arbitrary Units (AU) per microgram of TGF- β3 protein, and, by comparing the MLEC-inhibitory-activity of a sample of interest with the MLEC-inhibitory-activity of Reference Reagent code 98/608, the biological activity of the sample of interest in AU can be readily determined.

Thus, a dose of 500 ng of TGF-β3 (when calibrated with the NIBSC Reference Reagent) provides 5,000 AU of TGF-β3 activity, and a dose of 1 ,000 ng of TGF-β3 provides 10,000 AU of TGF-β3 activity. References to the use of specific doses of TGF-β3 in the present disclosure should be construed accordingly.

Scars to be non-surgically improved

The medicaments or methods of the invention may be used to achieve non-surgical improvement of scars of both male and female patients. Furthermore, the inventors believe that the medicaments or methods of the invention may be of use in non-surgical improvement of scars located at any body site and in any tissue or organ. The skin represents a preferred location of scars that may be subject to non-surgical improvement using the medicaments or methods of the invention. It will be appreciated that nonsurgical scar improvement using the medicaments or methods of the invention will bring about a notable improvement in the cosmetic appearance of a scar. Accordingly, it is a preferred embodiment that the medicaments or methods of the invention be used to non- surgically improve scars located on visible body parts. These include the face, neck and hands, and may also encompass the legs and abdomen. In addition to use in the non-surgical improvement of scars of the skin, the inventors believe that the medicaments and methods of the invention may be used in non-surgical improvement of scars in a range of locations, including, but not limited to, those independently selected from the group consisting of: scars of blood vessels; scars of the central and peripheral nervous system; scars of tendons, ligaments or muscle; scars of the oral cavity, including the lips and palate; scars of the internal organs such as the liver, heart, brain, digestive tissues and reproductive tissues; and scars in body cavities such as the abdominal cavity, pelvic cavity and thoracic cavity.

"Treated scars" and "untreated scars"

The provision of a therapeutically effective amount of TGF-β3 to a scar will give rise to a "treated scar," in which scarring is improved (i.e., in which the extent of scarring is decreased). The extent of scarring will be decreased both compared to that which was present prior to treatment, and compared to the extent of scarring that may be found in a comparable "untreated scar." A single scar may comprise both "treated" and "untreated" portions if a therapeutically effective amount of TGF-β3 is provided only to a portion of the scar (which will, in this case become the "treated" portion) and not to another portion of the scar (in this case the "untreated" portion).

Comparison between the extent of scarring in treated scars and in untreated scars may be of benefit in assessing the efficacy of various medicaments or methods of the invention, including experimental medicaments or methods used to determine preferred aspects of the medicaments or methods of the invention, and particularly preferred therapeutically effective amounts of TGF-β3, able to bring about optimal non-surgical improvement of scars.

Untreated scars may typically be used as comparators in assessing the extent to which non-surgical improvement has been achieved in a treated scar. Suitable comparator untreated scars of this type may preferably be matched to the treated scar with reference to one or more criteria independently selected from the group consisting of: scar age; scar size; scar site; Body Mass Index of patients; patient age; patient race and patient gender. Models of non-surgical improvement of scars

Therapeutically effective amounts of TGF-β3 able to bring about non-surgical improvement of a scar may be assessed directly on a patient requiring such treatment. In this case, the necessary determination may be undertaken by any suitable party, such the patient or a clinician.

However, it will generally be preferred that therapeutically effective amounts of TGF-β3 be determined with reference to suitable models of non-surgical scar improvement. Such models may be used to determine therapeutically effective amounts of TGF-β3 that may be used in medicaments or methods applicable to a wide range of patients.

Scars can be visually assessed as described below under the heading relating to assessment of scarring. Thus the redness and/or contrast of scars before and after treatment with TGF-β3 can be assessed using the procedures described.

In vivo models may be utilised to assess non-surgical scar improvement. Such models will generally involve assessment and comparison of the extent of non-surgical scar improvement achieved in treated scars, with the level of scarring found in non-treated scars. Suitable models will include those using non-human animals, but it may be preferred to use models in which non-surgical scar improvement is assessed in human subjects.

Experimental models of non-surgical improvement of scars may also allow identification of particular effective routes or regimes by which TGF-β3 may be administered. These routes or regimes may provide significant advantages in the efficacy of medicaments and methods of the present invention, and these advantages may give rise to further aspects or embodiments of the invention.

Assessment of scarring, and of inhibition of scarring

The extent to which non-surgical scar improvement has been achieved in accordance with the present invention may be determined by assessing the extent of scarring found in treated scars, and comparing this with the extent of scarring found in either the same scar prior to treatment, or in an untreated portion of the same scar or in a comparator untreated or control-treated scar. Effective non-surgical scar improvement will be demonstrated when the appearance of a treated scar is assessed as more similar to unwounded tissue than is the appearance of the same scar prior to treatment, or the appearance of an untreated or control treated scar.

The extent of non-surgical scar improvement achieved using medicaments or methods of the invention may be assessed and/or measured with reference to the microscopic and/or macroscopic appearance of a treated scar compared to the microscopic and/or macroscopic appearance of the scar prior to treatment. The extent of non-surgical scar improvement achieved using medicaments or methods of the invention may also suitably be assessed with reference to microscopic and/or macroscopic appearance of a treated scar as compared to the microscopic and/or macroscopic appearance of an untreated scar.

While assessment of scarring may take into consideration the macroscopic appearance of scars and/or the microscopic appearance of scars, it may be preferred that assessment of scarring be conducted with reference to macroscopic appearance. Assessment with reference to macroscopic appearance may be preferred since the macroscopic appearance of a scar most directly reflects the way in which the scar is perceived by the patient or by others.

A number of methods for the assessment of scarring have been developed, primarily with regard to scarring of the skin (being the body's largest organ, and the organ in which scars have the greatest cosmetic impact). Accordingly, the following description of methods for assessing the scar inhibitory activity of medicaments and methods of the invention will primarily be described with reference to assessment of scarring in the skin. However, the skilled person will immediately appreciate that many of the factors that are relevant when assessing scarring in the skin are also relevant to assessment of scarring in other organs or tissues. Accordingly the skilled person will recognise that, except for where the context requires otherwise, the parameters suggested below, in the context of assessment of scars of the skin, may also be applicable to assessment of scarring in tissues other than the skin.

Preferably an assessment of scarring may be an assessment providing a quantifiable value indicative of the degree of scarring present. This allows ready quantification of the degree to which non-surgical scar improvement has been attained. The macroscopic appearance of a scar, whether treated, untreated or control treated, may be assessed with reference to a number of parameters. Suitable parameters may be considered individually or in combination.

The extent of scarring, and so the extent of any non-surgical scar improvement achieved, may be assessed by macroscopic clinical assessment of scars. This may be achieved by the direct assessment of scars upon a subject; or by the assessment of photographic images of scars; or of silicone moulds taken from scars, or positive plaster casts made from such moulds. Macroscopic characteristics of a scar which may be considered when assessing scarring include:

i) Colour of the scar. Scars may typically be hypopigmented or hyperpigmented with regard to the surrounding skin. Effective non-surgical revision of scarring may be demonstrated when the pigmentation of a treated scar more closely approximates that of unscarred skin than does the pigmentation of an untreated scar. Scars may often be redder than the surrounding skin. In this case effective non-surgical revision of scarring may be demonstrated when the redness of a treated scar fades earlier, or more completely, or to resemble more closely the appearance of the surrounding skin, compared to an untreated scar. Colour can readily be measured, for example by use of a number of non-invasive colourimetry devices which are able to provide data with respect to the pigment of scars and unscarred skin, as well as redness of the scar and unscarred skin. Colour can also readily be measured by analysis of standardised clinical photographs (as described in more detail elsewhere in the specification). Scar colour, and in particular scar redness, is a preferred parameter to be used in the assessment of scarring, whether as the sole parameter assessed, or in combination with other parameters. ii) Height of the scar. Scars may typically be either raised or depressed as compared to the surrounding skin. Effective non-surgical revision of scarring may be demonstrated when the height of a treated scar more closely approximates that of unscarred skin (i.e. is neither raised nor depressed) than does the height of an untreated scar. Height of the scar can be measured directly on the patient (e.g. by means of profilometry), or indirectly, (e.g. by profilometry of moulds taken from a scar). iii) Surface texture of the scar. Scars may have surfaces that are relatively smoother than the surrounding skin (giving rise to a scar with a "shiny" appearance) or that are rougher than the surrounding skin. Effective nonsurgical revision of scarring may be demonstrated when the surface texture of a treated scar more closely approximates that of unscarred skin than does the surface texture of an untreated scar. Surface texture can also be measured either directly on the patient (e.g. by means of profilometry), or indirectly (e.g. by profilometry of moulds taken from a scar).

A treated scar will preferably exhibit effective non-surgical improvement of scarring as assessed with reference to at least one of the parameters for macroscopic assessment set out in the present specification. More preferably a treated scar may demonstrate effective non-surgical improvement of scarring with reference to at least two of the parameters, even more preferably at least three of the parameters, and most preferably at least four of these parameters (for example, all four of the parameters set out above).

One preferred method for the macroscopic assessment of scars is holistic assessment. This may be accomplished by means of assessment of macroscopic photographs by an expert panel or a lay panel, or clinically by means of a macroscopic assessment by a clinician or by patients themselves. Assessments may be captured by means of a VAS (visual analogue scale) or a categorical scale. Examples of suitable parameters for the assessment of scarring (and thereby of non-surgical revision of scarring) are described below. Further examples of suitable parameters, and means by which assessment of such parameters may be captured, are described by Duncan et al. (2006), Beausang et al. (1998) and van Zuijlen et al. (2002).

Microscopic assessment of scars will generally employ histological analysis of the microscopic structure of scars. Suitable parameters for the microscopic assessment of scars may include: i) Thickness of extracellular matrix (ECM) fibres. Scars typically contain thinner ECM fibres than are found in unscarred skin. Effective non-surgical revision of scarring may be demonstrated when the thickness of ECM fibres in a treated scar more closely approximates the thickness of ECM fibres found in unscarred skin than does the thickness of fibres found in an untreated scar. ii) Orientation of ECM fibres. ECM fibres found in scars tend to exhibit a greater degree of alignment with one another than do those found in unscarred skin (which have a random "basket weave" orientation). Accordingly, effective nonsurgical revision of scarring may be demonstrated when the orientation of ECM fibres in a treated scar more closely approximates the orientation of ECM fibres found in unscarred skin than does the orientation of such fibres found in an untreated scar. iii) Abundance of ECM components. Scars typically contain an increased amount of ECM components such as collagen when compared to unscarred skin. In this case effective non-surgical revision of scarring may be indicated when a treated scar contains reduced abundance of ECM components when compared to untreated or control treated scars, or when a treated scar contains an abundance of ECM components that is more similar to unscarred skin than the abundance contained in an untreated or control scar. iv) ECM composition of the scar. The composition of ECM molecules present in scars shows differences from that found in normal skin. Thus effective nonsurgical revision of scarring may be demonstrated when the composition of ECM fibres in the dermis of a treated scar more closely approximates the composition of such fibres found in unscarred skin than does the composition found in an untreated scar. v) Cellularity of the scar. Scars tend to contain relatively fewer cells than unscarred skin. It will therefore be appreciated that effective non-surgical revision of scarring may be demonstrated when the cellularity of a treated scar more closely approximates the cellularity of unscarred skin than does the cellularity of an untreated scar.

One or more of the parameters suggested above may be used to form the basis of a visual analogue scale for microscopic assessment of scarring. Effective non-surgical improvement of scarring may be indicated when the quality of a treated scar is closer to that of unscarred skin than is the quality of an untreated or control scar.

It is surprising that the overall appearance of scars, such as those of the skin, is little influenced by the epithelial covering of the scar, even though this is the part of the scar that may be most readily seen by the observer. Instead, the inventors have found that the properties of the connective tissue (such as that making up the dermis, or neo-dermis) present within the scar have greater impact on the perception of extent of scarring, as well as on the function of the scarred tissue. Accordingly, assessments of criteria associated with the connective tissues such as the dermis, rather than epithelia such as the epidermis, may prove to be the most useful in assessing effective non-surgical revision of scarring. The thickness of ECM fibres and orientation of ECM fibres within the connective tissue may be favoured parameters for assessing non-surgical revision of scarring. It may be desirable to assess ECM organisation and abundance in the papillary dermis and the reticular dermis separately when considering scar quality.

A treated scar may preferably demonstrate effective non-surgical improvement of scarring as assessed with reference to at least one of the parameters for microscopic assessment set out above. More preferably, a treated scar may demonstrate nonsurgical improvement of scarring with reference to at least two of the parameters, even more preferably at least three of the parameters, and most preferably all four of these parameters. Macroscopic and microscopic parameters may be combined in assessing effective non-surgical improvement of scarring (i.e. assessing at least one parameter used in macroscopic assessment and at least one parameter used in microscopic assessment).

There are a number of ways in which assessments of scarring may be captured and quantified. Suitable methods may be used to capture macroscopic or microscopic assessments of scarring, and may generally be performed either directly (on the patient), or indirectly (on photographs or moulds taken from the patient). Without limitation, examples of means by which assessment of scarring may be captured include:

Assessment using Visual Analogue Scale (VAS) scar scores.

Assessments of scars may be captured using a scarring-based VAS. A suitable VAS for use in the assessment of scars may be based upon the method described by Duncan et a/. (2006) or by Beausang et al. (1998). This is typically a 10 cm line in which 0 cm is considered an imperceptible scar and 10 cm a very poor hypertrophic scar. Use of a VAS in this manner allows for easy capture and quantification of assessment of scarring. VAS scoring may be used for the macroscopic and/or microscopic assessment of scarring. Merely by way of example, a suitable macroscopic assessment of scarring may be carried out using a VAS consisting of a 0-10 cm line representing a scale, from left to right, of 0 (corresponding to normal skin) to 10 (indicative of a bad scar). A mark may be made by an assessor on the 10 cm line based on an overall assessment of the scar. This may take into account parameters such as the height, width, contour and colour of the scar. The best scars (typically of small width, and having colour, height and contour like normal skin) may be scored towards the "normal skin" end of the scale (the left hand side of the VAS line) and bad scars (typically large width, raised profile and with uneven contours and redder or whiter colour than normal skin) may be scored towards the "bad scar" end of the scale (the right hand side of the VAS line). The marks may then be measured from the left hand side to provide the final value for the scar assessment in centimetres (to 1 decimal place).

An alternative macroscopic assessment of scarring, involving comparison of two scars or two scar segments (one segment treated and the other segment untreated, or treated with placebo alone) to determine which one has a preferred appearance, may be carried out using a VAS comprising two 100 mm VAS lines intersected by a vertical line (The Global Scar Comparison Scale). In a VAS of this sort, the two VAS lines correspond to the two scars being compared, while the vertical line represents zero (indicating that there is no perceptible difference between the scars compared). The extremes of 100% (100 mm at the end of either VAS line) indicate one of the scars has become imperceptible in comparison to the surrounding skin.

When comparing a pair of scars using a VAS of this sort, an assessor must first decide which of the scars has the preferred appearance, or if there is no perceptible difference between the two. If there is no perceptible difference this is recorded by placing a mark at the zero vertical line. If there is a perceptible difference, the assessor uses the worse of the two scars as an anchor to determine the level of improvement found in the preferred scar, and then marks the score on the relevant section of the scale. The point marked represents the percentage improvement over the anchor scar.

The inventors have found that use of VAS measures of this sort in assessing the macroscopic appearance of scars offers a number of advantages. Since these VAS are intuitive in nature they, 1) reduce the need for extensive training using reference images of different scar severities in different skin types, making this tool relatively simple to deploy in a large phase 3 trial; 2) reduce variability of the data: one assessment of each scar pair is performed as opposed to two independent assessments of drug and placebo scars; 3) incorporate the well-established principles of VAS {i.e., a continuous distribution of data) and the benefits of ranking in the same scale; and 4) allow easier communication of drug effect (percentage improvement) to clinicians and patients.

For microscopic assessment, the scars may be excised from experimental subjects (preferably incorporating a small amount of surrounding normal tissue) and fixed (for example in formalin). The fixed tissue may then be processed for wax histology. Histological slides may be stained using a suitable protocol to allow assessment of scarring (such as Masson's trichrome or Mallory's trichrome), and scarring assessed by a assessor using a microscopic VAS. A suitable VAS may consist of a 0-10 cm line representing a scale, from left to right, of 0 (corresponding to normal skin) to 10 (indicative of a bad scar). A mark may be made on the 10cm line based on an overall assessment of the scar taking into account parameters such as collagen fibre spacing, orientation and thickness. The best scars (typically narrow scars with thick and randomly organised collagen fibres that have normal spacing between fibres, similar to that found in unscarred dermis) will be scored towards the "normal skin" end of the scale (the left hand side of the VAS line) and bad scars (typically wide scars with thin densely packed parallel collagen fibres) will be scored towards the "bad scar" end of the scale (the right hand side of the VAS line). The marks can then be measured from the left hand side to provide the final value for the scar assessment in centimetres (to 1 decimal place). Likewise two microscopic images can be assessed comparatively using the principles of The Global Scar Comparison Scale outlined above.

Assessment with regard to an overall categorical scale.

Assessment of scarring may be captured by allocating scars to different categories based on either textual descriptions of the scar assessed (e.g. "barely noticeable," "blends well with normal skin," "distinct from normal skin," etc.), or by comparing a treated scar and an untreated or control scar, noting any differences between these, and allocating the differences to selected categories (e.g. "mild difference," "moderate difference," "major difference," etc.). These assessments may be conducted with reference to the over-all appearance of the scar(s) assessed. Effective non-surgical improvement of scarring may be demonstrated when an assessment indicates that treated scars are allocated to at least one more favourable category than are untreated or control scars. Assessment of this sort may be performed by the patient, by an investigator, by an independent panel, or by a clinician.

Assessment of scar height, scar width, scar perimeter, scar area or scar volume. The height and width of scars can be measured directly upon the subject, for example by use of manual measuring devices such as callipers, or automatically with the use of profilometers. Scar width, perimeter and area may be measured either directly on the subject, by image analysis of photographs of the scar, or using plaster casts of impressions of the scar. The skilled person will also be aware of further non-invasive methods and devices that can be used to investigate suitable parameters, including silicone moulding, ultrasound, optical three-dimensional profilimetry and high resolution Magnetic Resonance Imaging. All such measurements are readily captured and quantified.

Non-surgical improvement of scarring may be demonstrated by a reduction in the height, width, area, perimeter or volume (or any combination thereof), of a treated scar as compared to an untreated scar.

Appearance and/or colour of scar compared to unscarred skin.

The appearance or colour of a treated scar may be compared to that of unscarred skin, and/or untreated or control scars and unscarred skin. The appearance of a scar may be compared with unscarred skin with reference to whether the scar is lighter or darker, or redder, than the unscarred skin.

The appearance or colour of the scars and skin may be categorised (e.g. perfectly matched to one another, slightly mismatched, obviously mismatched or grossly mismatched) and these categorisations recorded and/or quantified. Suitable comparisons may be made on the basis of a visual assessment of the respective scars and unscarred skin.

Alternatively or additionally to visual assessment, there are a number of non-invasive colorimetric devices which are able to provide data with respect to pigmentation of scars and unscarred skin, as well as redness of the skin (which may be an indicator of the degree of vascularity present in the scar or skin). Examples of such devices include the X-rite SP-62 spectrophotometer, Minolta Chronometer CR-200/300; Labscan 600; Dr. Lange Micro Colour; Derma Spectrometer; laser-Doppler flow meter; and Spectrophotometric intracutaneous Analysis (SIA) scope. The results obtained using such devices may also be recorded and quantified.

Appearance and/or colour of scar compared to the baseline scar before treatment

Using the apparatus, methods and instruments described in the above paragraph the appearance of the scar/scar segment after treatment can be compared with the appearance of the same scar/scar segment before treatment. Analysis before and after treatment, or control treatment, can be done for the treated scar/scar segment and for any control treatment e.g. placebo administration, and the differences compared.

Scar distortion

The differences between scars and unscarred skin may lead to distortion in the region of the scar. This may arise as a result of various factors, including the scar being above or below the surface of the surrounding skin. Scar distortion may be assessed by visual comparison of a scar and unscarred skin, and the degree of distortion categorised (e.g. as causing no distortion, mild distortion, moderate distortion or severe distortion).

Scar contour and scar texture

Scar contour may be investigated by means of visual assessment, and the contour and texture categorised using suitable parameters. Suitable parameters for categorisation may include whether or not a scar is flush with surrounding skin, slightly proud, slightly indented, hypertrophic or keloid.

The texture of a scar may be assessed with reference to the scar's appearance, and this may also be undertaken by a visual assessment and categorised accordingly (for example, whether a scar is matt or shiny, or has a roughened or smooth appearance as compared to unscarred skin).

In addition to the techniques set out above, there are a number of non-invasive profilimetry devices that use optical or mechanical methods for assessment of scar contour and/or texture. Such assessments may be carried out directly or indirectly. Assessments in this manner may give rise to a representative value by which the assessment may be readily captured. Photographic Assessments

Photographic assessment of treated and untreated scars may be performed by any suitable assessor. Examples of suitable assessors include independent lay or expert panels, clinicians, or the patients themselves. Treated or untreated scars may be assessed compared to standardised and calibrated photographs of scars.

An advantageous application of photographic assessment of scars lies in the assessment of scar colour, and in particular scar redness. This may preferably be carried out by comparison of standardised clinical photographs (e.g. photographs in which the parameters influencing colour are controlled from photograph to photograph). Suitable procedures by which clinical images may be standardised and calibrated for such applications are well known to those skilled in the art.

In a preferred embodiment, standardised images of scars (such as treated and non- treated scars, or treated scars at various time-points preceding, during or after treatment) may be loaded into any image analysis or suitable digital photo editing software, such as Adobe Photoshop, and the outline of the scar (or areas of scar to be analysed) traced. Global colour analysis measurements (ΔL, Δa and Δb) may then be calculated as the difference in L (lightness), a (red/green value) and b (yellow/blue) values between the area inside the traced outline (the scar area) and outside the traced line (the surrounding skin area). Comparison of the overall colour difference from the surrounding skin (ΔE_ab) allows a quantitative assessment of scar colour to be made, and this colour to be compared directly with the surrounding skin. These colour analysis measurements can also be used to provide information regarding scar redness, a reduction in which would be indicated by a shift in Δa value between the treated scar pre and post treatment, such that the treated scar recorded an Δa value closer to the Δa value of the surrounding skin.

Scars may also be assessed by a trained clinical or independent lay panel to provide categorical ranking data (e.g. that a given treated scar is "better," "worse," or "no different" when compared to an untreated scar) and/or quantitative data (such as by using a VAS) as described elsewhere in the specification. The capture of these data may make use of suitable software and/or electronic system(s) as described in the applicant's co-pending patent application PCT/GB2005/004787. Suitable assessments may consider differences in the appearance of a treated scar that occur over time. This may be achieved by comparison of a time-course of images of selected treated and untreated scars. Assessment of the progression of scarring with time may consider changes in the overall appearance of a scar, and/or changes in specific criteria such as those considered elsewhere in the specification (e.g., scar colour, scar texture, scar width).

The assessments and parameters discussed above are suitable for assessment of the ability of TGF-β3 (in particular doses or treatment regimes of interest) to non-surgically improve scars, as compared to control, placebo or standard care treatment in animals or humans. It will be appreciated that these assessments and parameters may be utilised in determining therapeutically effective amounts of TGF-β3. Appropriate statistical tests may be used to analyse data sets generated from different treatments in order to investigate the significance of results.

While the various parameters described above may be of use when assessing the extent of scarring (and hence the extent of any non-surgical improvement of scarring achieved) in skin, other parameters may be of particular relevance when scarring is to be assessed in sites other than the skin.

Merely by way of example, effective non-surgical improvement of scarring in tendons or ligaments may be indicated by restoration of function of tissues treated with the medicaments or methods of the invention. Suitable indicators of function may include the ability of the tendon or ligament to bear weight, stretch, etc. Such assessments may, for example, be made using electrophysiological reflex examination, surface electromyography, ultrasonography, ultrasound/MRI scan, and self reported symptom and pain questionnaires

Similarly, effective non-surgical improvement of scarring occurring in blood vessels can be measured directly e.g. using ultrasound, or indirectly by means of blood flow. Nonsurgical scar revision achieved using the medicaments or methods of the invention may lead to a reduction in narrowing of the blood vessel lumen and allow a more normal blood flow. Preferred formulations for use in accordance with the invention

Generally, medicaments of the invention may be formulated and manufactured in any form that allows for the medicament to be administered to a patient such that a therapeutically effective amount of TGF-β3 is provided to a scar that is to be non- surgically improved.

Suitable compositions or medicaments containing TGF-β3 may take a number of different forms depending, in particular, on the manner in which they are to be used. Thus, for example, they may be in the form of a liquid, ointment, cream, gel, hydrogel, powder or aerosol. All of such compositions are suitable for local administration to a scar requiring non-surgical improvement.

However, the inventors have found that non-surgical scar improvement in accordance with the invention is particularly effective when using compositions formulated for administration by injection. More preferably, medicaments the invention may be formulated for intradermal injection. Typical formulations suitable for intradermal injection will be well known to those skilled in the art.

Medicaments of the invention suitable for localised parenteral administration (e.g., intradermal, intramuscular and subcutaneous) may be prepared by mixing TGF-β3 with optional physiologically acceptable carriers, excipients or stabilizers (including sugars such as maltose or trehalose) in the form of; lyophilised and non-lyophilised powder formulations for reconstitution prior to use, non-aqueous and aqueous solutions, and semi-solid formulations. Acceptable carriers, including excipients, are non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; tonicity modifiers such as sodium chloride, glycerol, and the like; preservatives such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benazalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl and/or propyl and/or butyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (comprising less than about ten amino acid residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates including dextrins; chelating agents such as EDTA; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); anionic surfactants such as fatty acid soaps or acyl sulfates; cationic surfactants, such as alkyl primary, secondary, tertiary, or quaternary amines; non-ionic surfactants, for example, sorbitan esters or polyethoxylated esters of acyl acids, copolymers of polyethylene oxide and polypropylene oxide.

By way of example, a pharmaceutical composition of the invention in the form of a sterile solution suitable for parenteral administration, may include the following constituents in addition to the TGF-β3:

A sugar

Sodium Hydroxide Water for injection Nitrogen Gas

A lyophilized (freeze-dried) powder 'cake' of the above solution could be prepared.

Such a medicament of the invention could be presented in the form of a vial, an ampoule, or a pre-filled syringe of, either; a sterile solution, a sterile suspension or any other pharmaceutically acceptable form of presentation suited to localised parenteral drug delivery.

Techniques such as lyophilisation may extend the shelf life of medicaments of the invention, and may be used to produce, for example, a sterile lyophilised (freeze-dried) powder suitable for reconstitution.

Medicaments the invention may be formulated for use in scars of the eye (for instance as eye drops), or as intraperitoneal instillates.

Alternatively, or additionally, medicaments of the invention may be formulated for topical administration. The topical administration of medicaments of invention may be particularly preferred in the context of methods in which TGF-β3 is administered by a patient or non-medically trained practitioner as part of an ongoing non-surgical scar treatment. Medicaments of the invention suitable for topical administration (e.g. transdermal/cutaneous, ocular, otic, nasal, pharyngeal, buccal, rectal, vaginal, urethral) may be prepared by mixing TGF-β3 with optional physiologically acceptable carriers, excipients or stabilisers (including sugars such as maltose) in the form of lyophilised or non-lyophilised powder formulations, non-aqueous or aqueous solutions, non-aqueous or aqueous dispersions/suspensions, including emulsions and semi-solid formulations. Acceptable carriers, including excipients, are non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, purified water, saline, phosphate-buffered saline (PBS) Ringer's solution, Ringer"s-lactate solution, hydro- alcoholic solutions, polyethylene glycol (PEG), propylene glycol (PG), phosphates, acetates, gelatin, collagens, Carbopol 934™ (BF Goodrich Corp.), vegetable and synthetic oils and waxes, anionic surfactants such as fatty acid soaps or acyl sulfates; cationic surfactants, such as alkyl primary, secondary, tertiary, or quaternary amines; non-ionic surfactants, for example, sorbitan esters or polyethoxylated esters of acyl acids, copolymers of polyethylene oxide and polypropylene oxide, and the like. Pharmaceutical compositions of the invention may additionally include suitable preservatives, stabilisers, antioxidants, anti-microbials and buffering agents, for example, methyl and/or propyl and/or butyl parabens, butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), citric acid, ascorbic acid, and the like. Emulsion, cream or ointment bases useful in formulation may include aqueous-based creams and emulsions (oil-in-water), oil-based creams and emulsions (water-in-oil), ointments (emulsifying and non-emulsifying hydrocarbon), gels, hydrogels, and the like. Alternatively medicaments of the invention may be incorporated or encapsulated in a suitable polymer matrix or membrane, thus providing a sustained-release delivery device suitable for placement on a scar requiring non-surgical revision. Suitable encapsulation may use liposomes or transfersomes of the sort contemplated in Paul et.al. Vaccine. Jan-Feb; 16(2-3): 188- 95 1998.

Suitable medicament of the invention may be presented in the form of a bottle, a jar, a tube, a spray, of, either; a sterile solution; a sterile lyophilized or non-lyophilized powder for reconstitution, a sterile dispersion/suspension, a sterile semi-solid, or any other pharmaceutically acceptable form of presentation suited to topical drug delivery. Routes of administration

Therapeutically effective amounts of TGF-β3 may be administered by any suitable route capable of achieving the desired effect of providing the medicament comprising TGF-β3 to a scar requiring non-surgical improvement. However, it may generally be preferred that a medicament and hence TGF-β3 is provided to a scar to be improved by means of local administration.

Suitable methods by which such local administration may be achieved will depend on the identity of the tissue or organ in question, and may also be influenced by whether or not a tissue or organ to be treated is permeable to the chosen medicament. Suitable routes of administration may be selected from the group consisting of: injections; application of sprays, ointments, or creams; inhalation of medicaments; release from biomaterials or other solid medicaments. Generally, preferred routes of administration may include local injection (for example intradermal injection in the case where it is wished to non- surgically improve scars of the skin). Suitable formulations for use in these embodiments of the invention are considered elsewhere in the specification.

In the case of scars of blood vessels, TGF-β3 may be administered by direct injection into the scars, or by locally applied devices, such as stents.

Medicaments of the invention may be provided on a sterile dressing or patch, which may be used to cover a scar requiring non-surgical improvement.

It will be appreciated that the vehicle of medicament of the invention should be one that is well tolerated by the patient and allows release of TGF-β3 to a scar in need of nonsurgical improvement. Such a vehicle is preferably biodegradeable, bioresolveable, bioresorbable and/or non-inflammatory.

Medicaments of the invention may be incorporated within a slow or delayed release device. Such devices may be placed on a scar to be improved, and TGF-β3 from the devise may be released over days, weeks or even months.

Medicaments or methods of the invention may be used to non-surgically improve scars as a monotherapy (e.g. through use of medicaments or methods of the invention alone). Alternatively the methods or medicaments of the invention may be used in combination with other compounds or non-surgical treatments for the revision of scars.

It will be appreciated that many of the advantages that may be gained as a result of nonsurgical revision of scars of humans are also applicable to other animals, particularly veterinary or domestic animals (e.g. horses, cattle, dogs, cats etc). Accordingly it will be recognised that the medicaments and methods of the invention may also be used for the non-surgical improvement of scars of non-human animals.

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

Figure 1 is a diagrammatic representation of scar marking prior to treatment;

Figure 2 is a schematic representation of a preferred protocol by which medicaments of the invention may be administered to a scar requiring non-surgical improvement.

Experimental results and protocols

The ability of medicaments and methods of the invention to bring about effective nonsurgical improvement of scars may be demonstrated using a clinical study or trial as set out below. Trials of this sort may also be used in the investigation and identification of preferred therapeutically effective amounts of TGF-β3 suitable for use in the medicaments or methods of the invention.

Briefly, suitable trials may include optional screening visits (during which the suitability of patients for inclusion in the trial may be confirmed and relevant "baseline" values established), followed by one or more incidences of treatment. These incidences of treatment may involve provision of a therapeutically effective amount of TGF-β3, or a known proportion of such a therapeutically effective amount, and, alternatively or additionally, the administration of an amount of interest of TGF-β3. Such amounts of interest may be putative therapeutically effective amounts (in which case trials of this sort may be able to identify whether or not the amount in question provides a required therapeutic effect), or may be amounts that are to be compared to those known to be therapeutically effective. Following the selected number of incidences of treatment, individuals receiving TGF-β3 may be subject to a number of "follow up" visits, which may serve to investigate the extent of scarring present (and thus any non-surgical improvement of scarring that has occurred) and/or other relevant details, such as tolerance of the dose of TGF-β3 provided.

The list set out below constitutes a preferred set of variables and assessments to be investigated as part of a trial. However it will be appreciated that suitable trials may use only some of the assessments referred to above, or may use other additional assessments as appropriate

Screening Visit (Day -28 to Day-1)

The following assessments are performed during the screening period:

• Patient's informed consent to participate in the trial is obtained.

• Medical history (including concomitant medications taken in the month prior to the screening visit) smoking history and alcohol consumption is recorded.

• Assessment of patient inclusion/exclusion criteria.

• Record of demographic data.

• A 12-Lead ECG is performed.

• Physical examination, including height, weight, blood pressure and resting pulse.

• Blood and urine samples are collected for laboratory safety screening including the following parameters: haematology (including analysis of haemoglobin, red blood cells, haematocrit, mean cell volume, white blood cells, including differentials, and platelet count); clinical chemistry (including total bilirubin, total protein, albumin, aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase, alkaline phosphatase, urea, creatinine, sodium, potassium and calcium); urinalysis: (including pH, protein, glucose, ketones and blood; and drugs of abuse (including: cocaine, amphetamines, methamphetamines, cannabis, opiates and benzodiazepines). The results of these tests are reviewed prior to the subject progressing to Day 0.

• A blood sample is taken for immunogenicity analysis.

• A urine sample is collected from females of child-bearing potential for pregnancy testing.

• A skin patch test is performed on the patient using the semi-permanent cosmetic ink used for tattooing. • The following information is recorded regarding the scar that is to be treated: o Age of scar (for example, is the scar at least two months old?). o Scar site. o Scar orientation. o Cause of scar. o Previous treatment. o The length and maximum width of each scar segment will be recorded.

• On-Patient Clinical Scar Assessments is made for each scar segment by the Investigator.

Day O

The following assessments are performed on Day 0 prior to administration of TGF-β3:

• Routine physical examination, including blood pressure and resting pulse.

• Blood and urine samples are collected for laboratory safety screening.

• A urine sample is collected from female patients of child bearing potential for pregnancy testing. Only patients having a negative pregnancy test proceed to treatment.

• Any changes in concomitant medications, or changes in the condition of the patient since the screening visit are recorded.

• Silicone moulds of the scar segments to be treated are taken.

• The skin patch test area is reviewed and patients who have had an allergic response are not allowed to participate in the study.

• The scar to be treated is divided into two equal segments and marked using semi-permanent cosmetic ink. The mid-line of the scar is marked by making two dots with the semi-permanent cosmetic ink perpendicular to and 1 cm away from the scar, and each end of the scar will be marked with one dot (as shown schematically in Figure 1).

The two scar segments separated by the midline are labelled "A" and "B" according to the following allocation rule:

Wherever possible, segment A is allocated to the proximal end of the scar and B is allocated to the distal end. If the scar is completely horizontal and it is not possible to use the proximo-distal rule, A is allocated to the end on the patient's right and B to the patient's left side.

• Local tolerability is assessed for a baseline measurement. • The scar to be treated is photographed (following marking of the scar).

• Patients are asked to complete a questionnaire to assess their scars.

• The scar to be treated is prepared and anaesthetised.

• Following anaesthesia, the scar segments (A and B) are dosed by intradermal injection with either TGF-β3 or placebo, according to the randomisation schedule. Injections are made along the scar, such that 100μl is administered per linear centimetre of scar. Doses are administered by intradermal injection along the scar using 1.OmL syringe with a 0.3 x 13mm BD Microlance™ 3 needle or equivalent.

The preferred method of administration is set out schematically in Figure 2. As shown in this Figure, the scar is marked, using a surgical marker, with a series of dots 1 cm apart from one another. The dots at either end of the scar extend at least 0.5cm from either end of the scar. The needle is inserted intradermal^ at Site B and advanced to Site A along the centre of the scar. 100μL of dosing solution is then injected into the 1cm length of the dermis as the needle is withdrawn, ensuring that the trial dose is evenly distributed between Sites A and B. Blanching is seen where the solution has been injected into the dermis. The needle is then inserted at Site C in the direction of Site B and the dosing process repeated until all of one segment is dosed. The procedure is then repeated for the other segment. Administrations extend up to the scar mid-line and at least 0.5cm, but no more than 1cm, beyond the end of each scar segment (into normal skin).

• Preferred medicaments for use in this embodiment may comprise 500ng or IOOOng of TGF-β3 (when calibrated against the NISBC reference standard) per 10Oμl of the medicament

• Details of the TGF-β3 administered and concomitant medications used are recorded.

The following procedures and assessments are performed after dosing:

• Photographs are taken of the treated scar.

• Any adverse events (AEs) and or changes in concomitant medication following dosing are recorded. Day 1 (24+/-4h following first IMP administration)

The following procedures are performed:

• Any AEs and/or changes in concomitant medication are recorded.

• Local tolerability is assessed.

• Following appropriate anaesthesia, the scar segments (A and B) are dosed as described for Day 0 above.

• As before, preferred medicaments for use in this embodiment may comprise 500ng or IOOOng of TGF-β3 (when calibrated against the NISBC reference standard) per 100μl of the medicament

• Details of IMP and concomitant medications used are recorded

Day 7 (+/- 2 days)

The following assessments are performed:

• Any changes in concomitant medications are recorded.

• Any AEs are recorded.

• An assessment is made of local tolerability.

• Photographs are taken of the treated scar.

Day 14 (+/- 2days)

The following assessments are performed:

• Any changes in concomitant medications are recorded.

• Any AEs are recorded.

• Physical examination.

• Blood pressure and pulse.

• 12-lead ECG.

• An assessment is made of local tolerability.

• Photographs of the treated scar are taken.

• A blood sample is taken for laboratory safety.

Month 1 (4 weeks +/- 4 days)

The following assessments are performed: • Any AEs and/or changes in concomitant medication are recorded.

• A blood sample is taken for immunogenicity analysis.

• Local tolerability is assessed.

• Female subjects of child bearing potential have a urine pregnancy test.

• The treated scar is photographed.

• On-Patient Clinical Scar Assessments are made for each individual treatment segment.

• Patients are asked to complete a questionnaire to assess their treated scar segments.

Months 2, 3, 4 & 5 (+/-1 week)

The following assessments are performed:

• Any AEs and/or changes in concomitant medication are recorded.

• Local tolerability is assessed.

• The treated scar is photographed.

Month 6 (22-26 weeks +/- 2 weeks)

The following assessments are performed:

• Any AEs and/or changes in concomitant medication are recorded.

• Local tolerability is assessed.

• The treated scar segments are photographed.

• Silicone moulds of the treated scar segments are taken.

• Patients are asked to complete a questionnaire to assess their treated scar segments. Month 12 (+/- 2 weeks)

The following assessments are performed:

• Any AEs and/or changes in concomitant medication are recorded.

• Local tolerability is assessed.

• The treated scar segments are photographed.

• Silicone moulds of the treated scar segments are taken.

Sequence Information

TGF-β 3 (Sequence ID No. 1)

ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADT THSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMWKSCKCS

Claims

1. TGF-β3 for use as a medicament for provision to a scar to bring about the nonsurgical improvement thereof.

2. TGF-β3 according to claim 1 , for use as an injectable medicament.

3. TGF-β3 according to claim 1 or claim 2, wherein the medicament is for the provision of between approximately 100 ng and 10,000 ng of TGF-β3 per centimetre of scar to be non-surgically improved.

4. TGF-β3 according to claim 3, wherein the medicament is for the provision of between approximately 500 ng and 1000 ng of TGF-β3 per centimetre of scar to be non- surgically improved.

5. TGF-β3 according to claim 4, wherein the medicament is for the provision of approximately 500 ng of TGF-β3 per centimetre of scar to be non-surgically improved.

6. TGF-β3 according to claim 4, wherein the medicament is for the provision of approximately 1000 ng of TGF-β3 per centimetre of scar to be non-surgically improved.

7. TGF-β3 according to any preceding claim, wherein the non-surgical improvement comprises reducing redness of the scar.

8. TGF-β3 according to any preceding claim, for use in non-surgical improvement of scars of the skin.

9. TGF-β3 according to any preceding claim for use in scars that are at least two months post-wounding.

10. TGF-β3 according to any preceding claim for use in scars that are up to six months post-wounding.

11. TGF-β3 according to any preceding claim for use in scars that are undergoing scar remodelling.

12. TGF-β3 according to any preceding claim, wherein the medicament is for local administration to a scar to be non-surgically improved.

13. A method for improving a scar in a subject, the method comprising non-surgically administering a therapeutically effective amount of TGF-β3 to a scar of a subject in need of such improvement.

14. A method according to claim 13 comprising two or more incidences of treatment in which TGF-β3 is provided to the subject.

15. A method according to claim 14, comprising two incidences of treatment in which TGF-β3 is provided to the subject.

16. A method according to any one of claims 13 to 15, wherein the TGF-β3 is provided by injection.

17. A method according to claim 16, wherein the TGF-β3 is provided by intradermal injection.

18. A method according to any one of claims 13 to 17, wherein the TGF-β3 is provided by topical administration.

19. A method according to any one of claims 13 to 18, wherein the TGF-β3 is administered substantially along the centre of the scar.

20. TGF-β3 for reducing the redness and/or contrast of a pre-existing scar.