Molecular targets for healing or treating wounds

Abstract

Molecular target for healing or treating wounds and, in particular chronic, human wounds, are described. The molecular target is PTPRK, or a protein 50% homologous therewith, and which retains the same activity as PTPRK protein. Further, methods and novel therapeutics are described for treating said wounds.

Claims

1. A method for treating a mammalian non-parasitic wound, comprising administering to said wound a therapeutic comprising an inhibitor of PTPRK protein activity, wherein said inhibitor is Stibogluconate or a salt thereof.

2. The method according to claim 1, wherein said non-parasitic wound is a chronic wound.

3. The method according to claim 2, wherein said chronic wound is selected from the group consisting of venous ulcers, diabetic ulcers, and pressure ulcers.

4. The method according to claim 1, wherein said non-parasitic wound is a human wound.

5. The method according to claim 1, including formulating said therapeutic for topical application.

6. The method according to claim 1, including formulating said therapeutic for application to a dressing or impregnated in a dressing.

7. The method according to claim 1, including formulating said therapeutic with a pharmaceutically or veterinarily acceptable carrier or vehicle.

8. The method according to claim 5, wherein said topical formulation comprises a hydrogel.

9. A method for treating a chronic human non-parasitic wound, comprising administering a topical formulation comprising Stibogluconate or a salt thereof.

10. The method according to claim 9, including formulating said topical formulation for application to a dressing or impregnated in a dressing.

11. The method according to claim 10, including formulating said topical formulation with a pharmaceutically or veterinarily acceptable carrier or vehicle.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

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

(2) FIG. 1. Shows the secondary structure of human PTPRK mRNA;

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

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

(5) 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;

(6) 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;

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

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

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

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

(11) 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;

(12) FIG. 10. Shows the concentration related effect of PENTOSTAM™ (GlaxoSmithKline), a commercially available form of stibogluconate on the migration of the cells;

(13) FIG. 11. Shows the effect of systemic administration of sodium stibogluconate, via the I.P. route, on the rate of wound healing.

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

(15) FIG. 13. Shows the effect of weekly delivery of Stibogluconate on the rate of wound healing;

(16) FIG. 14. Shows the effect of twice weekly delivery of Stibogluconate on the rate of wound healing;

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

(18) FIG. 16. shows the amino acid and cDNA sequence structure of PTPRK;

(19) 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

(20) Table 1. shows the primers and oligonulceotides used for the Construction and verification of ant-human PTPRK ribozyme transgenes described herein.

DETAILED DESCRIPTION

(21) Materials and Procedure

(22) Cells

(23) 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.

(24) Construction of Ant-Human PTPRK Ribozyme Transgenes

(25) 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 pEF6/V5His-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 ug/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).

(26) Generation of Sublines of Human Keratinocytes and Endothelial Cells with Differential Expression of PTPRK

(27) 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.

(28) In Vivo Tolerance Test

(29) 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.

(30) In Vivo Efficacy Test and Wound Healing

(31) 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.

(32) Effects of Knocking Down PTPRK on the Function of Cells

(33) Three models of ECIS instrument were used: ECIS 9600 for screening and ECIS1600R and ECIS Z8 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 Z8), 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.

(34) Results

(35) Knocking Down PTPRK from HaCaT and Endothelial Cells Resulted in an Acceleration of Cell Adhesion and Migration

(36) It was found that after knocking down PTPRK in HaCaT cells, there was a rapid increase in cell adhesion, FIGS. 2A-2B. 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.

(37) Human Keratinocytes Showed a Dose Dependent Response to PTPRK Inhibitor Stibogluconate

(38) Using the ECIS Theta96 model, we 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.

(39) 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.

(40) Stibogluconate was Well Tolerated In Vivo

(41) 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.

(42) Stibogluconate Accelerates Wound Healing In Vivo.

(43) Formulation of the Compounds. 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. 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. 3. Sodium stibogluconate was well tolerated 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. 4. Sodium stibogluconate increased the rate of wound healing without producing any side effects. 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). However, topical application of Sodium stibogluconate showed no significant effect after one week, both in Bactroban and in Aquagel (FIGS. 2A-2B and 3).

(44) In Vivo Test on the Dosing Effect and Exploration of the Optimal Way of Applying the Stibogluconate

(45) 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.

(46) 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).

(47) 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).

(48) Summary

(49) The main findings of the present study can be summarised as follows:

(50) 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.

(51) 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.

(52) TABLE-US-00003 TABLE 1 Primer and oligo sequences used in the present study. Primer names Sense primers Anti-sense primers PTPRK pair Aattacaattgatggggaga Ccacttttccacctgaagta F11/R11 (SEQ ID NO: 10) (SEQ ID NO: 11) PTPRK pair Aattacaattgatggggaga Actgaacctgaccgtacacatattgtgtgacgat ZF11/ZR11 (SEQ ID NO: 10) gaaagc (SEQ ID NO: 12) PTPRK pair Gcgagtcaagttatcaaacc Tgtagctgtccataagagca F12/R12 (SEQ ID NO: 13) (SEQ ID NO: 14) PTPRK pair Gcgagtcaagttatcaaacc Actgaacctgaccgtacacactctttcagccatg ZF12/ZR12 (SEQ ID NO: 15) tctagc (SEQ ID NO: 16) Anti-PTPRK Ctgcagagtgagttacacagcctgatgagtccg Actagtgacaaaaactg accaggattt transgene-1 tgagga gtAtttcgtcctcacggact (SEQ ID NO: 1) (SEQ ID NO: 2) Anti-PTPRK Ctgcaggatgataggaccatcgccaatctgatg ActagtgatccaactaaatgccaactcgAtttcg transgene-2 agtccgtgagga tcctcacggact (SEQ ID NO: 3) (SEQ ID NO: 4) Anti-PTPRK Ctgcagtttgctcttttttacaattaatatctg ActagttcatcctccttctcctagttGtttcgtc transgene-3 atgagtccgtgagga ctcacggact (SEQ ID NO: 5) (SEQ ID NO: 6) T7F and Taatacgactcactataggg Tagaaggcacagtcgagg BGHR (SEQ ID NO: 17) (SEQ ID NO: 18) RBTPF and Ctgatgagtccgtgaggacgaa Ttcgtcctcacggactcatcag RBBMR (SEQ ID NO: 19) (SEQ ID NO: 20)