WO2000010592A2

WO2000010592A2 - Medicaments for preventing or reducing scarring

Info

Publication number: WO2000010592A2
Application number: PCT/GB1999/002634
Authority: WO
Inventors: Mark William James Ferguson; Salah Chettibi
Original assignee: The Victoria University Of Manchester
Priority date: 1998-08-22
Filing date: 1999-08-23
Publication date: 2000-03-02
Also published as: AU1168600A; WO2000010592A3; GB9818278D0

Abstract

Medicaments for use in the prevention or reduction of scarring comprising a therapeutically effective amount of an immunomodulator. Such medicaments are particularly useful for preventing or reducing dermal scarring. The immunomodulator may be Cyclosporin A, FK-506 or guanylhydrazone.

Description

MEDICAMENTS FOR PREVENTING OR REDUCING SCARRING

The present invention concerns the provision of medicaments for preventing or reducing scarring.

The reconstitution of tissue structural integrity in higher vertebral adult animals following, for example, surgical or accidental trauma, involves a broadly understood pattern of repair. The repair process has been categorised into three major phases; inflammation, granulation tissue formation and tissue remodelling although this is necessarily an artificial concept since it is further recognised that overlap exists between these three phases.

The reparative process begins with the recruitment of a variety of specialised cells to the effected tissue and involves extracellular matrix and basement membrane deposition, angiogenesis, selective protease activity and re-epithelialisation. An important component of the healing process in adult mammals is the stimulation of fibroblasts to generate the extracellular matrix. This extracellular matrix constitutes a major component of the connective tissue which develops to repair a wound area. The repair process, however, is not perfect and the connective tissue is often fibrous in nature and commonly forms into a connective tissue scar (a process known as fibrosis).

A scar is an abnormal morphological structure resulting from a previous injury or wound (e.g. an incision, excision or trauma). Scars are composed of a connective tissue which is predominately a matrix of collagen types 1 and 3 and fibronectin. The scar may consist of collagen fibres in an abnormal organisation (as seen in scars of the skin) or it may be an abnormal accumulation of connective tissue (as seen in scars of the central nervous system). Most scars consist of abnormally organised collagen and also excess collagen. A cutaneous or dermal scar may be defined as the macroscopic disturbance of normal skin structure and function arising as a consequence of wound repair (see: Clark R.A.F. (editor) The molecular & cellular biology of wound repair; 2^nd Edition, Plenum Press, New York, 1996, which is incorporated herein by reference).

In man, in the skin, scars may be depressed below the surface or elevated above the surface of the skin. Hypertrophic scars are a more severe form of normal scarring and are elevated above the normal surface of the skin and contain excessive collagen arranged in an abnormal pattern. A keloid is another form of pathological scarring which is not only elevated above the surface of the skin but also extends beyond the boundaries of the original injury. In a keloid there is excessive connective tissue which is organised in an abnormal fashion predominately in whirls of collagenous tissue. There are genetic predispositions to forming both hypertrophic scars and keloids. They are particularly common in Africo-Carribean and Mongoloid races.

There is a need to provide medicaments that promote the healing of wounds with reduced scarring. Examples of such situations are scars of the skin where excessive scarring may be detrimental to tissue function and particularly when scar contracture occurs (for instance skin burns and wounds which impair flexibility of a joint). The reduction of scarring to the skin when cosmetic considerations are important is also highly desirable. In the skin, hypertrophic or keloid scars (particularly in Africo-Caribbean and Mongoloid races) can cause functional and cosmetic impairment and there is a need to prevent their occurrence. Scarring resulting from skin grafts (e.g. following burn injury) in both donor and recipient sites and from the application of artificial skin can also be problematic and need to be minimised or prevented.

Whilst the above considerations mainly apply to scar development in man it will be appreciated that scarring can also be problematic in other animals, particularly veterinary or domestic animals (e.g. horses, cattle, dogs, cats etc). For instance abdominal wounds are a major reason for having to put down horses (particularly race horses).

There are a number of post-scarring agents that attempt to treat the scar once formed (e.g. CICA-CARE®, Smith & Nephew) but a need remains for the provision of further compositions for the treatment of scarring. In particular there is a need for medicaments which prevent the development of a scar or improve the quality of existing or developing scars.

Therefore, according to a first aspect of the present invention, there is provided the use of an immunomodulator for the manufacture of a medicament for the prevention or reduction of scarring.

According to a second aspect of the present invention, there is provided a method of preventing or reducing scarring comprising administering to a subject in need of treatment a therapeutically effective amount of an immunomodulator.

By "medicament" we mean a composition comprising an immunomodulator as an active agent that may be used clinically and/or for cosmetic purposes.

In accordance with the invention, the inventors have established that immunomodulators may be used to prevent or reduce the development of a scar (when given at the time of wounding or as a prophylactic) or to reduce the size of an existing scar.

The immunomodulators used according to the present invention may be known and safe for application to a mammal and broad acting (i.e. down regulates the immune system by affecting a broad range of immune effector cells). An effective amount of agent should be incorporated into the medicament (i.e. an amount sufficient to promote scar free healing).

It is preferred that the immunomodulator is an immunosuppressant.

The immunomodulator may be selected from a group consisting of Cyclosporin A, Cyclosporin G, Guanylhydrazone, CS-1, Terrapin 1, FK-506. We have found that these immunomodulators have a good efficacy for treating scarring (and dermal scarring in particular).

It is preferred that the immunomodulator is an immunosuppressant selected from Cyclosporin A, Guanylhydrazone (CN 1-1493) or FK-506 (Tacrolimus) and most preferred that the immunomodulator is Cyclosporin A.

Guanylhydrazone (also known as CNI-1493) is a tetravalent compound that belongs to a class of guanyl hydrazine compounds developed for the targeted inhibition of cytokine-inducible NO production by nitric oxide synthase II (iNOS). In addition, it blocks the production of pro-inflammatory cytokines from activated human monocytes and protects against lethal endotoxemia and carrageenan-induced inflammation.

Cyclosporin A (CsA ) is a known hydrophobic oligopeptide fungal metabolite with potent immunosuppressant properties currently used as a drug in transplant surgery and also in the treatment of systemic conditions that involve immunological components.

FK-506 (also known as Tacrolimus) is a macrolide immunosupressant, chemically unrelated to cyclosporin but has a similar mode of action and uses. The efficacy of these immunomodulators for treating scarring and fibrosis is surprising for a number of reasons including the following:

(1) The prior art teaches that the abovementioned immunomodulators have a negative effect on wound healing. Therefore a clinician would not consider giving immunomodulators according to the present invention to a subject to treat scarring because he would expect the rate of healing of the associated wound to be compromised. Furthermore he would also expect such a wound to be more susceptable to infection. For instance, Cohen et al. (at p351 in Wound Healing Biochemical and Clinical Aspects, W B Saunders CO., Philadelphia, 1992) state that immunosuppressives such as steroids, azathioprine, and cyclosporin A cause a potential deficiency in tissue repair. Furthermore Fishel et al. (J Surg Res 1983 Jun;34(6):572-5) published that Cyclosporin A impairs wound healing in rats. Schaffer et al. (at p815 Transplantation Vol 65(6) 1998) indicate that tacrolimus impairs wound healing. However the inventor observed, at the concentration of immunomodulator required to treat scarring, that there was no increased risk of infection or delay in wound healing.

(2) Immunosuppessives are also associated with potentially severe side effects (e.g.renal and hepatic damage, gastro-intestinal disturbances, infections and the like). Given the risk of such side effects, immunomodulators have only been used in a clinical context where the benefits outweigh the risks (e.g. transplant rejection). Therefore a clinician would not think to administer an immunomodulator to prevent scarring for cosmetic reasons nor for non-life threatening medical reasons. However, as explained in more detail below, the inventor has surprisingly found that the immunomodulators used according to the present invention may prevent or reduce scarring when administered (preferably topically) to a subject such that the systemic concentration of the immunomodulator is substantially lower than required to regulate transplant rejection etc. In fact the inventors have found that a dose of an immunomodulator approximately 1000 fold lower than required for systemic immunosuppression may be used to prevent or reduce scarring. At this sort of dose level the risks of side effects are eliminated or at least substantially reduced.

US 5,573,775 discloses methods and compositions for treatment of corneal haze. Several proposals are put forward to explain the appearance of this disorder (which occurs after photoablation of the cornea during opthalmic surgery). These proposals include that the haze may be caused by scar formation as well as that the fibroblasts in the stroma may be improperly activated or that the laser has damaged collagen fibrils, caused necrosis of fibroblasts in the stroma or corneal oedema. A broad range of compositions are proposed to treat corneal haze. To prevent scar formation, wound healing modulators such as steroids, growth factors, basement membrane components and regulators of collagen structure are proposed. In particular, basement membrane components are used to prevent scar formation. Immunomodulating agents are included in this disclosure but are specifically used to control corneal haze associated with inflammation. However, there is no disclosure in US 5,573,775 suggesting how or what kind of dose of an immunomodulator should be used to treat corneal haze. In particular there is no suggestion that immunomodulators may be used according to the present invention for reducing or preventing dermal scarring or fibrosis. Furthermore, other prior art such as Chang et al. (Exp. Eye Res. 1998 April 60(4) p389-396) and Filipec et al. (Cornea 1992 Nov;l l(6):p546-52) report that topical cyclosporin A has no effect on corneal wound healing or corneal haze. In fact these papers suggest that cyclosporin A has an adverse effect by causing conjunctival infections. Therefore a skilled person would doubt that immunomodulators are useful in the treatment of corneal haze and would certainly believe that Cyclosporin A had no role to play in the wound healing process or the regulation of scarring.

It is preferred that immunomodulators are used according to the invention in situations or conditions where dermal scarring needs to be prevented or reduced such as: (i) where scars of the skin may be excessive and/or detrimental to tissue function and particularly when scar contracture occurs or may occur (for instance skin burns and wounds may impair flexibility of a joint). In this respect scarring can be particularly detrimental in children as they grow;

(ii) scarring to the skin when cosmetic considerations are important;

(iii) when hypertrophic or keloid scars (particularly in Africo-Caribbean and Mongoloid races) may occur which can cause functional and cosmetic impairment; and

(iv) scarring resulting from skin grafts at both donor and recipient sites and from the application of artificial skin.

As well as scars of the skin, internal scarring or fibrosis can be highly detrimental. Specific examples of internal scarring which may be treated with immunomodulators according to the present invention include:

(v) Scarring within the central nervous system such as glial scarring which can prevent neuronal reconnection and/or loss of motor-sensory function (e.g. following neuro-surgery or penetrating injuries of the brain).

(vi) Scarring in the heart (e.g. following surgery or myocardial infarction) can give rise to abnormal cardiac function.

(vii) Operations involving the abdomen or pelvis, often result in adhesion between viscera. For instance, adhesions between elements of the gut and the body wall may form and cause twisting in the bowel loop leading to ischaemia, gangrene and the necessity for emergency treatment (untreated they may even be fatal). Likewise, trauma or incisions to the guts can lead to scarring and scar contracture to strictures which cause occlusion of the lumen of the guts which again can be life threatening.

(viii) Scarring in the pelvis in the region of the fallopian tubes can lead to infertility.

(ix) Scarring following injury to muscles can result in abnormal contraction and hence poor muscular function. (x) Scarring or fibrosis following injury to tendons and ligaments can result in serious loss of function.

The development of scarring for conditions (v) - (x) is similar to that observed in the dermis (e.g. following surgery or trauma). Therefore the use of immmunodulators for treating these conditions according to the present invention is distinguishable from the disclosure of US 5,573,775 for at least the same reasons as stated above.

Medicaments according to the invention 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 sterile solution (suitable for e.g. intradermal injection). Preferably the medicament is in a form suitable for topical application. In this case it may be in the form of a gel, hydrogel, aerosol, cream, lotion, ointment, liniment, paste, paint, powder or the like.

It will be appreciated that the vehicle of the composition of the invention should be one that is well tolerated by the patient and allows release of the active compound to the wound. Such a vehicle is preferably biodegradeable, bioresolveable and/or non-inflammatory.

Preferably, the immunomodulator is further combined with pharmacologically acceptable carriers or vehicles such as e.g. hyaluronic acid, cellulose, carboxymethylcellulose, e.g. INTRASITE (Trade mark, Smith & Nephew), various hexose sugars (for example, glucose or sucrose) or other naturally occurring polymers (e.g. fibrin and collagen). Hexose sugars (and in particular glucose) are preferred carriers or vehicles.

We have found that when Cyclosporin A is used as the immunomodulator that it has particular efficacy for preventing or reducing scarring when the carrier or vehicle is hyaluronic acid. Therefore it is preferred that the medicament comprises an immunomodulator and hyaluronic acid.

The medicament of the invention may be used in a number of ways. Thus, for example, a composition may be applied in, and/or around a wound of a patient to regulate wound healing. If the composition is to be applied to an "existing" wound, then the pharmaceutically acceptable vehicle will be one which is relatively "mild" i.e. a vehicle which is biocompatible, biodegradable, bioresolvable and noninflammatory.

Immunomodulators may be formulated with pharmaceutically acceptable vehicles, carriers or delivery systems such as creams, gels, films or liposome/transferasome preparations.

According to one embodiment of the present invention, the medicament is injected or otherwise placed into the wound site and covered with a conventional wound dressing or, where necessary, suturing. This can occur shortly after a trauma event.

In a further embodiment of the present invention, there is provided a film, dressing or patch for the treatment of skin scarring having incorporated therein, an effective amount of releasable immunomodulator. The film, dressing or patch may be used to cover or even pack a wound to be treated.

The immunomodulator may also be incorporated into a tissue graft of natural or artifically constructed skin to help prevent or reduce scarring as the graft heals.

The medicament of the present invention may be used in conjunction with skin replacement products e.g. Dermagraft™, Appligra ^M and Laserskin™. In other embodiments, the medicament may be used as a prophylactic in advance of anticipated wounding (e.g. particularly elective surgery) so as to provide for regulation of scarring of the subsequently formed surgical wound. In this case a subcutaneous injection around the incision site may be used to provide an effective localised concentration of the immunomodulator during and immediately after the surgical procedure.

The vehicle of a topically applied medicament may need to be one capable of promoting the transfer of the immunomodulator across the keratinous layer of the skin. Examples of suitable vehicles for this purpose include dimethyl sulphoxide and acetic acid. Liposomes and transfεrsomes are also useful as delivery vehicles across intact skin. The choice of such vehicles will obviously depend upon the physiochemical properties of the specific immunomodulator used. Such vehicles are useful when prophylactic treatment is required or after a wound site has re- epithelialised.

It will be appreciated that the amount of an immunomodulator to be applied to a tissue to prevent or reduce scarring depends on a number of factors such as its biological activity and bioavailability, which in turn depends on the mode of administration and the physicochemical properties of the immunomodulator. Other factors include:

A) The half-life of the immunomodulator in the subject being treated.

B) The specific type of treatment (e.g.prevention of dermal, scarring, prevention of a keloid, tendon repair, prevention of adhesions).

C) The age of the subject and other individual characteristics.

The frequency of administration will also be influenced by the above mentioned factors and particularly the half-life of the compound within the subject being treated. Generally when the medicaments are used to treat existing dermal wounds the immunomodulator may be administered as soon as the wound has occurred. Therapy with the medicament should continue until the wound has healed and a clinician is satisfied that scarring has been prevented or reduced. Scars and fibrosis can develop over days, weeks or months. Therefore the subject being treated may well benefit by administration of an immunomodulator (such as Cyclosporin A) even if it is administered days or even weeks after the wound occurred.

When used as a prophylactic (e.g. before surgery) the immunomodulators should be administered as soon as the risk of undesirable scarring or fibrosis has been recognised. For instance, a cream, ointment or liposome/transferasome gel containing Cyclosporin A may be applied to a site on the skin of subject where elective surgery is to be performed and there is a risk of a scar forming. In this case, the medicament may be applied during the preoperative preparation of the subject or it may even be desirable to apply the medicament in the hours or days preceding the surgery (depending upon the health status and age of subject as well as the size of the wound to be formed).

Frequency of administration will depend upon the biological half-life of the compound used. Typically a cream or ointment or film containing an immunomodulator should be administered to a target tissue such that the concentration of the immunomodulator at the wound site or tissue affected is maintained at a level suitable for having a therapeutic effect. This may require administration daily or even several times daily.

Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials etc), may be used to establish specific formulations of medicaments and precise therapeutic regimes (such as daily doses of the immunomodulators and the frequency of administration). The medicament of the present invention preferably comprises the immunomodulator at a level capable of delivering a dose to the target area which, whilst promoting scar- free healing of a trauma site, minimises or eliminates clinically- significant immune suppression or other side-effects associated with known uses of immunomodulators. Thus the medicament may comprise an ointment or the like that comprises the immunomodulator at concentrations of between 1 to 30% wt, preferably greater than 3% wt, more preferably less than 20% wt, typically between 5 to 15% wt.

Purely by way of example a medicament containing 0.1 - 1000 μg/ml of Cyclosporin A is suitable for application to an existing (i.e. "open") wound. Preferably 1 - 100 μg/ml of Cyclosporin A is used and most preferably about 10 μg/ml of Cyclosporin A. We have found that topical application of a cream or the like containing 10 μg/ml of Cyclosporin A when applied to a wound is particularly useful for preventing or reducing scarring. Furthermore such a cream does not lead to the systemic distribution of the immunomodulator at levels which are likely to cause significant systemic immunosupression or lead to the side-effects associated with such immunomodulators.

Also by way of example, a medicament may contain about 0.2 - 20 μM of guanylhydrazone and more preferably about 2μM to have the desired effect on scar development.

By way of further example, a medicament may contain between O.lmg/ml and 50mg/ml of FK-506 and more preferably between 1 and 5mg/ml and most preferrably about 5mg/ml FK-506.

The above considerations relate to the amount of immunomodulator which should be contained within a medicament. It will be appreciated that a suitable daily dose of an immunomodulator (i.e. the amount of a medicament required) depends upon various factors (see above) and particularly the size of the wound, or amount of tissue effected by scarring or fibrosis, which is to be treated.

The present invention will further be described in the following non-limiting Examples which refer to the accompanying drawings, in which:

Fig. l illustrates a photograph at xlOO magnification of the wound site at 70 days post-wounding following treatment with a control solution of 5% glucose only from Example 1 ;

Fig.2 illustrates a photograph of fig.l at x200 magnification from Example 1 ;

Fig.3 illustrates a photograph at xlOO magnification of the wound site at 70 days post- wounding following treatment with lOμg/ml cyclosporin A in 5% glucose carrier from Example 1 ;

Fig.4 illustrates a photograph of the view of fig.3 at x200 magnification from Example 1 ;

Fig.5 illustrates a photograph at x 100 magnification of the wound site at 70 days post-wounding following treatment with ethanol/tween/PBS control only from Example 1 ;

Fig.6 illustrates a photograph at xlOO magnification of a wound site at 70 days post- wounding following treatment with lmg/ml FK506 in ethanol/tween/PBS solution from Example 1 ;

Fig.7 illustrates a the view of fig.7 at x200 magnification from Example 1 ; Fig.8 illustrates a photograph at xlOO magnification of the wound site at 14 days post- wounding following treatment with (A) lOμg/ml cyclosporin A in 50μl PBS and (B) a control treated with 50μl PBS only from Example 2;

Fig.9 illustrates a photograph at xlOO magnification of the wound site at 70 days post-wounding following treatment with (A) Hyaluronic acid treated wounds, (B) Hyaluronic acid and lOμg/ml cyclosporin A treated wounds and (C) lOμg/ml cyclosporin A treated wounds from Example 3; and

Fig.10 illustrates a photograph at x 100 magnification of the wound site at 70 days post-wounding following treatment with (A) a control treated with 50μl PBS only and (B) lOμg/ml cyclosporin A in 50μl PBS from Example 4.

EXAMPLE 1

Experiments were performed to test the efficacy of the immunomodulators Cyclosporin A and FK-506 for preventing or reducing scaring in an animal model of wound healing.

1.1 Methods

1.1.1 Surgical Technique

CD1 adult murine male 8-12 weeks old were anaesthetised and shaven down the length of their backs. Two 1cm wounds were drawn onto the skin of the back and 25μl of test or control solution injected intradermally down each side of the wound site. Two standardised full thickness linear incisions were made using a scalpel blade on the flanks of the animals extending 3.5-4.5 cm from the base of the skull. The animals were allowed to recover from the anaesthesia. The injections were repeated daily for up to seven days post-wounding.

1.1.2 Test and control solutions

Cyclosporin A (50mg/ml), was reconstituted in 5% glucose to give a test solution of lOμg/ml Cyclosporin A in 5% glucose. FK-506 (lmg/ml) was made up in a solution of 2% ethanol, 0.5% Tween and 97% PBS. Solutions of the glucose and ethanol/tween/PBS without added immunomodulator were used as controls.

Each test and control solution was administered as described above to a separate group of mice prepared as described above.

1.1.3 Wound Harvesting and Histology

Wounds were harvested 70 days post-wounding from the animals and prepared for histological analysis. This was carried out by fixing the wounds in freshly prepared 4% paraformaldehyde and embedded in paraffin wax according to standard techniques well known to those skilled in the art. The embedded samples were then stained using Lillie modification of Masson's trichrome stain as well known to those skilled in the art.

1.2 Results

1.2.1 Control Treatments (vehicle for Cyclosporin A)

In figs.1 and 2, the edges of the wound can be identified by the presence of skin appendages and hair follicles. The wound site itself lacks any of these structures. The collagen at the wound site is less mature having narrower bundles and lighter staining than the surrounding uninjured dermal tissue.

1.2.2 Treatment with 1 Oμg/ml Cyclosporin A

In fig.3 and 4 again the wound area can be easily identified due to the lack of appendages. However, there is very little difference in the collagen bundle structures and orientation when compared with the uninjured surrounding skin. Mature, dense staining, collagen bundles can be seen extending from the wound site into the surrounding tissue. Fig.4 in particular illustrates that the wounded tissue is more similar to that of normal skin and dermis than that of scar tissue.

1.2.3 Control Treatments (vehicle for FK-506)

In fig.5 less dense staining with smaller collagen bundle diameter can be seen in the central wound area. Hair follicles and sweat glands can be seen either side of the wound margins but are almost entirely absent from the central wound area itself.

1.2.4 Treatment with lOmg/ml FK-506

In fig.6 and 7, closely packed mature looking collagen fibres can be observed and the overall appearance is similar to that of normal uninjured skin.

These data illustrate that both Cyclosporin A and FK-506 have beneficial effects on the quality of a scar formed after a healing of a dermal wound. The experiments performed above were expanded to further investigate the usefulness of immunomodulators for preventing or reducing dermal scarring.

The methods described for Example 1 were followed except where outlined as different below.

EXAMPLE 2: Scar modulation by Cyclosporin A (CsA

Twelve adult male CD1 mice were divided into three groups each containing four animals. All animals were injected intradermally with CsA for eight consecutive days starting injection immediately pre-wounding.

The following concentrations of CsA were used 1 Oμg/ml, 1 μg/ml and 100 ng/ml per 50 μl of PBS per wound. In each animal one wound was treated with CsA and the other wound was treated with PBS as a control. All animals were left for 14 days so that any changes in the extracellular matrix could be easily identified. All wounds were processed in accordance with standard protocols for histology and immunocytochemistry.

Results

Results showed that all four wounds treated with 10 μg/ml of CsA were markedly different from their relevant controls with wounds being narrower and with a greater proportion of fibroblastic cells to inflammatory cells than the controls showing accelerated wound healing (Fig. 8a and b).

The lower concentrations of CsA had less, but observable, effects on scarring than 10 μg/ml of CsA. These data are important as they demonstrate that CsA can modulate scar formation at relatively low doses (compared to doses required for systemic immunosuppression). Therefore the immunomodulators may be used according to the invention without a significant risk of developing the kind of side-effects associated with usage as an immunosupressent (e.g. to prevent rejection of transplanted organs).

EXAMPLE 3: Scar modulation by Cyclosporin A (CsA)

Six animals were divided to three groups each containing two animals:

(i) animals were injected for eight days with 10% hyaluronic acid;

(ii) animals were injected for eight days with 10% hyaluronic acid + 1 Oμg/ml

CsA reconstituted in 20% ethanol and 5% glucose; and

(iii) animals were injected for eight days with 1 Oμg/ml CsA reconstituted in

20% ethanol and 5% glucose.

All wounds were harvested at day 70 post-wounding and processed in accordance with standard protocols for histology for scar assessment.

Results

This study showed that intradermal injection of 1 Oμg/ml CsA for eight days at the wound margin resulted in a clear improvement in scar formation when compared with control wounds (Fig. 9 a, b and c). Treatments in which CsA was administered with hyaluronic acid (Fig. 9 b) resulted in a good reduction in scar formation.

EXAMPLE 4: Scar modulation by Cyclosporin A (CsA)

30 CD1 male adult mice were divided to 4 groups Group-1. 18 animals were injected intradermally with 1 Oμg/ml CsA dissolved in PBS containing 0.005% ethanol/tween-80 at 5:1 v/v respectively. Group-2. 4 animals were injected intradermally with PBS containing 0.005% ethanol/tween-80 at 5:1 v/v/ respectively. Group-3. 4 animals were injected intradermally with lOOμg/ml CsA dissolved in PBS containing 0.05% ethanol/tween-80 at 5:1 v/v respectively. Group-4. 4 animals were injected intradermally with PBS containing 0.05% ethanol/tween-80 at 5:1 v/v respectively.

All 30 animals were wounded and each wound was injected with 50μl of the relevant solution for eight consecutive days starting injections immediately pre- wounding. Wounds were harvested at day 70 post-wounding and processed in accordance with standard histology protocols for scar assessment.

In this study we confirmed that wounds treated with 1 Oμg/ml of CsA were markedly different from their relevant controls with wounds being narrower, healing faster and with a greater proportion of fibroblastic cells to inflammatory cells at later time points (Fig 10a, b). The scars in the CsA treated wounds were markedly improved compared to the controls.

EXAMPLE 5: Scar modulation by Guanylhydrazone (CNI-1493)

CD1 mice aged 8-12 weeks were wounded using the standard model as previously shown. Intradermal injection of CNI-1493 was performed everyday for the first 7 days of wound healing.

The doses used were 2, 20, 200 μM in isotonic sucrose, injecting 50 μl each day per wound at the wound margin. Control animals were injected with isotonic sucrose only. The wounds were harvested 14 and 70 days post wounding and processed for wax histology.

Results

Intradermal injection of 50μl per wound of CNI-1493 reconstituted in isotonic sucrose showed that its effect on wound healing was dose dependent. At 70 days post wounding there was a difference in scarring firstly from control and secondly between doses. The 2μM injected wounds appeared to be much narrower and less cellular and result in a better scar than the relevant control (isotonic sucrose treated wounds)

The 20 μM wounds appeared better than controls, however at this dosage there was an increased number of inflammatory cells at the sites of injection which resulted in less improved scar.

The 200 μM wounds macroscopically were worse than controls. Microscopically the wound was large and very cellular and the epidermis was thick. The sites of injection had even more inflammatory cells than the 20 μM wounds, to an extent that the areas of injection resembled a wound site.

These data indicate that CNI-1493 may have a toxic effect at higher concentrations which results in more damage to the tissues at the wound area leading to major influx of leukocytes. Therefore it is preferred that the medicament contains 20 μM or less of CNI-1493.

EXAMPLE 6: Scar modulation by FK-506

14 CD1 male adult mice were divided into 7 groups Group-1. 2 animals were injected intradermally with 5mg/ml FK506 dissolved in PBS containing 25% methanol/tween-80 at 5:1 v/v respectively. Group-2. 2 animals were injected intradermally with lmg/ml FK506 dissolved in PBS containing 2.5% methanol/tween-80 at 5:1 v/v respectively. Group-3. 2 animals were injected intradermally with 0. lmg/ml FK506 dissolved in PBS containing 0.25% methanol/tween-80 at 5:1 v/v respectively. Group-4. 2 animals were injected intradermally with 0.0 lmg/ml FK506 dissolved in PBS containing 0.025% methanol/tween-80 at 5:1 v/v respectively. Group-5. 2 animals were injected intradermally with PBS containing 25% methanol/tween-80 at 5:1 v/v respectively. Group-6. 2 animals were injected intradermally with PBS containing 2.5% methanol/tween-80 at 5: 1 v/v respectively.

Group-7. 2 animals were injected intradermally with PBS containing 0.25% methanol/tween-80 at 5: 1 v/v respectively.

All animals were wounded and each wound was injected with 50μl of the relevant solution for eight consecutive days starting injections immediately pre- wounding. Wounds were harvested at day 70 post-wounding and processed in accordance with standard histology protocols for scar assessment.

Results

The effect of each concentration of the immunomodulator was assessed relative to the respective control treatments. The effect of FK-506 on murine adult scarring was dose dependent with the maximum effect observed at the highest concentration (5mg/ml).

Claims

1. The use of an immunomodulator for the manufacture of a medicament for the prevention or reduction of scarring or fibrosis.

2. The use according to claim 1 wherein the scarring or fibrosis is associated with a burn or a penetrative injury.

3. The use according to claim 1 or 2 wherein the medicament is for the prevention or reduction of dermal scarring or fibrosis.

4. The use according to claim 3 for preventing or reducing hypertrophic or keloid scarring.

5. The use according to claim 3 for preventing or reducing scarring for cosmetic purposes.

6. The use according to claim 1 or 2 wherein the medicament is for the prevention or reduction of neural, cardiac, abdominal, pelvic or muscular scarring or fibrosis.

7. The use according to claim 1 or 2 wherein the medicament is for the prevention or reduction of scarring or fibrosis following injury to tendons and ligaments.

8. The use according to claim 1 or 2 wherein the medicament is for the prevention or reduction of scarring or fibrosis leading to abdominal adhesions.

9. The use according to any preceding claim for preventing or reducing scarring to prevent loss of tissue function.

10. The use according to any preceding claim wherein the immunomodulator is selected from Cyclosporin A, Guanylhydrazone (CNI-1493) or FK-506 (tacrolimus).

1 1. The use according to claim 10 wherein the immunomodulator is Cyclosporin A.

12. The use according to claim 1 1 wherein the medicament comprises 0.1 - 1000 ╬╝g/ml of Cyclosporin A.

13. The use according to claim 12 wherein the medicament comprises 1 - 100 ╬╝g/ml of Cyclosporin A.

14. The use according to claim 13 wherein the medicament comprises 10 ╬╝g/ml of Cyclosporin A.

15. The use according to claim 10 wherein the immunomodulator is Guanylhydrazone (CNI-1493) and the medicament comprises less than 20╬╝M Guanylhydrazone (CNI-1493).

16. The use according to claim 10 wherein the immunomodulator is FK-506 (tacrolimus) and the medicament comprises about 1 - 5mg/ml of FK-506.

17. The use according to any preceding claim wherein the immunomodulator is for topical application.

18. The use according to any one of claims 1 - 16 wherein the medicament is for local injection.

19. The use according to any preceding claim wherein the immunomodulator is combined with a pharmaceutically acceptable carrier or delivery system.

20. The use according to claim 19 wherein the carrier or delivery system is one of a cream, gel, film or liposome/transf╬╡rasome preparation.

21. The use according to claim 19 wherein the carrier or delivery system is hyaluronic acid.

22. The use according to any preceding claim wherein the immunomodulator is formulated into a film, bandage or patch.

23. The use according to any one of claims 1 - 16 wherein the immunomodulator is for incorporation into a graft of natural or artifically constructed skin.

24. A method of preventing or reducing scarring or fibrosis comprising administering to a subject in need of treatment a therapeutically effective amount of an immunomodulator.