MOLECULAR TARGETS FOR HEALING OR TREATING WOUNDS

Abstract

The invention relates to at least one molecular target for healing or treating wounds and, in particular chronic, human wounds. The molecular target is PTPRK, or a protein 50% homologous therewith, and which retains the same activity as PTPRK protein. Further, the invention concerns a novel therapeutic for treating said wounds and a novel gene therapy approach, involving said molecular target, for treating said wounds.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to at least one molecular target for healing or treating wounds and, in particular, human wounds. More particularly still, the molecular target has application in the treatment of chronic wounds. Further, the invention concerns a novel therapeutic for treating said wounds and a novel gene therapy approach, involving said molecular target, for treating said wounds. Additionally, the invention concerns a method for treating wounds using said therapeutic or said gene therapy.

BACKGROUND OF THE INVENTION

[0002] In one form or another, chronic and poorly healing wounds constitute a major burden on the UK health system. Moreover, in certain member countries of the EU health expenses relating to wound healing are already approaching the third most expensive drain on health care funding.

[0003] A Chronic wound is herein defined as one exhibiting delayed or defective healing which does not progress through the predictable stages of the healing process (as described below). Commonly, chronic wounds are classified into three broad categories: venous ulcers, diabetic, and pressure ulcers. Long-term venous insufficiency accounts for 70% to 90% of chronic wounds and commonly affects the elderly. Venous insufficiency results in venous hypertension, in which blood flow is abrogated resulting in subsequent ischaemia. Venous insufficiency can occur as a result of obstructions to venous outflow or reflux due to valve damage. Following a period of ischaemia, tissue reperfusion can result in reperfusion injury, causing the tissue damage that leads to wound formation.

[0004] Chronic foot ulcers are a major complication of diabetes, accounting for up to 25% of all hospital admissions involving diabetes, and at a cost to the UK National Health Service of .English Pound.250M annually. Chronic foot ulcers cause substantial morbidity, impair the quality of life, and are the major cause of lower limb amputation. Despite careful attention to foot care, as many as 25% of diabetics develop foot ulcers in their lifetimes. The causes of lower limb ulceration are the same in diabetics as in non-diabetics, namely neuropathy, ischaemia and trauma. However, this "pathogenic triad" predisposes wounds to infection, which can also contribute to the non-healing nature of the wounds.

[0005] Pressure wounds are another major resource health cost. They are typically caused by failure to provide routine nursing or medical care. In the UK 412,000 people are affected annually by this sort of wound at a cost of .English Pound.1.4-2.1 billion.

[0006] Furthermore, chronic wounds can also be categorised by whether they are caused by surgery, burns, dermatitis, vasculitis or radiation.

[0007] Current wound treatment strategies involve removing pressure from the area, debridement, wound dressing and management of infection: surgical resection and vascular reconstruction may be required in more advanced disease, which ultimately may necessitate amputation. These stragegies commonly seek to address problems that are associated with chronic wounds, such as bacterial load, ischaemia, and imbalance of proteases, all of which can further affect the wound healing process.

[0008] The healing of a wound is controlled by complex biological processes that involve a diverse number of cell types; complex interactions between cells and tissues; the activation of the immune system and the activation of the angiogenic process. Moreover, all of these processes involve a large number of molecules.

[0009] A typical healing process can be divided into 5 distinct, but closely related, stages: clotting stage, acute inflammation stage, matrix deposition stage, capillary formation stage and re-epithelialisation stage. A diverse number of factors are involved in and control each of these stages. Deficiencies in any aspect of the process may result in defective wound healing. Thus, a `normal` healing process may be defective as a result of either intrinsic or external factors, which manifest as `abnormal non-healing` or `chronic` wounds. It is these chronic or `non-healing` wounds that present the greatest challenge to the quality of a patient's life and mounting expenses to the healthcare system.

[0010] A chronic wound often arises from failure to progress through the normal stages of wound healing, whereby an initial injury resulting in a wound cannot subsequently be repaired. Changes occur within the molecular environment of a chronic wound, such as high levels of inflammatory cytokines or proteases, and low levels of growth factors, these changes detain or terminate the healing process and increase the potential for septic infections. By enhancing or manipulating factors that contribute to wound healing it may therefore be possible to correct the process, thereby reducing the likely occurrence of a chronic wound, or accelerate its subsequent repair.

[0011] PTPRK

[0012] Protein tyrosine phosphatise receptor type K, PTPRK, is also known as DKFZp686C2268, DKFZp779N1045 and R-PTP-kappa. It is a member of the protein tyrosine phosphatase (PTP) family. PTPs are known to be signalling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. PTPRK possesses an extracellular region, a single transmembrane region, and two tandem catalytic domains, and thus represents a receptor-type PTP. The extracellular region contains a meprin-A5 antigen-PTP mu (MAM) domain, an Ig-like domain and four fibronectin type III-like repeats. Moreover, PTPRK has been shown to mediate homophilic intercellular interaction, possibly through the interaction with beta- and gamma-catenin at adherens junctions. Expression of the PTPRK gene has been found to be stimulated by TGF-beta 1, which may be important for the inhibition of keratinocyte proliferation. In cancer, PTPRK has been found to be suppressed in aggressive tumours as shown by our recent study in breast cancer (Sun et al, SABCS, Cancer Res 2010).

[0013] While the biochemical functions of the PTP family is known to some degree, the therapeutic implication of the PTPRK enzyme has rarely been explored, particularly in relation to wound healing.

[0014] We, therefore, have surprisingly discovered that PTPRK has a role to play in wound healing. Indeed, we have discovered that the expression of this protein impedes the wound healing process. Moreover, the inhibition of PTPRK promotes wound healing.

[0015] Inhibitors of PTPRK are known. The most readily available is a salt of stibogluconate. Sodium stibogluconate is a medicine used to treat leishmaniasis, a disease resulting from infection by one of over 20 different species of the Leishmania species of parasite. Sodium Stibogluconate belongs to the class of medicines known as the pentavalent antimonials. Whilst its exact paracidal effect on the Leishmania parasite is unknown it is thought that the parasite is killed by inhibition of glucose catabolism resulting in reduced ATP synthesis, thereby decreasing subsequent macromoleular synthesis and preventing replication.

[0016] Sodium stibogluconate is sold in the United Kingdom as Pentostam® (manufactured by GlaxoSmithKline) and is currently only available for administration by injection. Unfortunately, widespread resistance to this medicine has limited the utility of sodium stibogluconate, and in many parts of the world, amphotericin or miltefosine is used instead to treat leishmaniasis.

[0017] In summary, we have identified at least one molecular target for treating wounds and in particular human wounds. More particularly, but not exclusively, said molecular target has application in the treatment of chronic wounds. The molecular target is PTPRK and therefore the invention relates to a novel therapeutic comprising an inhibitor of either, or both, PTPRK expression or PTPRK activity. In the former instance, the invention involves a novel gene therapy approach and in the latter instance a novel protein therapy approach. Accordingly, the invention also relates to a novel therapeutic comprising an inhibitor of either, or both, PTPRK expression or PTPRK activity. In the former instance, the invention involves a novel gene therapy approach and in the latter instance a novel protein therapy approach.

[0018] Reference herein to PTPRK, is reference to a gene or protein whose identity is shown in FIG. 16.

[0019] Our invention can improve the quality of a patient's life by ensuring that new wounds do not deteriorate into a chronic state and existing chronic wounds can be treated in a way that actively promotes healing.

STATEMENTS OF INVENTION

[0020] Accordingly, in one aspect of the invention there is provided a therapeutic comprising an inhibitor of either, or both, PTPRK gene expression or PTPRK protein activity for use in the treatment of wounds.

[0021] In the former instance, the invention involves a novel gene therapy approach and in the latter instance a novel protein therapy approach. Thus, in one embodiment the novel therapeutic comprises an inhibitor of PTPRK gene expression, this inhibitor can be either anti-sense DNA or RNA, siRNA, or ribozymes, either naked or in the form of plasmid and viral vectors. Those skilled in the art are aware of the aforementioned inhibitory molecules and so would be able to work the invention once they knew that expression of PTPRK contributed to the chronic wound phenotype. However, in another embodiment the novel therapeutic comprises an inhibitor of PTPRK protein function, this inhibitor can be either a PTPRK binding agent that binds, either reversibly or irreversibly, to inhibit protein function such as an antibody or a known, or synthesized, PTPRK antagonist; or an agent that works upstream or downstream of the PTPRK signalling mechanism to inhibit PTPRK signalling and so negate the effects of expression of PTPRK protein in wound tissue. Those skilled in the art are aware of the aforementioned inhibitory molecules and so would be able to work the invention once they knew that expression of PTPRK contributed to the chronic wound phenotype.

[0022] In a preferred embodiment of the invention the therapeutic comprises a PTPRK gene inhibitor such as transgene 1 or transgene 2 or transgene 3 described herein. These molecules are termed anti-PTPRK ribozyme/RNA transgenes. Transgene 1 is produced by transcription of the PTPRK gene using the following short oligos:

TABLE-US-00001 Anti-PTPRK transgene1F Ctgcagagtgagttacacagcctgatgagtccgtgagga And Anti-PTPRK transgene1R ActagtgacaaaaactgaccaggatttgtAtttcgtcctcacggact.

[0023] Transgene 2 is produced by transcription of the PTPRK gene using the following short oligos:

TABLE-US-00002 Anti-PTPRK transgene2F Ctgcaggatgataggaccatcgccaatctgatgagtccgtgagga and Anti-PTPRK transgene2R ActagtgatccaactaaatgccaactcgAtttcgtcctcacggact.

[0024] Transgene 3 is produced by transcription of the PTPRK gene using the following short oligos:

TABLE-US-00003 Anti-PTPRK transgene3F Ctgcagtttgctcttttttacaattaatatctgatgagtccgtgagga and Anti-PTPRK transgene3R ActagttcatcctccttctcctagttGtttcgtcctcacggact.

[0025] These products are antisense-hammerhead ribozyme also known as antisense-hammerhead RNA, ideally they are flanked by selected restriction sites such as pstI and SpeI and more ideally still they are cloned into a cloning vector such as pEF6N5His-TOPO vector (Invitrogen),

[0026] The sequence structure of transgene 1 is:

TABLE-US-00004 5'Ctgcagagtgagttacacagcctgatgagtccgtgaggacgaaa tac aaatcctggtcagtttttgttactagt'3

[0027] The sequence structure of transgene 2 is:

TABLE-US-00005 5'Ctgcaggatgataggaccatcgccaatctgatgagtccgtgaggacga aatcgagttggcatttagttggatcactagt'3

[0028] The sequence structure of transgene 3 is:

TABLE-US-00006 Ctgcagtttgctcttttttacaattaatatctgatgagtccgtgaggacg aaacaactaggagaaggaggatgaactagt'3

[0029] In a preferred embodiment of the invention the therapeutic comprises a commercially available PTPRK protein inhibitor such as, without limitation, Stibogluconate (GSK) or (Santa Cruz Biotechnologies Inc., Tocris and Sigma-Aldrich).

[0030] In a further preferred embodiment of the invention the therapeutic comprises a commercially available PTPRK protein inhibitor, such as Pentostam® (GlaxoSmithKline).

[0031] The therapeutic of the invention is for use in treating, ideally, mammalian wounds, more ideally chronic mammalian wounds, and, more ideally still, chronic human wounds. Chronic wounds that are preferably treated using the invention are venous ulcers, diabetic ulcers, and pressure ulcers. Preferably, the wounds to be treated are non-parasitic i.e. not caused--by or occupied by parasites.

[0032] An antibody for use in the invention is most ideally a monoclonal antibody or a humanised antibody.

[0033] In the above aspects and embodiments of the invention the therapeutic is formulated for topical application.

[0034] Alternatively, in the above aspects and embodiments of the invention the therapeutic is formulated for oral application.

[0035] Alternatively again, in the above aspects and embodiments of the invention the therapeutic is formulated for application to a dressing or impregnation in a dressing.

[0036] The therapeutic of the invention may be administered in combination with an antibiotic or antibacterial agent. Numerous such agents are known and suitable choices will be familiar to skilled practitioners.

[0037] In yet another aspect of the invention, there is provided a pharmaceutical composition for use in treating wounds comprising a therapeutic of the invention together with a pharmaceutically acceptable carrier.

[0038] Other active materials may also be present in the pharmaceutical composition, as may be considered appropriate or advisable for the wound being treated. For example, the composition may also contain an emollient, or the like.

[0039] The carrier, or, if more than one be present, each of the carriers, must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.

[0040] The formulations include those suitable for topical (including eye drops), oral (including buccal and sublingual), rectal, nasal or vaginal administration and may be prepared by any methods well known in the art of pharmacy.

[0041] The composition may be prepared by bringing into association the therapeutic of the invention and the carrier. In general, the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. The invention extends to methods for preparing a pharmaceutical composition comprising bringing a therapeutic of the invention in conjunction or association with a pharmaceutically or veterinarily acceptable carrier or vehicle.

[0042] For topical application to the skin, compounds of conventional use may be made up into a cream, ointment, jelly, solution or suspension etc. Cream or ointment formulations that may be used for the composition are conventional formulations well known in the art, for example, as described in standard text books of pharmaceutics such as the British Pharmacopoeia.

[0043] Formulations for oral administration in the present invention may be presented as: discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution or a suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water in oil liquid emulsion; or as a bolus etc.

[0044] For compositions for oral administration (e.g. tablets and capsules), the term "acceptable carrier" includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring and the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.

[0045] Other formulations suitable for oral administration include lozenges comprising the active agent in a flavoured or inert base base and mouthwashes comprising the active agent in a suitable liquid carrier.

[0046] In a further aspect of the invention there is provided a method for treating a mammalian wound, typically a chronic wound, which method comprises: administering to said wound a therapeutic that inhibits either, or both of, PTPRK gene expression or PTPRK protein activity.

[0047] Additionally, or alternatively, the further aspect of the invention also, or alternatively, comprises a novel method for treating a mammalian wound, typically a chronic wound, which method comprises:

[0048] administering to said wound a therapeutic that inhibits either, or both of, PTPRK gene expression or PTPRK protein activity.

[0049] According to yet a further aspect of the invention there is provided a kit for treating a wound, preferably a chronic wound, wherein said kit comprises:

[0050] (a) at least one therapeutic as above described; and

[0051] (b) at least one dressing for applying to said wound.

[0052] According to a yet further aspect of the invention there is provided a combination therapeutic for treating a wound comprising an inhibitor of PTPRK gene expression and an inhibitor of PTPRK protein activity.

[0053] According to a further aspect of the invention there is provided a therapeutic for treating a wound comprising an inhibitor of PTPRK protein, or a homologoue thereof.

[0054] According to a further aspect of the invention there is provided use of an inhibitor of PTPRK protein, or a homologoue thereof, in the manufacture of a medicament for treating a wound.

[0055] According to a further aspect of the invention there is provided use of an inhibitor of PTPRK, or a homologue thereof, for treating a wound.

[0056] The term "homologue" as used herein refers to amino acid sequences which have a sequence at least 50% homologous to the amino acid sequence of PTPRK and which retain the biological activity of the PTPRK sequence. It is preferred that homologues are at least 75% homologous to the PTPRK peptide sequence and, in increasing order of preference, at least 80%, 85%, 90%, 95% or 99% homologous to the PTPRK peptide sequence.

[0057] Treatment of a wound described herein includes reference to human or veterinary use.

[0058] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprises", or variations such as "comprises" or "comprising" is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

[0059] Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

[0060] Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

[0061] Moreover, unless stated otherwise; any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

[0062] The present invention will now be described by way of the following examples with particular reference to FIGS. 1-21 wherein:

[0063] FIG. 1. Shows the secondary structure of human PTPRK mRNA;

[0064] FIG. 2. Shows HaCaT cells after lost PTPRK by way of anti-PTPRK transgenes showed an increase in cell adhesion. Shown are traces (Top two panels) at 4000 Hz and 32,000 Hz and 3D modelling at 4,000 Hz and 500 Hz (bottom two panels);

[0065] FIG. 3. Shows effects of knocking down PTPRK in endothelial cells on the adhesion of the cells and their response to PTPRK inhibitor, stibogluconate. Left: traces of cells, response in ECIS assays. Right: A: HECV WT, B: HECV/PTPRKrib; C: HECV wt plus stibogluconate; and D: HECV/PTPRKrib plus stibogluconate;

[0066] FIG. 4. Shows effects of knocking down PTPRK in endothelial cells on cellular migration s and their response to PTPRK inhibitor, stibogluconate. Left: traces of cells response in ECIS assays. Right: A: HECV WT, B: HECV/PTPRKrib; C: HECV wt plus stibogluconate; and D: cHECV/PTPRKrib plus stibogluconate. Cell were wounded at 6 v for 30 seconds and traced immediately after wounding;

[0067] FIG. 5. Shows Traces (in triplicate) of HaCaT (WT) response to stibogluconate over an arrange of concentrations;

[0068] FIG. 6. Shows 3D modelling of HaCaT (WT) adhesion response to stibogluconate over an arrange of concentrations;

[0069] FIG. 7. Shows traces (in duplicate) of HaCaT (WT) response to stibogluconate over an arrange of concentrations. Shown are traces at 100 hZ;

[0070] FIG. 8. Shows 3D modelling of HaCaT (WT) migration response to stibogluconate over an arrange of concentrations. Shown at 1000 Hz;

[0071] FIG. 9. Shows Using Rb modelling methods, a concentration dependent stimulation of cellular migration was also demonstrated. Shown are a 5-hour wounding assay, with mean plus SD displayed in the graph;

[0072] FIG. 10. Shows the concentration related effect of Pentostam® (GlaxoSmithKline), a commercially available form of stibogluconate on the migration of the cells;

[0073] FIG. 11. Shows the effect of systemic administration of sodium stibogluconate, via the I.P. route, on the rate of wound healing.

[0074] FIG. 12 Shows effects of stibogluconate on wound healing in the db/sb model. The compound was given topically every other day;

[0075] FIG. 13. Shows the effect of weekly delivery of Stibogluconate on the rate of wound healing;

[0076] FIG. 14. Shows the effect of twice weekly delivery of Stibogluconate on the rate of wound healing;

[0077] FIG. 15. Shows a scatter plot of stibogluconate concentration vs size of the wounds after two weeks of treatment (weekly);

[0078] FIG. 16. shows the amino acid and cDNA sequence structure of PTPRK;

[0079] FIG. 17 shows the effect of removing treatment between the third and fourth week in either a weekly dosage regimen or a twice weekly dosage regimen; and Table 1. shows the primers and oligonulceotides used for the Construction and verification of ant-human PTPRK ribozyme transgenes described herein.

MATERIALS AND PROCEDURE

[0080] Cells

[0081] HaCaT, a human keratinocyte cell line was purchased from the German Cancer Centre, HECV, a human vascular endothelial cells from Interlab, Milan, Italy, DB/DB mice from Harlan UK.

[0082] Construction of Ant-Human PTPRK Ribozyme Transgenes

[0083] The transgenes were based on the human PTPRK mRNA secondary structure (FIG. 1). Three transgenes were generated, targeting ATC and GTC sites, using respective oligos listed in Table 1. Ribozymes were generated by way of touchdown PCR, followed by verification using 2% agarose gels. The correct ribozymes were ligated into a pEF6N5His-TOPO vector (Invitrogen), followed by transformation of the ligated product to Top10 E. Coli. After heat shock for 30 seconds and recover over ice for 2 minutes, the bacteria was resuspended in SOC medium and allow to grow on a shaker (200 rpm) for 1 hour. The transformed bacteria were then plated over LB agar dishes which contained 100 μg/ml Ampicillin. After incubating the plate at 37° C. overnight, discreet colonies were identified and screened for the presence of the ribozyme and the orientation of the insert, by using orientation specific PCR, using T7F primers vs RBBMR and RBTPF primers. Correct colonies were picked, grew up in LB medium in the presence of Ampicillin. Plasmids were extracted, purified and further verified by direction specific PCR (using RBTOP vs T7F and RBBMR).

[0084] Generation of Sublines of Human Keratinocytes and Endothelial Cells with Differential Expression of PTPRK

[0085] HaCaT and HECV cells, which were positive for PTPRK, were transfected with anti-PTPRK transgenes by way of electroporation (270 v). After selection with a selection medium (DMEM with 10 ug/ml blasticidin) for 10 days, clones of selected cells were pooled and used for subsequent analysis.

[0086] In Vivo Tolerance Test

[0087] The first tolerance test was conducted on the CD-1 athymic (Charles River Laboratories). Briefly, CD-1 of 4-6 weeks old, 20 g in weight, were housed in filter topped cages. Sodium stibogluconate a known PTPRK inhibitor was injected, via the intraperitoneal route, on a daily basis. The compound was given at 100 final concentration (equivalent to ˜10 mg/kg body weight) in 100 ul in volume. CD-1 were observed daily, weighed twice weekly. An additional tolerance and efficacy test was carried out using the db/db strain.

[0088] In Vivo Efficacy Test and Wound Healing

[0089] The diabetic strain of db/db was obtained from Harlan. 4-6 weeks old with body weight at 20 g were used. Creation of a wound was according to a recently described method. Briefly, after being housed for a week, the db/db were first ear-pieced using an puncher, in order to create a wound (hole) of 1 mm in diameter. The following day after wound creation, all the db/db were weighed and the wound was photographed using a digital camera. Treatment was given systemically (by IP injection) or topically (by manually applying the compound in gel into the wound area). Both treatments were given every other day, twice weekly or weekly. Images were obtained weekly. The size of the wounds was determined using an image analysis software and is shown here as the area in pixels.

[0090] Effects of Knocking Down PTPRK on the Function of Cells

[0091] Three models of ECIS instrument were used: ECIS 9600 for screening and ECIS1600R and ECIS Zθ for modelling. In all systems, 8W10 arrays were used (Applied Biophysics Inc., Troy, N.Y., USA) (Giaever and Keese 1991, Kees et al 2004). Following treating the array surface with a Cysteine solution (or array stabilization procedure for ECIS Zθ), the arrays were incubated with complete medium for 1 hr Electric changes were continuously monitored for up to 24 hrs. In the 9600 system, the monitoring was at fixed 30 Hz. In the 1600R and ECIS Zθ systems, cells were monitored at 62.5, 125, 250, 500, 1,000, 2,000, 4,000, 8,000, 16,000, 32,000 and 64,000 Hz. The adhesion was analysed by the integrated Rb modelling method.

[0092] Results

[0093] Knocking Down PTPRK from HaCaT and Endothelial Cells Resulted in an Acceleration of Cell Adhesion and Migration

[0094] It was found that after knocking down PTPRK in HaCaT cells, there was a rapid increase in cell adhesion, FIG. 2. Endothelial cells, after loss of PTPRK, showed a high rate of adhesion using an ECIS assay. Likewise, HECV/WT when treated with stibogluconate, also showed a rapid adhesion to the surface of the electrode. It is interesting to observe that HECV/PTPRKrib cells' response to stibogluconate was markedly reduced compared with that of HECV/WT. The similar changes in cellular migration were seen using the electric wounding assay of the endothelial cell model, FIG. 3 and FIG. 4.

[0095] Human Keratinocytes Showed a Dose Dependent Response to PTPRK Inhibitor Stibogluconate

[0096] Using the ECIS Theta96 model tested the response of cells to stibugluconate over a range of concentrations. HaCaT cells responded over the range of concentrations tested in that there was an increase in cell adhesion between 0.16-20 uM with 20 uM showing the maximum effects, FIGS. 5 & 6. Likewise, the cells also responded to stibogluconate by increasing their migration from concentrations as low as 160 nM to 100 uM, FIGS. 7, 8 & 9.

[0097] We have also tested the concentration related effect of Pentostam® (GlaxoSmithKline), a commercially available form of stibogluconate, on the migration of the cells, FIG. 10.

[0098] Stibogluconate was Well Tolerated In Vivo

[0099] The first tolerance test was conducted the CD-1 athymic (Charles River Laboratories). Briefly, CD-1 of 4-6 weeks old, 20 g in weight, were housed in filter topped cages. Sodium stibogluconate was injected, via the intraperitoneal route, on a daily basis. The compound was given at 100 final concentration (equivalent to 10 mg/kg body weight) in 100 ul in volume. CD-1 were observed daily, weighed twice weekly. An additional tolerance and efficacy test was carried out using the db/db strain.

[0100] Stibogluconate Accelerates Wound Healing In Vivo.

[0101] Formulation of the Compounds.

[0102] 1. For systemic application, Sodium stibogluconate was dissolved in BSS and diluted in the same for the required concentration. The solutions were prepared that each 100 ul contained the correct amount of compounds and was aliquatted and stored as such at -20° C. until used. The compound was injected every other day by the IP route.

[0103] 2. For topical application, we used two carrier gels that are currently used in wound care, namely Bactroban and Aquagel. From the concentrated master stock of Sodium stibogluconate, 100 ul of the stock solution was mixed with 2 grams of the respective gels, followed by low speed homogenisation using a hand held homogeniser, for 2 minutes. The newly formulated gels which showed no signed of changes of the strength and consistency, were stored at 4° C. until use. For use, small amount (150 ul) of the gel was applied to the wound area and gently rubbed in using fingers:

[0104] 3. Sodium stibogluconate was well tolerated

[0105] We have delivered the compounds systemically every other day, for a two week period in db/db. Throughout the study, we did not observe any side effects. There was no weight loss in any of the groups.

[0106] 4. Sodium stibogluconate increased the rate of wound healing without producing any side effects.

[0107] Sodium stibogluconate was given systemically, at 100 uM. After one week, wounds in the treated were smaller than the control group as shown in FIG. 1 (p=0.0927 vs control).

[0108] However, topical application of Sodium stibogluconate showed no significant effect after one week, both in Bactroban and in Aquagel (FIGS. 2 and 3).

[0109] In Vivo Test on the Dosing Effect and Exploration of the Optimal Way of Applying the Stibogluconate

[0110] Using the same db/db mice, we further tested the possible dose response by applying stibogluconate at 2 mg/ml. 20 mg/ml and 100 mg/ml, using topical applications. At the same time, we tested two treatment methods: applying the agent on a weekly basis or twice weekly basis. We determined the size of the wound on a weekly basis. It was clear that both weekly and twice weekly application resulted in a rapid rate of wound healing. It was also clear that the therapeutic effects of stibogluconate is dependent on the dosage, in that the highest concentration used, namely 100 mg/ml appear to be most effective of all the concentrations using in the present study. Using a Two-way ANOVA (Holm-Sidak model), it was shown that in both treatment regimes, there was a highly significant difference between the treatment group and control group, p=0.013, 0.10 and 0.009, control vs 2 mg/ml, 20 mg/ml and 100 mg respectively, for the twice weekly treatment, and p=0:05, 0.013, 0.009 for the weekly treatment group.

[0111] Using Spearman correlation coefficient, we have found that after two weeks treatment, the size of the wounds was significantly correlated with the concentration (p=0.049, r=-0.950).

[0112] Further, we have also shown that interrupting treatment, in either a weekly or twice weekly dosing regimen, prior to complete healing had a significant effect on the healing process, resulting a noticeable reduction in wound closure (FIG. 17).

[0113] Summary

[0114] The main findings of the present study can be summarised as follows:

[0115] In wound tissue PTPRK is an important regulator of the migration of keratinocytes. PTPRK responds to a PTPRK inhibitor, stibogluconate, by way of increasing the adhesion and in particular migration of keratinocytes and also the migration of vascular endothelial cells. Moreover, Stibugluconate has a concentration dependent effect on the migration of keratinocytes. In vivo, both topical and systemic administration of stibogluconate increased the rate of wound healing, without noticeable side effects. The effect of stibogluconate on wound healing in vivo appears to be dose dependent. Both weekly and twice weekly administration of stibogluconate significantly increased the rate of wound healing, although twice weekly appears to be marginally more effective. Interrupting the treatment regimen adversely affects the healing process.

[0116] These findings collectively show that PTPRK is critical in controlling the migration and healing of wounds. Thus, both in vitro and clinical data point to PTPRK being an important therapeutic target in wounds.

TABLE-US-00007 TABLE 1 Primer and oligo sequences used in the present study. Primer names Sense primers Anti-sense primers PTPRK pair aattacaattgatggggaga Ccacttttccacctgaagta F11/R11 PTPRK pair aattacaattgatggggaga actgaacctgaccgtacacat ZF11/ZR11 attgtgtgacgatgaaagc PTPRK pair Gcgagtcaagttatcaaacc Tgtagctgtccataagagca F12/R12 PTPRK pair gcgagtcaagttatcaaacc actgaacctgaccgtacacactcttt ZF12/ZR12 cagccatgtctagc Anti-PTPRK Ctgcagagtgagttacacagcctg Actagtgacaaaaactg accagg transgene-1 atgagtccgtgagga atttgtAtttcgtcctcacggact Anti-PTPRK Ctgcaggatgataggaccatcgcc Actagtgatccaactaaatgccaact transgene-2 aatctgatgagtccgtgagga cgAtttcgtcctcacggact Anti-PTPRK Ctgcagtttgctcttttttacaat Actagttcatcctccttctcctagt transgene-3 taatatctgatgagtccgtgagga tGtttcgtcctcacggact, T7F and taatacgactcactataggg tagaaggcacagtcgagg BGHR RBTPF and ctgatgagtccgtgaggacgaa ttcgtcctcacggactcatcag RBBMR

Sequence CWU 1

1

22139DNAHomo sapiens 1ctgcagagtg agttacacag cctgatgagt ccgtgagga 39247DNAHomo sapiens 2actagtgaca aaaactgacc aggatttgta tttcgtcctc acggact 47345DNAHomo sapiens 3ctgcaggatg ataggaccat cgccaatctg atgagtccgt gagga 45446DNAHomo sapiens 4actagtgatc caactaaatg ccaactcgat ttcgtcctca cggact 46548DNAHomo sapiens 5ctgcagtttg ctctttttta caattaatat ctgatgagtc cgtgagga 48644DNAHomo sapiens 6actagttcat cctccttctc ctagttgttt cgtcctcacg gact 44774DNAHomo sapiens 7ctgcagagtg agttacacag cctgatgagt ccgtgaggac gaaatacaaa tcctggtcag 60tttttgttac tagt 74879DNAHomo sapiens 8ctgcaggatg ataggaccat cgccaatctg atgagtccgt gaggacgaaa tcgagttggc 60atttagttgg atcactagt 79980DNAHomo sapiens 9ctgcagtttg ctctttttta caattaatat ctgatgagtc cgtgaggacg aaacaactag 60gagaaggagg atgaactagt 801020DNAHomo sapiens 10aattacaatt gatggggaga 201120DNAHomo sapiens 11ccacttttcc acctgaagta 201240DNAHomo sapiens 12actgaacctg accgtacaca tattgtgtga cgatgaaagc 401320DNAHomo sapiens 13gcgagtcaag ttatcaaacc 201420DNAHomo sapiens 14tgtagctgtc cataagagca 201520DNAHomo sapiens 15gcgagtcaag ttatcaaacc 201640DNAHomo sapiens 16actgaacctg accgtacaca ctctttcagc catgtctagc 401720DNAHomo sapiens 17taatacgact cactataggg 201818DNAHomo sapiens 18tagaaggcac agtcgagg 181922DNAHomo sapiens 19ctgatgagtc cgtgaggacg aa 222022DNAHomo sapiens 20ttcgtcctca cggactcatc ag 22214320DNAHomo sapiens 21atggatacga ctgcggcggc ggcgctgcct gcttttgtgg cgctcttgct cctctctcct 60tggcctctcc tgggatcggc ccaaggccag ttctccgcag gtggctgtac ttttgatgat 120ggtccagggg cctgtgatta ccaccaggat ctgtatgatg actttgaatg ggtgcatgtt 180agtgctcaag agcctcatta tctaccaccc gagatgcccc aaggttccta tatgatagtg 240gactcttcag atcacgaccc tggagaaaaa gccagacttc agctgcctac aatgaaggag 300aacgacactc actgcattga tttcagttac ctattatata gccagaaagg actgaatcct 360ggcactttga acatattagt tagggtgaat aaaggacctc ttgccaatcc aatttggaat 420gtgactggat tcacgggtag agattggctt cgggctgagc tagcagtgag ctccttttgg 480cccaatgaat atcaggtaat atttgaagct gaagtctcag gagggagaag tggttatatt 540gccattgatg acatccaagt actgagttat ccttgtgata aatctcctca tttcctccgt 600ctaggggatg tagaggtgaa tgcagggcaa aacgctacat ttcagtgcat tgccacaggg 660agagatgctg tgcataacaa gttatggctc cagagacgaa atggagaaga tataccagta 720gcccagacta agaacatcaa tcatagaagg tttgccgctt ccttcagatt gcaagaagtg 780acaaaaactg accaggattt gtatcgctgt gtaactcagt cagaacgagg ttccggtgtg 840tccaattttg ctcaacttat tgtgagagaa ccgccaagac ccattgctcc tcctcagctt 900cttggtgttg ggcctacata tttgctgatc caactaaatg ccaactcgat cattggcgat 960ggtcctatca tcctgaaaga agtagagtac cgaatgacat caggatcctg gacagaaacc 1020catgcagtca atgctccaac ttacaaatta tggcatttag atccagatac cgaatatgag 1080atccgagttc tacttacaag acctggtgaa ggtggaacgg ggctcccagg acctccacta 1140atcaccagaa caaaatgtgc agaacctatg agaaccccaa agacattaaa gattgctgaa 1200atacaggcaa gacggattgc tgtggactgg gaatccttgg gttacaacat tacgcgttgc 1260cacactttta atgtcactat ctgctaccat tacttccgtg gtcacaacga gagcaaggca 1320gactgtttgg acatggaccc caaagcccct cagcatgttg tgaaccatct gccaccttat 1380acaaatgtca gcctcaagat gatcctaacc aatccagagg gaaggaagga gagtgaagag 1440acaattattc aaactgatga agatgtgcct ggtcccgtac cagtaaaatc tcttcaagga 1500acatcctttg aaaataagat cttcttgaac tggaaagaac ctttggatcc aaatggaatc 1560atcactcaat atgagatcag ctatagcagt ataagatcat ttgatcctgc agtcccagtg 1620gctggacctc cccagactgt atcaaattta tggaacagta cacaccatgt ctttatgcat 1680ctccaccctg gaaccacgta ccagtttttc ataagagcca gcacggtcaa aggctttggt 1740ccagccacag ccatcaatgt caccaccaat atctcagctc caactttacc tgactatgaa 1800ggagttgatg cctctctcaa tgaaactgcc accacaataa ctgtattgtt gagaccagca 1860caagccaaag gtgctcctat cagtgcttat cagattgttg tggaagaact gcacccacac 1920cgaaccaaga gagaagccgg agccatggaa tgctaccagg ttcctgtcac ataccaaaat 1980gccatgagtg ggggtgcacc gtattacttt gctgcagaac tacccccggg aaacctacct 2040gagcctgccc cgttcactgt gggtgacaat cggacctacc aaggcttttg gaaccctcct 2100ttggctccgc gcaaaggata caacatctat ttccaggcga tgagcagtgt ggagaaggaa 2160actaaaaccc agtgcgtacg cattgctaca aaagcagcaa cagaagaacc agaagtgatc 2220ccagatcccg ccaagcagac agacagagtg gtgaaaatag caggaattag tgctggaatt 2280ttggtgttca tcctccttct cctagttgtc atattaattg taaaaaagag caaacttgct 2340aaaaaacgca aagatgccat ggggaatacc cggcaggaga tgactcacat ggtgaatgca 2400atggatcgaa gttatgctga tcagagcact ctgcatgcag aagatcctct ttccatcacc 2460ttcatggacc aacataactt tagtccaaga tatgagaacc acagtgctac agcagagtcc 2520agtcgccttc tagacgtacc tcgctacctc tgtgagggga cggaatcccc ttaccagaca 2580ggacagctgc atccagccat cagggtagct gatttactgc agcacattaa tctcatgaag 2640acatcagaca gctatgggtt caaagaggaa tatgagagct tttttgaagg acagtcagca 2700tcttgggatg tagctaaaaa agatcaaaat agagcaaaaa accgatatgg aaacattata 2760gcatatgatc actccagagt gattttgcaa cccgtagagg atgatccttc ctcagattat 2820attaatgcca actatattga tggctaccag agaccaagtc attacattgc aacccaaggt 2880cccgttcatg aaacagtgta tgatttctgg aggatgattt ggcaagaaca atctgcttgc 2940attgtgatgg ttacaaattt agttgaggtt ggccgggtta aatgctataa atattggcct 3000gatgatactg aagtttatgg tgacttcaaa gtaacgtgtg tagaaatgga accacttgct 3060gaatatgtag ttaggacatt caccctggaa aggagggggt acaatgaaat ccgtgaagtt 3120aaacagttcc atttcacggg ctggcctgac catggagtgc cctaccatgc tacagggctg 3180ctttccttta tccggcgagt caagttatca aaccctccca gtgctggccc catcgttgta 3240cattgcagtg ctggtgctgg acgaactggc tgctacattg tgattgacat catgctagac 3300atggctgaaa gagagggtgt tgttgatatt tacaattgtg tcaaagcctt aagatctcgg 3360cgtattaata tggtccagac agaggaacag tacattttta ttcatgatgc cattttagaa 3420gcctgcttat gtggagaaac tgccatacct gtctgtgaat ttaaagctgc atattttgat 3480atgattagaa tagactccca gactaactct tcacatctca aggatgaatt tcagactctg 3540aattcagtca cccctcgact acaagctgaa gactgcagta tagcgtgcct gccaaggaac 3600catgacaaga accgtttcat ggacatgctg ccacctgaca gatgtctgcc ttttttaatt 3660acaattgatg gggagagcag taactacatc aatgctgctc ttatggacag ctacaggcaa 3720ccagctgctt tcatcgtcac acaataccct ctgccaaaca ctgtaaaaga cttctggaga 3780ttagtgtatg attatggctg tacctccatt gtgatgttaa acgaagtcga cttgtcccag 3840ggctgccctc agtactggcc agaggaaggg atgctacgat atggccccat ccaagtggaa 3900tgtatgtctt gttcaatgga ctgtgatgtg atcaaccgga tttttaggat atgcaatcta 3960acaagaccac aggaaggtta tctgatggtg caacagtttc agtacctagg atgggcttct 4020catcgagaag tgcctggatc caaaaggtca ttcttgaaac tgatacttca ggtggaaaag 4080tggcaggagg aatgcgagga aggggaaggc cggacgatta tccactgcct aaatggtggc 4140gggcgaagtg gcatgttctg tgctataggc atcgttgttg aaatggtgaa acggcaaaat 4200gttgtcgatg ttttccatgc agtaaagaca ctgaggaaca gcaagccaaa catggtggaa 4260gccccggagc aataccgttt ctgctatgat gtagctttgg agtacctgga atcatcttag 4320221428PRTHomo sapiens 22Met Asp Thr Thr Ala Ala Ala Ala Leu Pro Ala Phe Val Ala Leu Leu 1 5 10 15 Leu Leu Ser Pro Trp Pro Leu Leu Gly Ser Ala Gln Gly Gln Phe Ser 20 25 30 Ala Gly Gly Cys Thr Phe Asp Asp Gly Pro Gly Ala Cys Asp Tyr His 35 40 45 Gln Asp Leu Tyr Asp Asp Phe Glu Trp Val His Val Ser Ala Gln Glu 50 55 60 Pro His Tyr Leu Pro Pro Glu Met Pro Gln Gly Ser Tyr Met Ile Val 65 70 75 80 Asp Ser Ser Asp His Asp Pro Gly Glu Lys Ala Arg Leu Gln Leu Pro 85 90 95 Thr Met Lys Glu Asn Asp Thr His Cys Ile Asp Phe Ser Tyr Leu Leu 100 105 110 Tyr Ser Gln Lys Gly Leu Asn Pro Gly Thr Leu Asn Ile Leu Val Val 115 120 125 Asn Lys Gly Pro Leu Ala Asn Pro Ile Trp Asn Val Thr Gly Phe Thr 130 135 140 Gly Arg Asp Trp Leu Arg Ala Glu Leu Ala Val Ser Ser Phe Trp Pro 145 150 155 160 Asn Glu Tyr Gln Val Ile Phe Glu Ala Glu Val Ser Gly Gly Arg Ser 165 170 175 Gly Tyr Ile Ala Ile Asp Asp Ile Gln Val Leu Ser Tyr Pro Cys Asp 180 185 190 Lys Ser Pro His Phe Leu Arg Leu Gly Asp Val Glu Val Asn Ala Gly 195 200 205 Gln Asn Ala Thr Phe Gln Cys Ile Ala Thr Gly Arg Asp Ala Val His 210 215 220 Asn Lys Leu Trp Leu Gln Arg Arg Asn Gly Glu Asp Ile Pro Val Ala 225 230 235 240 Gln Thr Lys Asn Ile Asn His Arg Arg Phe Ala Ala Ser Phe Leu Gln 245 250 255 Glu Val Thr Lys Thr Asp Gln Asp Leu Tyr Arg Cys Val Thr Gln Ser 260 265 270 Glu Arg Gly Ser Gly Val Ser Asn Phe Ala Gln Leu Ile Val Arg Glu 275 280 285 Pro Pro Arg Pro Ile Ala Pro Pro Gln Leu Leu Gly Val Gly Pro Thr 290 295 300 Tyr Leu Leu Ile Gln Leu Asn Ala Asn Ser Ile Ile Gly Asp Gly Pro 305 310 315 320 Ile Ile Leu Lys Glu Val Glu Tyr Arg Met Thr Ser Gly Ser Trp Thr 325 330 335 Glu Thr His Ala Val Asn Ala Pro Thr Tyr Lys Leu Trp His Leu Asp 340 345 350 Pro Asp Thr Glu Tyr Glu Ile Arg Val Leu Leu Thr Arg Pro Gly Glu 355 360 365 Gly Gly Thr Gly Leu Pro Gly Pro Pro Leu Ile Thr Arg Lys Cys Ala 370 375 380 Glu Pro Met Arg Thr Pro Lys Thr Leu Lys Ile Ala Glu Ile Gln Ala 385 390 395 400 Arg Arg Ile Ala Val Asp Trp Glu Ser Leu Gly Tyr Asn Ile Thr Arg 405 410 415 Cys His Thr Phe Asn Val Thr Ile Cys Tyr His Tyr Phe Arg Gly His 420 425 430 Asn Glu Ser Lys Ala Asp Cys Leu Asp Met Asp Pro Lys Ala Pro Gln 435 440 445 His Val Val Asn His Leu Pro Pro Tyr Thr Asn Val Ser Leu Lys Met 450 455 460 Ile Leu Thr Asn Pro Glu Gly Arg Lys Glu Ser Glu Glu Thr Ile Ile 465 470 475 480 Gln Thr Asp Glu Asp Val Pro Gly Pro Val Pro Val Lys Ser Leu Gln 485 490 495 Gly Thr Ser Phe Glu Asn Lys Ile Phe Leu Asn Trp Glu Pro Leu Asp 500 505 510 Pro Asn Gly Ile Ile Thr Gln Tyr Glu Ile Ser Tyr Ser Ser Ile Arg 515 520 525 Ser Phe Asp Pro Ala Val Pro Val Ala Gly Pro Pro Gln Thr Val Ser 530 535 540 Asn Leu Trp Asn Ser Thr His His Val Phe Met His Leu His Pro Gly 545 550 555 560 Thr Thr Tyr Gln Phe Phe Ile Arg Ala Ser Thr Val Lys Gly Phe Gly 565 570 575 Pro Ala Thr Ala Ile Asn Val Thr Thr Asn Ile Ser Ala Pro Thr Leu 580 585 590 Pro Asp Tyr Glu Gly Val Asp Ala Ser Leu Asn Glu Thr Ala Thr Thr 595 600 605 Ile Thr Val Leu Leu Arg Pro Ala Gln Ala Lys Gly Ala Pro Ile Ser 610 615 620 Ala Tyr Gln Ile Val Val Glu Glu Leu His Pro Arg Thr Lys Arg Glu 625 630 635 640 Ala Gly Ala Met Glu Cys Tyr Gln Val Pro Val Thr Tyr Gln Asn Ala 645 650 655 Met Ser Gly Gly Ala Pro Tyr Tyr Phe Ala Ala Glu Leu Pro Pro Gly 660 665 670 Asn Leu Pro Glu Pro Ala Pro Phe Thr Val Gly Asp Asn Arg Thr Tyr 675 680 685 Gln Gly Phe Trp Asn Pro Pro Leu Ala Pro Arg Lys Gly Tyr Asn Ile 690 695 700 Tyr Phe Gln Ala Met Ser Ser Val Glu Lys Glu Thr Lys Thr Gln Cys 705 710 715 720 Val Arg Ile Ala Thr Lys Ala Ala Thr Glu Glu Pro Glu Val Ile Pro 725 730 735 Asp Pro Ala Lys Gln Thr Asp Arg Val Val Lys Ile Ala Gly Ile Ser 740 745 750 Ala Gly Ile Leu Val Phe Ile Leu Leu Leu Val Val Ile Leu Ile Val 755 760 765 Lys Lys Ser Lys Leu Ala Lys Lys Arg Lys Asp Ala Met Gly Asn Thr 770 775 780 Arg Gln Glu Met Thr His Met Val Asn Ala Met Asp Arg Ser Tyr Ala 785 790 795 800 Asp Gln Ser Thr Leu His Ala Glu Asp Pro Leu Ser Ile Thr Phe Met 805 810 815 Asp Gln His Asn Phe Ser Pro Arg Tyr Glu Asn His Ser Ala Thr Ala 820 825 830 Glu Ser Ser Arg Leu Leu Asp Val Pro Arg Tyr Leu Cys Glu Gly Thr 835 840 845 Glu Ser Pro Tyr Gln Thr Gly Gln Leu His Pro Ala Ile Arg Val Ala 850 855 860 Asp Leu Leu Gln His Ile Asn Leu Met Lys Thr Ser Asp Ser Tyr Gly 865 870 875 880 Phe Lys Glu Glu Tyr Glu Ser Phe Phe Gly Gln Ser Ala Ser Trp Asp 885 890 895 Val Ala Lys Lys Asp Gln Asn Arg Ala Lys Asn Arg Tyr Gly Asn Ile 900 905 910 Ile Ala Tyr Asp His Ser Arg Val Ile Leu Gln Pro Val Glu Asp Asp 915 920 925 Pro Ser Ser Asp Tyr Ile Asn Ala Asn Tyr Ile Asp Gly Tyr Gln Arg 930 935 940 Pro Ser His Tyr Ile Ala Thr Gln Gly Pro Val His Glu Thr Val Tyr 945 950 955 960 Asp Phe Trp Arg Met Ile Trp Gln Glu Gln Ser Ala Cys Ile Val Met 965 970 975 Val Thr Asn Leu Val Glu Val Gly Arg Val Lys Cys Tyr Lys Tyr Trp 980 985 990 Pro Asp Asp Thr Glu Val Tyr Gly Asp Phe Lys Val Thr Cys Val Glu 995 1000 1005 Met Glu Pro Leu Ala Glu Tyr Val Arg Thr Phe Thr Leu Glu Arg 1010 1015 1020 Arg Gly Tyr Asn Glu Ile Arg Glu Val Lys Gln Phe His Phe Thr 1025 1030 1035 Gly Trp Pro Asp His Gly Val Pro Tyr His Ala Thr Gly Leu Leu 1040 1045 1050 Ser Phe Ile Arg Arg Val Lys Leu Ser Asn Pro Pro Ser Ala Gly 1055 1060 1065 Pro Ile Val Val His Cys Ser Ala Gly Ala Gly Arg Thr Gly Cys 1070 1075 1080 Tyr Ile Val Ile Asp Ile Met Leu Asp Met Ala Glu Arg Glu Gly 1085 1090 1095 Val Val Asp Ile Tyr Asn Cys Val Lys Ala Leu Arg Ser Arg Arg 1100 1105 1110 Ile Asn Met Val Gln Thr Glu Glu Gln Tyr Ile Phe Ile His Asp 1115 1120 1125 Ala Ile Leu Glu Ala Cys Leu Cys Gly Glu Thr Ala Ile Pro Val 1130 1135 1140 Glu Phe Lys Ala Ala Tyr Phe Asp Met Ile Arg Ile Asp Ser Gln 1145 1150 1155 Thr Asn Ser Ser His Leu Lys Asp Glu Phe Gln Thr Leu Asn Ser 1160 1165 1170 Val Thr Pro Arg Leu Gln Ala Glu Asp Cys Ser Ile Ala Cys Leu 1175 1180 1185 Pro Arg Asn His Asp Lys Asn Arg Phe Met Asp Met Leu Pro Pro 1190 1195 1200 Asp Arg Cys Leu Pro Phe Leu Ile Thr Ile Asp Gly Glu Ser Ser 1205 1210 1215 Asn Tyr Ile Asn Ala Ala Leu Met Asp Ser Tyr Arg Gln Pro Ala 1220 1225 1230 Ala Phe Ile Val Thr Gln Tyr Pro Leu Pro Asn Thr Val Lys Asp 1235 1240 1245 Phe Trp Arg Leu Val Tyr Asp Tyr Gly Cys Thr Ser Ile Val Met 1250 1255 1260 Leu Asn Glu Val Asp Leu Ser Gly Cys Pro Gln Tyr Trp Pro Glu 1265 1270 1275 Glu Gly Met Leu Arg Tyr Gly Pro Ile Gln Val Glu Cys Met Ser 1280 1285 1290 Cys Ser Met Asp Cys Asp Val Ile Asn Arg Ile Phe Arg Ile Cys 1295 1300 1305 Asn Leu Thr Arg Pro Gln Glu Gly Tyr Leu Met Val Gln Gln Phe 1310 1315 1320 Gln Tyr Leu Gly Trp Ala Ser His Arg Glu Val Pro Gly Ser Lys 1325 1330 1335 Arg Ser Phe Leu Lys Leu Ile Leu Gln Val Glu Lys Trp Gln Glu 1340 1345 1350

Glu Cys Glu Glu Gly Glu Gly Arg Thr Ile Ile His Cys Leu Asn 1355 1360 1365 Gly Gly Gly Arg Ser Gly Met Phe Cys Ala Ile Gly Ile Val Val 1370 1375 1380 Glu Met Val Lys Arg Gln Asn Val Val Asp Val Phe His Ala Lys 1385 1390 1395 Thr Leu Arg Asn Ser Lys Pro Asn Met Val Glu Ala Pro Glu Gln 1400 1405 1410 Tyr Arg Phe Cys Tyr Asp Val Ala Leu Glu Tyr Leu Glu Ser Ser 1415 1420 1425

Claims

1. A therapeutic comprising an inhibitor of one or both of PTPRK gene expression or PTPRK protein activity or an inhibitor of a protein that is at least 50% homologous to PTPRK for use in the treatment of wounds.

2. The therapeutic according to claim 1 wherein said inhibitor is an inhibitor of PTPRK gene expression.

3. The therapeutic according to claim 2 wherein said inhibitor is selected from the group consisting of: anti-sense DNA or RNA, siRNA, or ribozymes, either naked or in the form of plasmid or viral vectors.

4. The therapeutic according to claim 3 wherein said inhibitor is anti-PTPRK ribozyme/RNA transgene selected from the group comprising: transgene 1 5'Ctgcagagtgagttacacagcctgatgagtccgtgaggacgaaa tacaaatcctggtcagtttttgtt actagt'3 SEQ ID NO: 7; transgene 2 5'Ctgcaggatgataggaccatcgccaatctgatgagtccgtgaggacgaaa tcgagttggcatttagttggatcactagt'3 SEQ ID NO: 8; and transgene 3 Ctgcagtttgctcttttttacaattaatatctgatgagtccgtgaggacgaaa caactaggagaaggaggatgaactagt'3 SEQ ID NO: 9.

5. The therapeutic according to claim 1 wherein said inhibitor is an inhibitor of PTPRK protein function.

6. The therapeutic according to claim 5 wherein said inhibitor is selected from the group consisting of: a PTPRK binding agent that binds, either reversibly or irreversibly, to inhibit protein function such as an antibody or a known, or synthesized, PTPRK antagonist; or an agent that works upstream or downstream of the PTPRK signalling mechanism to inhibit PTPRK function.

7. The therapeutic according to claim 6 wherein said inhibitor is Stibogluconate, or a salt thereof, or Pentostam® (GlaxoSmithKline).

8. The therapeutic according to claim 1 wherein the therapeutic is formulated for use in treating mammalian wounds.

9. The therapeutic according to claim 1 wherein the therapeutic is formulated for use in treating chronic wounds comprising venous ulcers, diabetic ulcers, and pressure ulcers

10. The therapeutic according to claim 1 wherein the therapeutic is formulated for use in treating human wounds.

11. The therapeutic according to claim 1 wherein the therapeutic is formulated for topical application.

12. The therapeutic according to claim 1 wherein the therapeutic is formulated for application to a dressing or impregnation in a dressing.

13. A pharmaceutical composition comprising a therapeutic according to claim 1 together with a pharmaceutically acceptable carrier.

14. A method for preparing a pharmaceutical composition according to claim 13 comprising bringing said therapeutic in conjunction or association with a pharmaceutically or veterinarily acceptable carrier or vehicle.

15. A method for treating a mammalian wound which method comprises: administering to said wound a therapeutic that inhibits one or both of PTPRK gene expression or PTPRK protein activity.

16. A kit for treating a wound wherein said kit comprises: (a) at least one therapeutic according to claim 1; and (b) at least one dressing for applying to said wound.

17. A combination therapeutic for treating a wound comprising an inhibitor of PTPRK gene expression and an inhibitor of PTPRK protein activity.

18. A combination therapeutic for treating a wound comprising: a) either an inhibitor of PTPRK gene expression or an inhibitor of PTPRK protein activity; and b) at least one further therapeutic.

19. (canceled)

20. A kit for treating a wound wherein said kit comprises: (a) a composition according to claim 13; and (b) at least one dressing for applying to said wound.

Images