Tissue repair

Abstract

The present invention relates to a hypoxia-inducible factor (HIF) activator, or a pharmaceutically acceptable salt or solvate thereof, for use in promoting tissue repair. The HIF activator, or the pharmaceutically acceptable salt or solvate thereof, is for use with a prototypical tissue-protective cytokine, or a pharmaceutically acceptable salt or solvate thereof, which is administered exogenously to the body being treated.

Claims

1. A method of promoting tissue repair in dermal epithelial cells in a subject, the method comprising administering to a subject in need thereof a synergistic combination of (a) a therapeutically effective amount of a hypoxia-inducible factor (HIF) activator, or a pharmaceutically acceptable salt or solvate thereof, and (b) a therapeutically effective amount of a prototypical tissue-protective cytokine, or a pharmaceutically acceptable salt or solvate thereof, wherein the HIF activator of the pharmaceutically acceptable salt or solvate thereof is administered exogenously to the body of the subject being treated, wherein the HIF activator, or a pharmaceutically acceptable salt or solvate thereof, is selected from a group consisting of: dimethyloxalylglycine (DMOG); N-[[1,2-Dihydro-4-hydroxy-2-oxo-1-(phenylmethyl)-3-quinolinyl]carbonyl] glycine (IOX); N-[(1-Chloro-4-hydroxy-3-isoquinolinyl)carbonyl]glycine (BIQ); desferrioxamine (DFX); o-phenanthroline; iodochlorohydroxyquinoline; and cobalt chloride heptahydrate and the prototypical tissue-protective cytokine is erythropoietin (EPO) or an analogue or derivative thereof.

2. The method according to claim 1, wherein the HIF activator, or the pharmaceutically acceptable salt or solvate thereof, is administered locally to the site where promotion of tissue repair is desired.

3. The method according to claim 1, wherein the method is a method of promoting wound healing.

4. The method according to claim 1, wherein the HIF activator, or the pharmaceutically acceptable salt or solvate thereof, is a HIF-1 activator and/or a HIF-3 activator, or a pharmaceutically acceptable salt or solvate thereof.

5. The method according to claim 4, wherein the HIF activator, or the pharmaceutically acceptable salt or solvate thereof, is a HIF-1 activator, or a pharmaceutically acceptable salt or solvate thereof.

6. The method according to claim 5, wherein the HIF activator, or the pharmaceutically acceptable salt or solvate thereof, is a HIF-1α activator, or a pharmaceutically acceptable salt or solvent thereof.

7. The method according to claim 1, wherein the analogue or derivative of EPO is desialylated EPO, carbamylated EPO (CEPO), asialoerythropoietin (asialoEPO), glutaraldehyde EPO (GEPO), epopeptide AB, an isoform of EPO, recombinant EPO, mutant S100E of EPO or pyroglutamate helix B surface peptide (pHBSP).

8. The method according to claim 1, wherein the HIF activator or a pharmaceutically acceptable salt or solvate thereof, is dimethyloxalylglycine (DMOG) or N-[[1,2-Dihydro-4-hydroxy-2-oxo-1-(phenylmethyl)-3-quinolinyl] carbonyl] glycine (IOX).

9. The method according to claim 1, wherein the prototypical tissue-protective cytokine is erythropoietin (EPO).

Description

(1) For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which:—

(2) FIG. 1 is a graph showing the in vitro effects of erythropoietin (EPO) (1 ng/mL) on wound closure in aortic endothelial cells (BAECs) when incubated in 1% oxygen or 21% oxygen. Results are expressed as % wound healing after 24 h. Each data point represents the mean value ±SEM (n=6). Different samples were tested for significant difference using t-test where *p<0.05;

(3) FIG. 2 is a graph showing the cell viability of rat aortic endothelial cells (RAECs) is not affected when exposed to different concentrations of dimethyloxalylglycine (DMOG) between 0-500 μM. Each data represents the mean value ±SEM (n=3). Different samples were tested for significance difference using t-test;

(4) FIG. 3 is a graph showing the effect of different concentrations of dimethyloxalylglycine (DMOG) on a) hypoxia inducible factor-1 (HIF-1) levels in cell lysates from rat aortic endothelial cells and b) gene expression of vascular endothelial growth factor (VEGF), one of the proteins whose production is regulated by HIF. Each data represents the mean value ±SEM (n=3), different samples were tested for significant difference using t-test where *p<0.05, **p<0.01;

(5) FIG. 4 is a graph showing the effects of erythropoietin (EPO, 1 ng/mL) and dimethyloxalylglycine (DMOG; 100 μM) on wound closure in a scratch assay model in rat aortic endothelial cells (RAECs) when incubated separately or together in cell culture medium containing 5% serum in 21% oxygen for 24 h. Results are expressed as % wound healing after 24 h. Each data point represents the mean value ±SEM (n=3). Different samples were tested for significant difference using one-way ANOVA where **p<0.01, ***p<0.01;

(6) FIG. 5 is a graph showing the effect different concentrations of dimethyloxalylglycine (DMOG) had on a) hypoxia inducible factor-1 (HIF-1) levels in plasma and b) gene expression of vascular endothelial growth factor (VEGF) in carotid artery. Each data represents the mean value ±SEM (n=.sub.3), different samples were tested for significant difference using t-test where *p<0.05, **p<0.01;

(7) FIG. 6a shows representative images of rat carotid arteries after injury with balloon catheter (angioplasty) and treatment with erythropoietin (EPO, 100 μg/Kg) and dimethyloxalylglycine (DMOG, 5 mg/Kg), separately and in combination, applied locally on the left carotid artery using a hydrogel (30% w/v Pluronic acid gel) immediately after injury. Evans Blue (1% w/v) was used to assess the repair response; blue stained area represents de-endothelialised injured areas; and FIG. 6b is a graph showing quantitative analysis of the injured areas by measuring the blue stained 5 days post injury. Each data point represents mean±SEM (n=3). Different treatments were tested for significant difference using t-test where *p<0.001;

(8) FIG. 7 is a graph showing hypoxia inducible factor (HIF) levels in plasma taken from rats at 0, 1 and 5 days post injury using an ELISA kit. Each data point represents mean±SEM (n=3). Different treatments were tested for significant difference using one-way ANOVA where *p<0.001 compared to arteries immediately after injury;

(9) FIG. 8 shows the effect different concentrations of several hypoxia inducible factor-1 (HIF-1) inducers: N-[[1,2-Dihydro-4-hydroxy-2-oxo-1-(phenylmethyl)-3-quinolinyl]carbonyl]glycine (IOX), N-[(1-Chloro-4-hydroxy-3-isoquinolinyl)carbonyl]glycine (BIQ) or desferrioxamine (DFX) had on a) HIF levels in cell lysates from rat aortic endothelial cells and b) gene expression of vascular endothelial growth factor (VEGF). Each data represents the mean value ±SEM (n=.sub.3), different samples were tested for significant difference using t-test where *p<0.05, **p<0.01;

(10) FIG. 9 shows the cell viability of rat aortic endothelial cells (RAECs) exposed to different hypoxia inducible factor (HIF) inducers: N-[[1,2-Dihydro-4-hydroxy-2-oxo-1-(phenylmethyl)-3-quinolinyl]carbonyl]glycine (IOX), N-[(1-Chloro-4-hydroxy-3-isoquinolinyecarbonyl]glycine (BIQ) or desferrioxamine (DFX) at concentrations ranging from 0-200 μM. Each data represents the mean value ±SEM (n=3);

(11) FIG. 10 shows how erythropoietin (EPO, 1 ng/mL) and N-[[1,2-Dihydro-4-hydroxy-2-oxo-1-(phenylmethyl)-3-quinolinyl]carbonyl]glycine (IOX, 100 μM) effected wound closure in a scratch assay model in rat aortic endothelial cells (RAECs) when incubated separately or together in cell culture medium containing 5% serum in 21% oxygen for 24 h. Results are expressed as % wound healing after 24 h. Each data point represents the mean value ±SEM (n=.sub.3). Different samples were tested for significant difference using one-way ANOVA where **p<0.01, ***p<0.01.

(12) FIG. 11 shows how different hypoxia inducible factor (HIF) inducers: dimethyloxalylglycine (DMOG) and N-[(1-Chloro-4-hydroxy-3-isoquinolinyecarbonyl]glycine (BIQ) a) enhanced the proliferation of rat aortic endothelial cells (RAECs) at the concentrations 0.1 mM but b) had no effect on the proliferation of rat aortic smooth muscle cells (SMCs). Effect of different HIF inducers on cell proliferation was done using MIT test. Each data represents the mean value ±SEM (n=3), different samples were tested for significant difference using t-test, *p<0.05 and **p<0.01; and

(13) FIG. 12 shows how dimethyloxalylglycine (DMOG) primes the reparative effect of erythropoietin (EPO) in a scratch assay model in human keratinocyte cells (HaCaT) and Human fibroblast cells. EPO (1 ng/mL) and DMOG (100 μM) enhanced wound closure in HaCaT and fibroblasts when incubated together in cell culture medium containing 3% serum in 21% oxygen for 24 h. Results are expressed as % wound healing after 24 h. Each data point represents the mean value ±SEM (n=3), *p<0.05.

EXAMPLES

(14) The inventors have explored the effects of a low oxygen environment on the response to a prototypical tissue-protective cytokine, e.g. erythropoietin (EPO), and how the effects of a low oxygen environment can be replicated in the body using HIF-1 activators.

(15) Finally, the inventors have investigated the effects of applying a combination of a prototypical tissue-protective cytokine and a HIF-1 activator to a wound.

Example 1—Effect of Hypoxia on the Response to Erythropoietin (EPO)

(16) Materials and Methods

(17) Scratch assay: This assay was used as an in vitro model of injury in rat aortic endothelial cells (RAECs). The effect of EPO on wound closure was investigated in either 21% or 1% O.sub.2. A reproducible scratch was produced in the endothelial monolayer and thereafter, either EPO (1 ng/mL) or saline (control) was added to the cells (in rat endothelial growth medium containing 5% serum) and subsequently incubated in 21% or 1% O.sub.2 for 24 hours. The defined area of the scratch was photographed under an inverted microscope (Olympus CKX41; Olympus Corporation, Tokyo, Japan) at lox magnification using a Micropix 5 megapixel colour complementary metal-oxide semiconductor digital camera (Olympus Cooperation). The position of the wound image was standardized each time against a horizontal line drawn on the base of the plate passing through the centre of each well. The scratch area was quantified using ImageJ software (National institutes of Health, Bethesda, Md., USA).

(18) Results

(19) As shown in FIG. 1, the presence of EPO has little effect on wound healing when the cells are incubated under normal oxygen levels (21% oxygen). However, when the cells were incubated under a low oxygen level (1% oxygen), the presence of EPO was shown to significantly improve wound healing, both when compared to cells which were not treated with EPO and to cells treated with EPO and incubated under 21% oxygen.

(20) The inventors hypothesised that hypoxia activates a molecular switch that makes cell responsive to the reparative effect of EPO. This could be due to activation of the adaptive response to low oxygen mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1).

(21) However, it will be appreciated that it is undesirable to lower oxygen concentration in patients. Accordingly, the inventors decided to see if this effect could be replicated using a HIF-1 activator, dimethyloxalylglycine (DMOG), as discussed in the further examples below.

Example 2—Determination of the Optimum Effective Concentration of a Standard HIF-1 Inducer, Dimethyloxalylglycine (DMOG), in Vitro

(22) Materials and Methods

(23) Cell culture: Rat aortic endothelial cells (RAECs) were seeded into 24-well plates and cultured until ˜80% confluent after which they were incubated with or without DMOG for 18 h at normal oxygen levels (21% O.sub.2). Cells incubated at 1% O.sub.2 where used as a positive control for HIF-1.

(24) Enzyme linked immunosorbent assay (ELISA): HIF-1 protein levels were measured using ELISA. Treated cells were lysed in 80 μL lysis buffer (25 mmol/L Tris HCl pH 7. 6, 0.1% SDS, deoxycholate, 1% NP40, 0.5 mol/L EDTA, 40 mmol/L EGTA and protease inhibitors). Lysates were then centrifuged at 11,000 g for 15 min at 4° C. and the supernatant was collected. Protein concentrations were quantified using a BCA reagent kit (Pierce Biotechnology). HIF was measured using an ELISA kit (R&D systems and biotechne, UK) and expressed as pg/mg protein.

(25) Real time qPCR: The gene expression of vascular endothelial growth factor (VEGF), a HIF-1 target gene, was analysed by quantitative polymerase chain reaction (qPCR). Cells were lysed using TRIzol (Invitrogen, Life Technologies) and RNA was then extracted and purified. RNA quality and concentration were determined using a NanoDrop ND-1000 (NanoDrop Technologies). Reverse transcription and real-time quantitative PCR (qPCR) were carried out on RNA samples for VEGF and β2-microglobulin (a housekeeping gene not affected by change in oxygen levels), using TaqMan gene expression assays (Applied Biosystems/Life Technologies). For gene expression quantification, the comparative threshold cycle (ΔΔCT) method was used following manufacturer's guide-lines. Results were normalized to β2-microglobulin expression and expressed as arbitrary units using one of the normoxic untreated samples as a calibrator.

(26) Viability assay: Cell viability was evaluated using MIT viability assay. Cells were seeded into 96-well plates at a density of 1×10.sup.5 cells/mL (0.15 mL/well) in culture medium. After 24 h, medium was removed and replaced by 150 μL fresh medium containing different concentrations of DMOG (0-500 μM) and incubated for 0 and 24 h. At each time point, MTT reagent was added, incubated for 3 hours. Formazan crystals formed only from living cells where then dissolved in DMSO and absorbance was measured at wavelength 540 and 630 nm. Results were expressed as number of cells/mL.

(27) Results

(28) As shown in FIG. 2, DMOG was not toxic to rat aortic endothelial cells (RAECs) when used in concentrations of up to 500 μM.

(29) As shown in FIG. 3, a concentration of 100 μM of DMOG was found to increase the HIF levels and gene expression of vascular endothelial growth factor (VEGF), one of the downstream effectors of HIF, in cell lysates from rat aortic endothelial cells to a level which is similar to that observed for cells incubated at low oxygen levels (1% oxygen).

Example 3—In Vitro Study of Effect of Combination of DMOG and EPO

(30) Materials and Methods

(31) Scratch assay: Scratch assay was used as described in example 1. Cells where treated with DMOG (100 μM) and/or EPO (at 1 ng/mL). Cells incubated with medium containing 10% serum where used as positive control to reflect the maximum wound closure the cells could attain after 24 h.

(32) Results

(33) As shown in FIG. 4, when cells were treated with just 100 μM of DMOG or just 1 ng/ml of EPO the % wound closure is similar to that observed for untreated cells.

(34) However, when cells were treated with a combination of 100 μM of DMOG and 1 ng/ml of EPO then a significant improvement was observed getting close to the maximum wound closure the cells could get to after 24 h.

Example 4—Determination of the Optimum Effective DMOG Concentration in Vivo

(35) Materials and Methods

(36) Male animal study: Sprague Dawley rats (450 g) were used in this study. Animals were anesthetized using 2% isoflurane where the rats were placed in a supine position. The right carotid artery was exposed and DMOG was applied locally at different concentrations (0-7.5 mg/Kg) on the artery using pluronic gel (30% w/w). 3 rats were used in each concentration group.

(37) ELISA: After 24 h, blood was taken from the rat's tail in tubes containing EDTA as an anti-coagulant. Blood was kept on ice and centrifuged to separate the plasma which was immediately treated with a protease inhibitor. HIF-1 was quantified in the collected plasma using the ELISA assay as described in example 2. After 24 h, treated animals were culled and both the right (treated) and left (untreated) carotid arteries were isolated and snap frozen for further analysis.

(38) Real time qPCR: RNA was extracted and purified from the frozen artery sections using TRIzol (Invitrogen, Life Technologies). RNA quality and concentrations were determined using a NanoDrop ND-1000 (NanoDrop Technologies). Reverse transcription and real-time quantitative PCR (qPCR) were carried out on RNA samples for VEGF and β2-microglobulin as described in example 2.

(39) Results

(40) As shown in FIG. 5, the inventors observed that DMOG showed a concentration dependent effect on increasing HIF levels in plasma and increasing gene expression of VEGF. The inventors identified that the optimal dose of DMOG to cause an increase in HIF-1 and VEGF as 5 mg/kg.

Example 5—In Vivo Study of Effect of Combination of DMOG and EPO

(41) Materials and Methods

(42) Angioplasty model: Balloon catheter injury (angioplasty) was the model used as an in vivo injury model using Sprague Dawley rats (450 g). Animals were anesthetized using 2% isoflurane where the rats were placed in a supine position. The right carotid artery was exposed and a (2F Fogarty catheter) was used to cause injury. The catheter was inserted into the common carotid artery via the external carotid to cause complete removal of the vascular endothelium from the common carotid artery down to its junction with the aortic arch. DMOG (5 mg/Kg) and EPO (100 ug/Kg) were administered either alone or in combination in the form of a hydrogel (30% w/v pluronic F127 gel) and applied to the artery after injury. Endothelial regrowth was then assessed using Evan's blue staining (injected IV in the tail vein 30 min before culling the animal) 0 and 5 days after injury to assess the area of de-endothelialised surface; re-endothelialised regions remain unstained. Images of stained arteries were quantified using Image J software.

(43) ELISA: Blood was collected from venous tail at 0, 1 and 5 days post injury. Plasma was separated and used for quantitative measurement of HIF-1 using ELISA method as described earlier.

(44) Results

(45) As shown in FIG. 6, the repair of the endothelial lining of rat carotid artery 5 days after injury was much improved by the combination of DMOG and EPO compared to either drug when used alone.

(46) Furthermore, the inventors noted that DMOG significantly increased HIF-1 levels in rat plasma 24 h post injury, see FIG. 7.

Example 6—Determination of the Optimum Effective Concentration of Alternative HIF Inducers in Vitro

(47) Materials and Methods

(48) Different HIF inducers were purchased from Sigma Aldrich (Dorset, UK):

(49) 1. BIQ: N-[(1-Chloro-4-hydroxy-3-isoquinolinyl)carbonyl]glycine

(50) 2. IOX: N-[[1,2-Dihydro-4-hydroxy-2-oxo-1-(phenylmethyl)-3-quinolinyl]carbonyl]-glycine

(51) 3. DFX: Desferrioxamine

(52) Similar to the methods described in example 2, HIF-1 protein expression and VEGF gene expression were quantitatively measured in vitro using ELISA and real time qPCR respectively using different the different HIF-1 inducers at different concentrations (0-500 uM).

(53) Viability test: Effect of different HIF-1 inducers on cell viability was tested at different concentrations (0-500 μM) using MTT method as described previously in example 2.

(54) Results

(55) As shown in FIG. 9, none of the HIF inducers used were toxic to rat aortic endothelial cells (RAECs) when used in concentrations of up to 500 μM.

(56) As shown in FIG. 8, a concentration of 100 μM was the optimum concentration for all of the HIF-1 inducers to increase the HIF levels and gene expression of vascular endothelial growth factor (VEGF) in cell lysates from rat aortic endothelial cells to a level which is similar to that observed for cells incubated at low oxygen levels (1%).

Example 7—In Vitro Study of Effect of Combination of IOX and EPO

(57) Materials and Methods

(58) Scratch assay: Similar to example 3, the reparative effect of EPO (1 ng/mL) and the HIF-1 inducer; IOX (100 uM) were tested either alone or in combination in an in vitro scratch injury model.

(59) Results

(60) As shown in FIG. 10, no change was observed when cells were treated with just 100 μM of IOX or just 1 ng/ml of EPO when compared to untreated cells. However, when cells were treated with a combination of 100 μM of IOX and 1 ng/ml of EPO, a significant improvement was observed compared to the maximum closure the cells could achieve when incubated with 10% serum.

Example 8—Effect of HIF Inducers on Proliferation of Endothelial Cells and Smooth Muscle Cells

(61) Materials and Methods

(62) Proliferation assay: Proliferation of rat aortic endothelial cells (RAECs) and rat aortic smooth muscle cells (isolated from rats at animal unit at Sussex University) was evaluated using Trypan Blue exclusion test. Cells were seeded in 96 well plates at a seeding density of 1×10.sup.4 cells/mL (0.15 mL/well). After 24 h, cells where treated with or without different concentrations of 2 HIF-1 inducers; DMOG and BIQ (0-200 μM). After another 24 hours, Trypan Blue was added and the cells that were stained (dead) and unstained (live) were counted. Results were expressed as number of cells/mL.

(63) Results

(64) As shown in FIG. 11, the 2 HIF-1 inducers DMOG and BIQ enhanced the proliferation of rat aortic endothelial cells at an optimum concentration of 100 μM. The same HIF-1 inducers had no effect on the proliferation of smooth muscle cells at the concentrations used.

Example 9—In Vitro Study of Effect of Combination of DMOG and EPO on Skin Cells

(65) Materials and Methods

(66) An in vitro injury model using cells relevant to wound injury were used. Two different cell lines; human keratinocytes (HaCaT) and human fibroblasts were used as skin injury models.

(67) Scratch assay: As described in example 3, the reparative effect of EPO (1 ng/mL) and DMOG (100 uM) were tested alone or in combination on HaCaT and fibroblasts incubated in cell culture medium (Dulbeccos cell culture medium) containing 3% serum in 21% oxygen for 24 h.

(68) Results

(69) As shown in FIG. 12, similar to the results obtained with endothelial cells, there was no significant different in % wound closure when human fibroblasts and keratinocyte cells were treated with just 100 μM of DMOG or just 1 ng/ml of EPO compared to untreated cells. However, when cells were treated with a combination of 100 μM of DMOG and 1 ng/ml of EPO, a significant improvement was observed in the % wound closure.

CONCLUSIONS

(70) The inventors have shown that low oxygen (hypoxia) activates a repair switch that makes cells responsive to the reparative action of a prototypical tissue-protective cytokine (TPCs), e.g. EPO. Furthermore, this priming can be achieved at normal oxygen concentrations by administration of chemical inducers of the transcription factor HIF-1α. The inventors have also produced evidence that suggests that this triggering of the endothelial repair response can occur in vivo in a model of vascular injury. This suggests that local application of a HIF-1 inducer and tissue-protective cytokines, such as by using a coated stent, could promote wound repair and promote the repair of the endothelial lining post-angioplasty.

(71) The inventors have shown that HIF-1 inducers in combination with TPCs enhance the proliferation of endothelial cells with no effect on smooth muscle cells. This is very important in terms of retarding the process of restenosis.

(72) The inventors have also shown that the combination of the prototypic TPC; EPO and the HIF-1 inducer; DMOG not only works in injury models of the endothelial cells but also works on injury models of skin cells. This is very promising as the same approach could be extended to wound healing as well as cardiovascular disease.