Treatment for cutaneous scars

Abstract

A medicament for treating a cutaneous scar, comprising a combination of Nintedanib and Propranolol.

Description

[0001] TITLE

[0002] Treatment for Cutaneous Scars

[0003] FIELD OF INVENTION

[0004] This invention relates to a treatment of cutaneous scars in humans, for example keloid, hypertrophic or atrophic scars, etc.

[0005] BACKGROUND

[0006] Cutaneous scars may occur when normal skin tissue is replaced by fibrotic tissue during the healing process, and can arise after trauma, surgery, infection and / or inflammation. Examples of cutaneous scars can include, but are not limited to keloid scars, hypertrophic scars, atrophic scars and acne scars.

[0007] Keloid scars are an abnormal overgrowth of fibrous tissue at the site of a healed wound in the skin. The scars may appear as a raised area in relation to the surrounding skin, which tends to extend further than just the initial site of the wound. Apart from the obvious adverse aesthetic issues, keloid scars may be associated with symptoms such as itching and pain. Hypertrophic scars on the other hand tend to be contained within the site of injury and may regress over time.

[0008] Public data indicates that people with darker skin tones are more likely to develop keloids. However, people that have albinism in African populations have been shown to have a similar prevalence rate to those that do not, suggesting that factors other than skin pigmentation are involved in the pathophysiology of keloid development.

[0009] Currently, there is no definitively known cause or pathology for keloid development. Consequently, known treatments for keloid scars such as excision, cryosurgery, triamcinolone acetonide (TAC), 5-fluorouracil (5FU), silicone gel, and pressure therapy have had limited success. There is no internationally accepted standard of care for the condition. OBJECT OF THE INVENTION

[0010] It is an object of preferred embodiments of the invention to go at least some way towards treating, preventing, or restricting the development of cutaneous scars in humans, for example keloid or hypertrophic scars.

[0011] DEFINITIONS

[0012] The term “comprises” or “has”, if / when used in this document in relation to one or more features, should not be seen as excluding the option of additional unmentioned features. The same applies to derivative terms such as “comprising” and “having”.

[0013] References in this document to pharmaceutically active agent incorporate pharmaceutically active salts thereof. In the event that a weight amount is given for the agent, the figure concerned should be taken as adjusted when it is applied to the salt. For example, a reference to Fosinopril should be taken to also include pharmaceutically acceptable salt forms of Fosinopril and, in that case, any weight amounts expressed, including in the claims, should be read as increased to account for the extra mass of the salt. For example, 332.3 grams (1 mol) of Propranolol Hydrochloride may be taken as equivalent to 259.3 g (1 mol) of Propranolol. And as a further example, 563.7 g (1 mol) of Fosinopril may be taken as substantially equivalent to 585.6 grams (1 mol) of Fosinopril Sodium.

[0014] The term FTU denotes “fingertip unit”, or in other words the amount of medicament normally carried on an adult fingertip when the fingertip is comfortably laden with medicament. One FTU comprises approximately 450 mg to 550 mg of medicament, and preferably about 500 mg of medicament.

[0015] SUMMARY OF THE INVENTION

[0016] First Aspect - A Medicament

[0017] According to one aspect of the invention there is provided a medicament for treating a cutaneous scar, comprising any of the following combinations of actives: a) Nintedanib and Propranolol; b) Propranolol and Tranilast; c) Nintedanib and Fosinopril; d) Fosinopril and Tranilast; e) Verapamil and Propranolol; or f) Verapamil and Nintedanib.

[0018] Optionally the medicament is for topical treatment or local injection at or adjacent to the scar.

[0019] Optionally the actives are in the following molar ratios respectively: a) 1 molar part Nintedanib and 27-33 molar parts Propranolol; b) 1 molar part Propranolol and 4-6 molar parts Tranilast; c) 1 molar part Nintedanib and 36-44 molar parts Fosinopril; d) 1 molar part Fosinopril and 1 .8 - 2.2 molar parts T ranilast; e) 1 molar part Verapamil and 1.1 - 1.5 molar parts Propranolol; or f) 1 molar part Verapamil and 0.05 - 0.08 molar parts Nintedanib.

[0020] Optionally the actives are in the following molar ratios respectively: a) 1 molar part Nintedanib and 28 - 32 molar parts Propranolol; b) 1 molar part Propranolol and 4.5 - 5.5 molar parts Tranilast; c) 1 molar part Nintedanib and 38 - 42 molar parts Fosinopril; d) 1 molar part Fosinopril and 1 .9 - 2.1 molar parts T ranilast; e) 1 molar part Verapamil and 1.2 - 1 .4 molar parts Propranolol; or f) 1 molar part Verapamil and 0.06 - 0.07 molar parts Nintedanib.

[0021] Optionally the actives are in approximately the following molar ratios respectively: a) 1 molar part Nintedanib and 30 molar parts Propranolol; b) 1 molar part Propranolol and 5 molar parts Tranilast; c) 1 molar part Nintedanib and 40 molar parts Fosinopril; d) 1 molar part Fosinopril and 2 molar parts Tranilast; e) 1 molar part Verapamil and 1.33 molar parts Propranolol; or f) 1 molar part Verapamil and 0.066 molar parts Nintedanib. Optionally the actives are present in the following % weight amounts respectively: a) 0.01 - 2 % Nintedanib and 0.05 - 30 % Propranolol; b) 0.05 - 3 % Propranolol and 0.5 - 30 % Tranilast; c) 0.01 - 0.5 % Nintedanib and 0.4 - 20 % Fosinopril; d) 0.1 - 8 % Fosinopril and 0.12 - 10 % Tranilast; e) 0.065 - 6.5 % Verapamil and 0.05 - 5% Propranolol; or f) 0.12 - 24 % Verapamil and 0.01 - 2% Nintedanib.

[0022] Optionally the actives are present in following the % weight amounts respectively: a) 0.05 - 2 % Nintedanib and 0.7-28 % Propranolol; b) 0.1 - 2 % Propranolol and 1 - 20 % Tranilast; c) 0.05 - 0.5 % Nintedanib and 2 - 20 % Fosinopril; d) 0.5 - 5 % Fosinopril and 0.6 - 6 % Tranilast; e) 1 - 4 % Verapamil and 2 - 4% Propranolol; or f) 1 - 10 % Verapamil and 0.5 - 1 .2% Nintedanib.

[0023] Optionally the actives are present in the following % weight amounts respectively: a) 0.1 - 1.52 % Nintedanib and 1.4 - 21 % Propranolol; b) 0.5 - 1.5 % Propranolol and 5 - 15 % Tranilast; c) 0.1 - 0.25 % Nintedanib and 4 - 10 % Fosinopril; d) 1 - 5 % Fosinopril and 1.2 - 6 % Tranilast; e) 2 - 3 % Verapamil and 2 - 3% Propranolol; or f) 2 - 8 % Verapamil and 0.8 - 1% Nintedanib.

[0024] Optionally the medicament is in the form of a topical lotion, cream or gel.

[0025] Optionally the medicament is for application to a cutaneous scar at a rate of approximately 0.5, 1 , 1.5 or 2 fingertip units (FTU) for up to approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 cm2of scar surface area.

[0026] Optionally the cutaneous scar comprises a keloid scar. Optionally the cutaneous scar comprises a hypertrophic scar.

[0027] Optionally the cutaneous scar comprises an atrophic or acne scar.

[0028] Second Aspect - Use for Manufacturing

[0029] According to a second aspect the invention relates to the use of any of the following combinations of actives in the manufacture of a medicament for treating cutaneous scars: a) Nintedanib and Propranolol; b) Propranolol and Tranilast; c) Nintedanib and Fosinopril; d) Fosinopril and Tranilast; e) Verapamil and Propranolol; or f) Verapamil and Nintedanib.

[0030] Optionally the actives are in the respective molar ratios, and / or respective weight amounts, set out in any respective option for the First Aspect of the invention mentioned above.

[0031] Optionally the medicament is for topical administration or local injection.

[0032] Optionally the medicament is in the form of a topical lotion, cream, ointment or gel.

[0033] Optionally the medicament is for application to a cutaneous scar at a rate of approximately 0.5, 1 , 1.5 or 2 fingertip units (FTU) for up to approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 cm2of scar surface area.

[0034] Optionally the cutaneous scar comprises a keloid scar.

[0035] Optionally the cutaneous scar comprises a hypertrophic scar.

[0036] Optionally the cutaneous scar comprises an atrophic or acne scar. Third Aspect - Method of Treatment

[0037] According to a third aspect of the invention there is a method of treating a cutaneous scar in a human subject, comprising administering to the scar a medicament comprising any of the following combinations of actives: a) Nintedanib and Propranolol; b) Propranolol and Tranilast; c) Nintedanib and Fosinopril; d) Fosinopril and Tranilast; e) Verapamil and Propranolol; or f) Verapamil and Nintedanib.

[0038] Optionally the medicament has the actives in the respective molar ratios, and / or respective weight amounts, set out in any respective option for the First Aspect of the invention mentioned above.

[0039] Optionally the medicament is applied topically to the surface of the scar or is administered at or immediately next to the scar by local injection.

[0040] Optionally the medicament is in the form of a topical lotion, cream, ointment or gel.

[0041] Optionally the medicament is applied to the scar at a rate of approximately 0.5, 1 , 1 .5 or 2 fingertip units (FTU) for up to approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 cm2of scar surface area.

[0042] Optionally the cutaneous scar comprises a keloid scar.

[0043] Optionally the cutaneous scar comprises a hypertrophic scar.

[0044] Optionally the cutaneous scar comprises an atrophic or acne scar. DRAWINGS / GRAPHS

[0045] Some preferred embodiments of the invention will now be described by way of example and with reference to the accompanying drawings / graphs, of which-

[0046] Figure 1 is a representation of the results of an MTS assay measuring the ability of various active pharmaceutical agents to suppress keloid cells;

[0047] Figure 2 is a representation of the results for an MTS assay involving a combination of Propranolol + Fosinopril to determine its ability to suppress keloid cells;

[0048] Figure 3 is a representation of the results for an MTS assay involving a combination of Propranolol + Tranilast to determine its ability to suppress keloid cells;

[0049] Figure 4 is a representation of the results for an MTS assay involving a combination of Propranolol + Nintedanib to determine its ability to suppress keloid cells;

[0050] Figure 5 is a representation of the results for an MTS assay involving a combination of Fosinopril + Tranilast to determine its ability to suppress keloid cells;

[0051] Figure 6 is a representation of the results for an MTS assay involving a combination of

[0052] Fosinopril + Nintedanib to determine its ability to suppress keloid cells;

[0053] Figure 7 is a representation of the results for an MTS assay involving a combination of Tranilast and Nintedanib to determine its ability to suppress keloid cells;

[0054] Figures 8-9 are representations of the results for a confluence assay involving a combination of Propranolol and Nintedanib (Figure 8) and Fosinopril and Tranilast (Figure 9) to determine its ability to suppress keloid cells at different dosage concentrations for the same ratio;

[0055] Figure 10-11 are representations of the results for an MTS assay involving a combination of Propranolol and Nintedanib (Figure 10) and Fosinopril & Tranilast (Figure 11) to determine its ability to suppress keloid cells at different dosage concentrations for the same ratio;

[0056] Figure 12 is a representation of the results of a wound closure assay involving a combination of Fosinopril and Tranilast to determine its ability to suppress keloid cells and wound closure at different dosage concentrations for the same ratio Figure 13 is a graphical representation illustrating the ability of a combination of Nintedanib + Propranolol to suppress keloid fibroblasts;

[0057] Figure 14 is a graphical representation illustrating the ability of a combination of Nintedanib + Propranolol to impact the viability of keloid fibroblasts;

[0058] Figure 15 is a graphical representation illustrating the ability of a combination of Verapamil + Propranolol to suppress keloid fibroblasts;

[0059] Figure 16 is a graphical representation illustrating the ability of a combination of Verapamil + Propranolol I to impact the viability of keloid fibroblasts;

[0060] Figure 17 is a graphical representation illustrating the ability of a combination of Verapamil + Nintedanib to suppress keloid fibroblasts;

[0061] Figure 18 is a graphical representation illustrating the ability of a combination of Verapamil + Nintedanib to impact the viability of keloid fibroblasts;

[0062] Figure 19 is a graphical representation illustrating the results of wound closure tests for a combination of Propranolol + Nintedanib;

[0063] Figure 20 is a graphical representation illustrating the results of wound closure tests for a combination of Verapamil + Propranolol;

[0064] Figure 21 is a graphical representation illustrating the results of wound closure tests for a combination of Verapamil + Nintedanib;

[0065] Figure 22 is a graphical representation illustrating the results of signaling pathway tests for various drugs and drug combinations; and

[0066] Figure 23 is a graphical representation illustrating the results of collagen production tests with various drugs and drug combinations.

[0067] DETAILED DESCRIPTION

[0068] Studies were conducted to develop a treatment for cutaneous scars, using Keloid scar tissue as a human-relevant disease model, as follows.

[0069] Development of a Primary Cell Line from Keloid Tissues

[0070] Keloid tissues were digested with physical shearing and a blend of collagenase type I, collagenase type II and DNase. After incubation for 3 hours at 37°C, the digested tissue was filtered to remove debris, followed by red blood cell lysis with a cold RBC lysis buffer for 10 minutes. The product was passed through a filter again and centrifuged at 500 x g for 5 minutes. The supernatant was removed, and the resulting pellet was resuspended with growth media and a cell count was performed with Trypan blue. The digest was either frozen with freezing media (70% Advanced DMEM, 20% FBS, 10% DMSO) or seeded immediately in Kelch’s media (3:1 DMEM:F12, 10% FBS, 1 % AA, 20 ng / mL KGF, 10 uM ROCK inhibitor) for the selection of keratinocytes or DMEM (10% FBS, 1% AA) for the selection of fibroblasts.

[0071] Confirmation of cell types was achieved using light microscopy and protein expression via immunofluorescence. Under light microscopy, fibroblasts appear elongated and in a wavy pattern when growing towards confluent, while keratinocytes appear as small pebble stones in clusters. Cytokeratin 14 was used as a marker for proliferative keratinocytes, which can be seen in keratinocytes but not in fibroblasts. Vimentin was used as a marker for fibroblasts, which can be seen in fibroblasts and not in keratinocytes. Lastly, cytokeratin 10 was used as a marker for differentiated keratinocytes, and this expression was found to be low for the keratinocytes and not present for the fibroblasts.

[0072] Testing the Cell Lines using Pharmaceutical Agents

[0073] The following drugs were selected for trialing their ability to suppress the above cell lines:

[0074] Tranilast, Pirfenidone, Sorafenib, Nintedanib, Metformin, Cromoglycate, Propranolol, Fosinopril, Fasudil, EMA401.

[0075] The tests involved use of an MTS assay as a measure of cell suppression. Various concentrations of each drug were applied on keratinocytes and fibroblasts (n=3). The cells received a daily dose of these for three consecutive days. At the end of the third day, the test media was switched with fresh basal media containing MTS reagent and was incubated for 2 hours at 37°C. The absorbance for the test samples were measured with a spectrophotometer at 490nm. For all values and blank, was subtracted a blank, then expressed as a fold change from the control. Referring to Figure 1 , the drugs most effective across both cell lines were Propranolol, Fosinopril, Nintedanib, Sorafenib, WZB117, and Tranilast. Of these, Propranolol, Fosinopril, Nintedanib, and Tranilast were selected for further testing. Synergistic Interactions

[0076] Propranolol, Fosinopril, Nintedanib, and Tranilast were tested in various combinations for their ability to suppress or inhibit cell lines prepared as above. Again, an MTS assay was used.

[0077] More specifically, the cells were treated with a range of concentrations of one drug alongside a range of concentrations of the other drug. The cells received a single daily dose, and at the end of the third day they underwent the MTS assay as described previously. The test results were plotted as intensity maps and the various combinations assessed for any synergistic interactions. Darker areas in each map indicate zones of greatest synergy, and overlapping zones were used to gauge the effectiveness of the combinations.

[0078] All six combinations showed some high and some low synergy scores, that is based on the concentrations used. Referring to the figures mentioned below, the grids contained in these, in each case on the right side of the page, highlight areas of high synergy with the colour red

[0079] Propranolol + Fosinopril

[0080] Referring to Figure 2, this displays results for Propranolol + Fosinopril (Prop + Fos) for fibroblasts (top) and keratinocytes (bottom). The synergy score was weak with fibroblasts but high with keratinocytes.

[0081] Synergistic zones:

[0082] • Fibroblasts: Prop: 40-60 pM Fos: 80-100 pM

[0083] • Keratinocytes: Prop: 40-60 pM Fos: 10-20 pM

[0084] Propranolol + Tranilast

[0085] Figure 3 displays results for Propranolol + Tranilast (Prop+Tran) for fibroblasts (top) and keratinocytes (bottom). Synergy scores were low with fibroblasts and higher with keratinocytes.

[0086] Synergistic zones:

[0087] • Fibroblasts: Prop: 20-60 pM Tran: 100-300 pM

[0088] • Keratinocytes: Prop: 40-60 pM Tran: 150-300 pM

[0089] Propranolol + Nintedanib Figure 4 displays results for Propranolol + Nintedanib for fibroblasts (top) and keratinocytes (bottom). Synergy scores were good with fibroblasts and high with keratinocytes.

[0090] Synergistic zones:

[0091] • Fibroblasts: Prop: 20-60 pM Nin: 1-2 pM

[0092] • Keratinocytes: Prop: 40-80 pM Nin: 1.25-2.5 pM

[0093] Fosinopril + Tranilast

[0094] Figure 5 shows results for Fosinopril + Tranilast (Fos +Tran) for fibroblasts (top) and keratinocytes (bottom). Synergy scores were high with fibroblasts and low with keratinocytes.

[0095] Synergistic zones:

[0096] • Fibroblasts: Fos: 80 pM Tran: 100-200 pM

[0097] • Keratinocytes: Fos: 10-20 pM Tran: 75-150 pM

[0098] Fosinopril + Nintedanib

[0099] Figure 6 shows results for Fosinopril + Nintedanib (Fos + Nin) for fibroblasts (top) and keratinocytes (bottom). The synergy score was average with fibroblasts and low with keratinocytes.

[0100] Synergistic zones:

[0101] • Fibroblasts: Fos: 80 pM Nin: 2-4 pM

[0102] • Keratinocytes: Fos: 10-20 pM Nin: 2-5 pM

[0103] Tranilast and Nintedanib

[0104] Figure 7 shows results for Tranilast and Nintedanib (Tran + Nin) for fibroblasts (top) and keratinocytes (bottom). Synergy scores were low for both.

[0105] Synergistic zones:

[0106] Fibroblasts: Tran: 100-200 pM Nin: 2-4 pM

[0107] Keratinocytes: Tran: 75-150 pM Nin: 2-5 pM Effectiveness of drug combinations at various concentrations - ratios

[0108] The following combinations were selected for further investigation with fibroblasts as above, following screening, and at the mole ratios indicated below at various concentrations for each range:

[0109] • Prop : Nin 30:1

[0110] • Prop : Tran 1 :5

[0111] • Fos : Nin 40:1

[0112] • Fos : Tran 1 :2

[0113] For each sample of the cells a daily dose of the drug combinations was dosed over 3 days in a similar manner to the dosing described above. Other assays were used, such as administering a single dose over 3 days.

[0114] Propranolol and Nintedanib (Prop-Nin)

[0115] Referring to Figure 8, a range of concentrations for Propranolol and Nintedanib at the 30:1 drug ratio was tested. A synergistic effect was observed on growth curves for all concentrations of the combination, except for a triple dose at a high 90 pM : 3 pM concentration) where the amount of the drugs given individually was sufficiently high to inhibit growth.

[0116] Referring to Figure 9, this further illustrates the synergistic inhibitory effect observed for 30 : 1 pM and 60 : 2 pM doses for both the single dose and triple dose model. While the single dose of 90 : 3 pM showed synergy, the amount of each individual drug was considered sufficiently high to achieve suppression regardless of the synergy.

[0117] Fosinopril & Tranilast (Fos-Tran)

[0118] Referring to Figure 10, the results for a combination of Fosinopril & Tranilast showed a synergistic effect for a 100:200 pM single dose. And, as shown in Figure 11 , a synergistic effect was observed for inhibition at this concentration and dosing regimen. Further, as indicated in Figure 12, synergy was observed in a wound healing assay for this combination at 80:160 pM. Further Tests / Comparisons

[0119] A series of tests / comparisons was conducted to assess the ability of various drugs or combinations to suppress proliferation of keloid scar tissue.

[0120] To that end, primary cells were extracted from keloid tissues via physical shearing with scissors followed by immersing the tissue in a solution of enzymatic digestion buffer containing 0.1 % collagenase type I, 0.25% collagenase type II, 0.2 mg / ml DNase I, 1% FCS, and 1 % antibiotic / antimycotic in DMEM. The solution was mixed every 30 mins for 3 hours at 37°C, and digestion was then halted with the addition of 1 ml FCS. The sample was passed through a 100 pM filter and then a 40 pM filter to remove debris, followed by a 10-minute incubation period with cold red blood cell lysis buffer. After centrifuging for 5 minutes at 500 x g, the resulting pellet was resuspended using cell type specific growth media for the selection of keratinocytes (3:1 DMEM:F12, 10% FBS, 1 % AA, 20 ng / mL KGF, 10 pM ROCK inhibitor) or fibroblasts (DMEM, 10% FBS, 1% AA).

[0121] Confirmation of cell types was done by light microscopy and immunofluorescence. Under light microscopy, fibroblasts appeared elongated in a wavy pattern, while keratinocytes appeared as small pebble stone clusters. Cytokeratin 14 was used as a marker for proliferative keratinocytes which can be seen in cells grown in keratinocyte growth media (KGM) but not observed in the cells grown with fibroblast growth media (FGM). Vimentin was used as a marker for fibroblasts which can be seen in cells grown with FGM and low expression in cells grown with KGM. Cytokeratin 10 was used as a marker for differentiated keratinocytes and this expression was found to be low in the cells in keratinocyte growth media and not present for the cells in fibroblast growth media.

[0122] Primary cell extraction from fresh keloid tissues yielded two major cell types, keratinocytes and fibroblasts, which were selected for growth using different growth media formulations. The KGM was based on Kelch’s medium and incorporated necessary growth factors for propagating keratinocytes while inhibiting fibroblasts. The FGM used is a common cell culture media that propagates fibroblasts but, on its own, is insufficient for keratinocyte growth. Each growth media selected their cells with high purity. Using this method of digestion and the growth media, primary cells were determined as keloid-derived keratinocytes or fibroblasts. Only the fibroblasts were evaluated for their response to exposure to the various drugs as described below.

[0123] The drug candidates in Table below were screened via the MTS assay to measure suppression of cell proliferation.

[0124] In each case the drugs were prepared in either ddH2O or dimethyl sulfoxide (DMSO) depending on solubility and stored in -80°C in aliquots. A range of concentrations were tested across 3 different primary cell lines with a vehicle control matching the highest dose (n=3). Cells received a daily dose of the drugs over 72 hours, then switched to fresh media containing the MTS tetrazolium. After 2 hours at 37°C, to allow the reduction of MTS tetrazolium to a coloured formazan dye from viable cells, the optical density was measured with a spectrophotometer at 490 nm. Suppression of cell proliferation was calculated with the following equation [1]:

[0125] Suppression 100

[0126] Suppression of keloid fibroblast cellular proliferation by at least 50% was determined to be a potent drug candidate for further investigation. Six of the fourteen drugs tested reached this threshold, being:

[0127] 1. Tranilast;

[0128] 2. Sorafenib;

[0129] 3. Nintedanib;

[0130] 4. Propranolol;

[0131] 5. Verapamil; and

[0132] 6. Fosinopril.

[0133] All six of these drugs, excluding Tranilast, achieved almost 100% suppression. While Tranilast did not achieve such high suppression, it was selected for further investigation as it is currently used in therapy in some countries.

[0134] Drug Combinations

[0135] Next, tests were run on various combinations of the drugs to find possible synergistic interactions between them. This involved using an MTS assay for measuring suppression of keloid fibroblast cell proliferation as the primary outcome. Cells received a daily dose of each combination across a concentration range over 72 hours. Suppression of cell proliferation was measured as previously described (see equation [1]). More specifically, the tests were repeated across 3 independent primary keloid-derived fibroblasts. Values of suppression were input into SynergyFinder software1to show overlapping ‘zones of most synergistic areas’ for determining

[0136] 1https: / / synergyfinder.fimm.fi / synergy / synfin_docs / #welcome the effectiveness of the combinations tested. The Table below shows the drug combinations and concentrations together with the results obtained.

[0137] Table

[0138] Out of the ten drug combinations shown in the above Table the following combinations were selected for further testing (the ratios below are molar):

[0139] • 30 Propranolol + 1 Nintedanib

[0140] • 1 Propranolol + 5 Tranilast,

[0141] • 40 Fosinopril + 1 Nintedanib,

[0142] • 1 Fosinopril + 2 Tranilast,

[0143] • 3 Verapamil + 4 Propranolol

[0144] • 15 Verapamil + 1 Nintedanib

[0145] The six drug combinations were given as a one off (a single dose) or every 24 hours (a triple dose) over 72 hours. In each case the combination was compared with the individual drugs alone and with a control. So, for example, the combination Propranolol + Nintedanib was compared with Propranolol alone, with Nintedanib alone, and with the control.

[0146] Daily images of the cells were taken using the Incucyte (Sartorius), and keloid fibroblast cell proliferation was measured using analysis software. The MTS assay was used again as the endpoint measure for cell viability. Experiments were repeated with at least 3 independent primary keloid derived fibroblast cell lines. Statistical analysis was performed with GraphPad Prism 9. Two-way ANOVA RM was performed on cell proliferation data for concentration and time. Two-way ANOVA was also performed on cell viability for the drugs I concentrations. Post hoc analysis was carried out using Dunnett’s to correct for multiple comparisons with means against the combination treatment and a significant difference at P < 0.05. Data was presented as mean ± SEM.

[0147] Propranolol + Nintedanib

[0148] More specifically, and referring to Figure 13, for the 30:1 Propranolol + Nintedanib combination, keloid derived fibroblasts were treated with Propranolol (red), Nintedanib (blue), or Propranolol + Nintedanib in combination (green), with either a single or daily dose over 72 hours. Concentrations at a fixed ratio of 30:1 were tested, (A-B) 30pM : 1 M, (C-D) 60pM : 2pM, and (E-F) 90|JM : 3pM. A two-way ANOVA RM was performed between Propranolol, Nintedanib, and the combination for treatment and time. The significant difference (P < 0.05) between Propranolol + Nintedanib (combination) and Propranolol alone is indicated with the symbol “*”, and between Propranolol + Nintedanib (in combination) and Nintedanib alone is indicated with Data presented as mean ± SEM with n=3 for single dose and n=5 for triple dose.

[0149] Further, Figure 14 illustrates the cell viability of keloid derived fibroblasts with an MTS assay following 72 hours treatment with either (A) single or (B) daily dose of Propranolol (red), Nintedanib (blue), or PropNin combination (green). A two-way ANOVA was performed between Propranolol, Nintedanib, and the combination for treatment and concentration. The significant difference of P < 0.05 between the combination and Propranolol alone is indicated with the symbol “*” and between the combination and Nintedanib alone with the symbol “#”. Data are presented as mean ± SEM with n=3 for single dose and n=5 for triple dose.

[0150] The tests show that in the single dose experiment, the combination had a synergistic effect across all three concentration ranges.

[0151] Verapamil + Propranolol

[0152] Similar tests were run on the 3:4 Verapamil + Propranolol, at a low concentration (15pM : 20pM), a medium concentration (30pM : 40pM), and a high concentration (45pM : 60pM). Only the daily dose experiment was performed for this combination.

[0153] The results are shown at Figures 15 & 16 (the combination is the VP line). A significant synergistic effect was observed at the medium and high concentrations to suppress cell growth curve and cell viability.

[0154] Verapamil + Nintedanib

[0155] Similar tests were run on the 15:1 Verapamil + Nintedanib combination, at a low concentration (15pM : 1 M), a medium concentration (30pM : 2pM), and a high concentration (45pM : 3pM). Only the daily dose experiment was performed for this combination. The results are shown at Figures 17 & 18 (the combination is the VP line). A synergistic effect was observed at the medium and high concentrations in suppressing cell growth and cell viability.

[0156] The other combinations of drugs did not show results as strong as the three combinations above.

[0157] Cell Function & Signaling Pathway

[0158] The following combinations of drugs were tested for their effect on keloid fibroblast cell migration and protein expression.

[0159] 30 pM Propranolol + 1 pM Nintedanib

[0160] 3 pM Verapamil + 4 pM Propranolol

[0161] 15 pM Verapamil + 1 pM Nintedanib

[0162] One set of concentrations from each combination was selected for investigating protein signaling and collagen production. The concentrations were chosen for assays based on the above experiments in terms of the lowest concentration for which a synergistic effect was observed.

[0163] Wound Healing Assay

[0164] Further, for each combination, keloid fibroblast cells were seeded onto a 96-well plate and grown to confluency before being wounded using a specialised wounding tool to ensure consistency of wound sizes. Floating cells and media were removed and washed twice with PBS then replaced with fresh DM EM and treated with the various drugs once. Cell migration was monitored with Incucyte every 6 hours for 48 hours, and wound closure was measured with Imaged. Statistical analysis was performed with GraphPad Prism 9 for a two-way ANOVA RM for time-course data. The area under the curve was measured with GraphPad Prism 9.

[0165] Western Blotting

[0166] For each combination, keloid fibroblast cells were seeded onto a 6 well plate and grown to confluency. The cells were washed twice with PBS and replaced with fresh DM EM for overnight incubation. The next morning, the cells were treated with 10 ng TGF-p and the various drugs for 30 minutes. Immediately, the media was removed and washed twice with cold PBS and lysed with RIPA lysis buffer supplemented with Halt™ Protease and Phosphatase inhibitors. Samples were collected in a fresh microtube and sonicated for 2 minutes and then centrifuged at 13,000 x g at 4°C for 10 minutes. The supernatant was collected, and the protein concentration was determined with a Pierce BCA protein assay.

[0167] Samples were prepared for western blotting by equalizing all sample concentrations with a 4X Bolt LDS buffer, a 10X Bolt reducing agent, and water, and stored at -20°C. Samples were loaded into Bolt Tris gels for SDS-PAGE and transferred onto nitrocellulose membranes with Power Blotter (Invitrogen). Membranes were blocked with 5% non-fat skim milk in TBST for 1 hour and incubated with primary antibodies of interest overnight at 4°C. The next morning, membranes were incubated with the respective secondary antibody species for 1 hour and imaged with ECL Chemiluminescence with the ChemiDoc (Bio-rad). Membranes were stripped for the secondary antibodies and reprobed with another primary antibody. Densitometry bands were measured with Imaged. Statistical analysis was then performed with GraphPad Prism 9 for one-way ANOVA with the control and each drug combination group.

[0168] ELISA

[0169] For each combination, keloid fibroblast cells were seeded onto a 6 well plate and grown to confluency. Cells were washed twice with PBS and replaced with fresh DMEM and various drugs. After 24 hours, the media was collected and centrifuged at 300 x g for 10 minutes at 4°C The cells were washed twice with ice cold PBS and extracted protein as above. Both the media and cell lysates were stored in -80°C until all samples were collected. Human Pro-Collagen 1 alpha 1 DuoSet ELISA (#DY6220-05, R&D Systems) was performed according to manufacturer’s instructions. Optimisation of sample dilution were 1 : 100 for media and 1 :500 for cell lysates. Statistical analysis was performed with GraphPad Prism 9 with the control and each drug combination group.

[0170] Wound Healing Results

[0171] Referring to Figure 19 for wound closure analysis a synergistic effect was found for the Propranolol + Nintedanib combination at the low and medium concentrations in terms of inhibiting cell migration. Referring to Figure 20, the wound closure analysis for the low concentration for the Verapamil + Propranolol combination did not display a significant difference against the control, or against each drug alone, in both wound closure and total area. For the medium concentration, the combination was significantly different to Verapamil alone at 24 and 36 hours but showed no difference in total closure area. The high concentration of the combination displayed a significant difference to both drugs alone for wound closure over time and total area. It is considered that the high concentration of the combination showed a synergistic effect on inhibiting cell migration.

[0172] Referring to Figure 21 , in terms of wound closure analysis all three concentrations of the Verapamil + Nintedanib combination showed a significant difference against the control and Verapamil alone, but not against Nintedanib alone, for both wound closure over time and total closure area.

[0173] Signaling Pathway Assessment

[0174] The effects of the drug combinations on signaling pathways such as MAPK and TGFp / Smad pathways were assessed due to their role in wound-healing and scarring both in normal tissues and in keloids. Phosphorylation of ERK1 / 2 and Smad2 were measured as they are key targets in the respective pathways. In these assays, exogenous TGFp was required to stimulate phosphorylation of Smad, but not required for the phosphorylation of ERK1 / 2. Therefore, keloid fibroblast cells were treated with TGFp and drugs concurrently as the same sample was used to probed for both pathways. The results are shown at Figure 22.

[0175] As indicated, there was a reduction in phosphorylation of ERK1 and ERK2 with the Propranolol + Nintedanib combination against TGF+ control and Propranolol alone. This effect appeared to be mainly driven by Nintedanib as there was no difference in phosphorylation between the combination and Nintedanib alone, indicating the reduction in signal is mainly driven by Nintedanib.

[0176] There was a reduction in phosphorylation of ERK1 , ERK2, and Smad2 with in the Verapamil + Nintedanib combination against TGF+ control and Verapamil alone. This effect appeared to be mainly driven by Nintedanib as there was no difference in phosphorylation between VerNin and Nintedanib.

[0177] There was no significant difference in phosphorylation of ERK1 , ERK2, and Smad2 with the Verapamil + Propranolol combination.

[0178] Effect of drug combinations on collagen production

[0179] One of the hallmarks of keloid pathogenesis is the increased production of collagen type I and type III Click or tap here to enter text.. Therefore, it would be ideal to have a drug combination that can inhibit this effect. Tests were conducted to determine the effect of the drug combinations on human pro-collagen 1A1 via an ELISA kit. The results are shown at Figure 23.

[0180] As illustrated, in the media of the cells following drug treatment, there was no significant difference in the concentration of COL1A1 , suggesting that there is little effect on collagen secretion.

[0181] The COL1A1 concentration in keloid fibroblast cells following Propranolol (30pM) and Nintedanib (1 M) alone was not significantly different to the control. However, the PropNin combination showed a significant reduction in COL1A1 , although the combination was not significantly different to Propranolol and Nintedanib alone. This suggests that there is a synergistic interaction at play.

[0182] The COL1A1 concentration in cells following Verapamil, Propranolol (40pM), and VerProp combination had a significant difference to the control. However, the VerProp combination did not have significant difference to Verapamil or Propranolol alone.

[0183] The COL1A1 concentration in cells following Nintedanib (2pM) did not have a significant difference when compared to the control. The VerNin combination had a significant difference compared to the control, but not against Verapamil or Nintedanib alone.

[0184] Overall, for the three drug combinations, the wound healing assays showed that the PropNin combination gave the most prominent synergistic effect amongst the drug combinations. The VerProp combination had a synergistic effect at the highest concentration. Further, the VerNin combination showed the most inhibition of the signaling pathways, followed by PropNin. All three combinations showed a reduction in cellular COL1 A1 , which indicates that all combinations were effective at reducing collagen synthesis. Overall, the PropNin combination appeared to deliver the most significant effects across assays amongst the three combinations that surprisingly demonstrated significant impacts across multiple facets of cutaneous scar formation and healing.

[0185] While some forms of the invention have been described by way of example, it should be appreciated that modifications and improvements can be made without departing from the scope of the following claims.

[0186] In terms of disclosure, this document envisages and hereby posits any feature mentioned herein in combination with a repeat of itself (eg two of the same) and / or any other feature or features mentioned herein, even if the combination is not claimed below.

Claims

CLAIMS1. A medicament for treating a cutaneous scar, comprising a combination of Nintedanib and Propranolol.

2. A medicament according to claim 1 , for topical treatment or local injection at or adjacent to the scar.

3. A medicament according to claim 1 or 2, wherein the actives are in the ratio of 1 molar part Nintedanib and 27-33 molar parts Propranolol.

4. A medicament according to claim 1 or 2, wherein the actives are in the ratio of 1 molar part Nintedanib and 28 - 32 molar parts Propranolol.

5. A medicament according to claim 1 or 2, wherein the actives are in the ratio respectively 1 molar part Nintedanib and 30 molar parts Propranolol.

6. A medicament according to any one of the preceding claims, wherein the actives are present in the % weight amounts 0.01 - 2 % Nintedanib and 0.05 - 30 % Propranolol.

7. A medicament according to any one of the preceding claims, in the form of a topical lotion, cream, ointment or gel.

8. A medicament according to any one of the preceding claims, for application to a cutaneous scar at a rate of approximately 0.5, 1 , 1.5 or 2 fingertip units (FTU) for up to approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 cm2of scar surface area.

9. A medicament according to any one of the preceding claims, wherein the cutaneous scar comprises a keloid scar.

10. A medicament according to any one of claims 1 - 8, wherein the cutaneous scar comprises a hypertrophic scar.

11. A medicament according to any one of claims 1-8, wherein the cutaneous scar comprises an atrophic or acne scar.

12. The use of a combination of Nintedanib and Propranolol in the manufacture of a medicament for treating cutaneous scars.

13. The use according to claim 12, wherein the medicament has the actives in the respective molar ratio, and / or respective weight amount, set out in any one of claims 3-6.

14. The use according to claim 12 or 13, wherein the medicament is for topical administration or local injection.

15. The use according to claim 12 or 13, wherein the medicament is in the form of a topical lotion, cream, ointment or gel.

16. The use according to any one of claims 12 - 15, wherein the medicament is for application to a cutaneous scar at a rate of approximately 0.5, 1 , 1 .5 or 2 fingertip units (FTU) for up to approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 cm2of scar surface area.

17. The use according to any one of claims 12 - 16, wherein the cutaneous scar comprises a keloid scar.

18. The use according to any one of claims 12 - 16, wherein the cutaneous scar comprises a hypertrophic scar.

19. The use according to any one of claims 12 - 16, wherein the cutaneous scar comprises an atrophic or acne scar.

20. A method of treating a cutaneous scar in a human subject, comprising administering to the scar a medicament comprising Nintedanib and Propranolol.

21. A method according to claim 20, wherein the medicament has the actives in the respective molar ratios, and / or respective weight amounts, set out in any one of claims 3-6.

22. A method according to claim 20 or 21 , wherein the medicament is applied topically to the surface of the scar or is administered at or immediately next to the scar by local injection.

23. A method according to claim 20 or 21 , wherein the medicament is in the form of a topical lotion, cream, ointment or gel.

24. A method according to any one of claims 20-23 wherein the medicament is applied to the scar at a rate of approximately 0.5, 1 , 1.5 or 2 fingertip units (FTU) for up to approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 cm2of scar surface area.

25. A method according to any one of claims 20-24, wherein the cutaneous scar comprises a keloid scar.

26. A method according to any one of claims 20 - 24, wherein the cutaneous scar comprises a hypertrophic scar.

27. A method according to any one of claims 20 - 24, wherein the cutaneous scar comprises an atrophic or acne scar.

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