Patent Description:
Disruption of the integrity of skin and mucosal surfaces results in the formation of a wound. Wound healing is a complex biological process which restore the tissue integrity. Physiologically, wound healing can be schematically divided into four distinct phases: haemostasis, inflammation, proliferation and tissue remodelling. Immediately after an injury, the haemostasis process begins: the bleeding is controlled by the aggregation of platelets and the subsequent formation of the fibrin clot stops the bleeding and provides a scaffold for the attachment and proliferation of the cells. After <NUM>-<NUM> days, the inflammatory process progresses into the proliferative phase and fibroblasts are attracted into the wound to synthesize granulation tissue. This granulation tissue allows leukocytes to enter the wound site and once the wound is closed, the immature scar can move on to the final remodelling phase which can last up to a year. The main function of fibroblasts in tissue regeneration is to maintain the physical integrity of connective tissue by producing and remodelling the extracellular matrix. Regardless of the aetiology of the wound, whether acute or chronic, the above described repair processes are similar but while acute wounds, e.g. a surgical incision, usually pass through these phases relatively quickly, wounds undergoing delayed healing up to <NUM> weeks after the initial insult, often as a result of prolonged pathological inflammation, are defined chronic wounds.

Several insults like biofilm formation, microbial invasion, repeated trauma, ischemia, oedema, venous hypertension and effect of mechanical forces, including pressure, may cause healing impairment. These insults lead to poor migration of keratinocytes and the epithelization is also delayed. In these indolent cases, wound bed preparation remains the standard of therapy.

An effective wound bed preparation may involve enzymatic (<NPL>; <NPL>) and surgical debridement, resident fibroblast stimulation and stimulation of growth factor release (<NPL>), addition of extraneous growth factors to the wound (<NPL>), deployment of bioengineered extracellular matrix (<NPL>), collagen and alginates (<NPL>), cultured keratinocyte suspension and even bioengineered dermal preparation (<NPL>) in any combination and also the stem cell therapy holds promise (<NPL>).

The known treatment options remain only moderately effective and often fail to promote the closure of non-healing wounds in susceptible populations, such as aging and diabetic patients or represent a limited remedy in case of wounds by insect or spider bites (<NPL>). Moreover, some of these approaches, like for example the bioengineered skin substitutes, although representing a valid alternative to autografting, induce skin cells in repairing the wound rather than guiding a regeneration process.

Analogously, the surgical wounding of oral mucosa after surgical incision requires, without complications, long recovery times. In order to restore the original physiology of the oral cavity, up to five weeks are usually required but with some possible complications that could delay the complete healing of the wound. In particular, the normal healing response to tooth extraction results in a significant loss of bone and collapse of the surrounding gingiva. In normal healing, a substantial percentage of extraction sites suffers from postoperative complications.

In fact, after tooth extraction blood clots fill the socket and, after one week, the clots are replaced by granulation tissue; after <NUM> weeks, the granulation tissue is replaced by collagen, and bone formation begins at the base and the periphery of the extraction socket. At <NUM> weeks, it is estimated that on average two-thirds of the extraction socket is filled with bone. Epithelium was found to require a minimum of three weeks to completely cover the extraction socket, with some extraction sites requiring up to five weeks to completely cover the socket (<NPL>; <NPL>). Post-operative complications of the above-mentioned normal healing in response to tooth extraction may include dry socket, dysesthesia, severe infection, pain, swelling, trismus and haemorrhage. All these possible complications significantly increase the total healing time from a surgical incision of the oral mucosa.

Trichloroacetic acid alone or in association with other compounds may be used as "chemical peel" (<NPL>). A <NUM>% trichloroacetic (TCA) solution is commonly used as medium-depth chemoexfoliation which is a frequently used to treat fine rhytides, actinic photodamage, hyperpigmentation, and even actinic-related premalignant changes, such as actinic keratoses.

Chloroacetic acid has been disclosed for the treatment of chronic wounds together with an extracellular polymeric substance solvating system surfactant and buffering agent (<CIT>). Formulations containing trichloroacetic acid and hydrogen peroxide in a pH range of <NUM>-<NUM> were also proposed for the treatment of pathologies of the skin and mucosa membranes, such as acne, damage caused by the sun and sun freckles (<CIT>) with some limitations due to the fact that hydrogen peroxide, known as mild antiseptic, may be used on the skin to prevent infection of minor cuts, scrapes, and burns but it is endowed with potential negative effects on tissue repairs and should not be used to treat deep wounds, animal, insect or spider bites, or serious burns (Agency for Toxic Substances and Disease Registry, Buford Hwy NE Atlanta-Hydrogen Peroxide).

More recently, the biological effect of hydrogen peroxide during the wound-healing process was reconsidered: in relatively high concentrations (i.e. <NUM>%), hydrogen peroxide displays its strong ability of oxidization and pro-inflammation to disinfect wound tissue (<NPL>); however, in lower concentrations (i.e. <NUM>%) it shows good antimicrobial effect and skin tolerability (<NPL>). On the contrary, an oxidized form of trichloroacetic acid, namely trichloroperoxyacetic acid has never been considered for human use even if in vitro tests confirmed disinfectants properties of diluted aqueous solutions against Bacillus subtilis, Escherichia coli and Staphylococcus aureus (<NPL>).

There is thus a need for innovative pharmacological treatments of surgical wounds on skin and mucous membrane, avoiding the above mentioned post-operative complications and allowing an optimal wound bed preparation.

In order to solve the above-mentioned problems, the present invention provides formulations for repairing skin and mucous membrane wounds.

The wound healing compositions of the invention comprise:.

The components of these formulations display a synergic action in the treatment of wounds of skin and mucous membranes.

The formulations of the invention are devoid of the potential negative effects of hydrogen peroxide on tissue.

The formulations of the invention are applied topically in the form of cream, ointment, liquid, gel or similar administration forms. The formulations have a rapid effect, quickly reduce swelling and pain, promote wound healing and reduce the times of recovery of chronic wounds, like chronic ulcers bedsores. The use of the formulations is also effective in treating post-operative complications of normal healing in response to tooth extraction including dry socket, dysesthesia, severe infection, pain, swelling, trismus, haemorrhage and in the treatment of slowly healing wounds, spontaneous ulcerations, sores of various origins and necrotic sores including, pyogenic granulomas, chalazion, epulis, fistulas and abscesses.

The formulations of the invention are characterized by a concentration of hydrogen peroxide aqueous solution (<NUM> to <NUM> % by weight of a <NUM>-<NUM>%, hydrogen peroxide aqueous solution) remarkably lower than that disclosed in the prior art, particularly in EP <NUM> (<NUM>-<NUM>% by weight).

The concentration of trichloroacetic preferably ranges from <NUM> to <NUM> % by weight, more preferably from <NUM> to <NUM> % by weight.

The formulations of the invention may be diluted using a <NUM>,<NUM>% w/v aqueous NaCl solution. The diluted formulations secure the viability of the cells of the mucosal membrane and of the skin and are useful to accelerate the repair of the surgical wounds, post traumatic wounds, unhealed wounds, ulcers and bedsores. The pH value of the formulations is higher than <NUM>,<NUM>, typically in the range from <NUM> to <NUM>.

The liquid formulations of the invention may contain water in a range from <NUM> to <NUM>% by weight, EDTA disodium salt in a range from <NUM> to <NUM>% by weight, glycerol in a range from <NUM>,<NUM> to <NUM>% by weight, aqueous ammonia (<NUM>-<NUM>%) in a range from <NUM>,<NUM> to <NUM>% by weight, a thickening and texture agent in a range from <NUM>,<NUM> to <NUM>% by weight and optionally <NUM>,<NUM>-<NUM>,<NUM>% w/v of sodium chloride. Preferred thickening and texture agents include hydroxyethylcellulose, guar gum, locust bean gum, xanthan gum, gelatine and hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymers. More preferred thickening and texture agents comprise hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer (Sepineo™ d.

The undiluted ointment formulations of the invention may also contain water in a range between <NUM> and <NUM>% by weight, sodium laurylsulfate in a range from <NUM> to <NUM>% in weight, propylene glycol in a range from <NUM> to <NUM>% by weight, stearyl alcohol in a range between <NUM> and <NUM>% by weight, white petrolatum in a range between <NUM> and <NUM>% by weight, aqueous ammonia (<NUM>-<NUM>%) in a range between <NUM> and <NUM>% by weight and optionally one or more preservatives in a concentration between <NUM> and <NUM>%. Preferred preservatives include propylparaben, methylparaben, sodium benzoate and ethylhexylglycerin. Optionally, the formulation does not contain any preservative. Trichloroacetic acid and trichloroperoxyacetic acid can be alternatively used instead of trichloroacetic acid and hydrogen peroxide. These formulations can optionally be administered after dilution with an isotonic <NUM>,<NUM>% w/v NaCl aqueous solution.

The undiluted cream formulations of the invention may contain, in addition to the combination of trichloroacetic acid and hydrogen peroxide or of trichloroperoxyacetic acid and trichloroacetic acid in the proportions above defined, water in a range between <NUM> and <NUM>% by weight, liquid paraffin in a range between <NUM> and <NUM>% by weight, propylene glycol in a range from <NUM> to <NUM>% by weight, glycerine in a range from <NUM> to <NUM>% by weight, aqueous ammonia <NUM>-<NUM>% in a range from <NUM> to <NUM>% by weight and polysorbate <NUM> in a range from <NUM> to <NUM>% by weight. These formulations can optionally be administered after dilution with an isotonic <NUM>,<NUM>% w/v NaCl aqueous solution.

The undiluted gel formulations of the invention may contain ethanol in a range between <NUM> and <NUM>% by weight, propylene glycol in a range between <NUM> and <NUM>% by weight, aqueous ammonia (<NUM>-<NUM>% w/v) in a range of <NUM>-<NUM>%, diethylene glycol monoethyl ether in a range from <NUM> to <NUM>% by weight and myristyl alcohol in a range between <NUM> and <NUM>%. These formulations can optionally be administered after dilution with an isotonic <NUM>,<NUM>% w/v NaCl aqueous solution.

An ointment for repairing skin and mucous membranes wounds has the following composition:.

This concentrated ointment formulation can be diluted using a <NUM>,<NUM>% w/v NaCl aqueous solution to obtain a final liquid formulation with a pH value ≥ <NUM>,<NUM>.

A liquid for repairing wounds of skin and mucous membranes has the following composition (Formulation <NUM>-<NUM>-<NUM>: <NUM>/<NUM> dilution).

The concentrated liquid solution can be diluted up to <NUM>:<NUM> using a <NUM>,<NUM>% w/v NaCl aqueous solution to obtain a final liquid formulation with a pH value ≥ <NUM>.

A liquid for repairing skin and mucous membranes wounds has the following composition:.

This concentrated liquid solution can be diluted using a <NUM>,<NUM>% w/v NaCl aqueous solution to obtain a final liquid formulation with a pH value ≥ <NUM>,<NUM>. Alternatively, trichloroperoxyacetic acid can be prepared in situ by addition of equimolar amounts of hydrogen peroxide to trichloroacetic acid.

A cream for repairing skin and mucous membranes wounds has the following composition.

This concentrated formulation can be diluted using a <NUM>,<NUM>% w/v NaCl aqueous solution to obtain a final liquid formulation with a pH value ≥ <NUM>,<NUM>.

A gel for repairing skin and mucous membranes wounds has the following composition:.

The only difference from Example <NUM> (Formulation <NUM>-<NUM>-<NUM>) is that the components do not contain hydrogen peroxide.

This concentrated formulation was diluted <NUM>:<NUM>, using a <NUM>% w/v NaCl solution to obtain a final liquid formulation with a pH value ≥ <NUM>.

This concentrated liquid solution was diluted <NUM>:<NUM>, using a <NUM>% w/v aqueous NaCl solution to obtain a final liquid formulation with a pH value ≥ <NUM>.

The liquid formulations <NUM>-<NUM>-<NUM> (TCA plus H<NUM>O<NUM>), <NUM>-<NUM>-<NUM> (TCA only) and <NUM>-<NUM>-<NUM> (H<NUM>O<NUM> only) were then biologically evaluated in in vivo and in vitro tests. These tests confirmed the synergic action of trichloroacetic acid and hydrogen peroxide in accelerating the healing of wounds and ulcers of mucous membranes and skin, even after dilution up to <NUM> time with a <NUM>,<NUM>% NaCl aqueous solution and at a pH value ≥ <NUM>.

This study was divided in two steps: in the first, the effectiveness of solutions was verified on an in vitro cell model using NHEK cells (normal human epidermal cell); in the second the effectiveness of solutions was carried out on a mouse animal model mimicking a slow-healing human wound. The solutions tested in both steps included the following combinations:.

and their effects were compared to an untreated control sample.

In an in vitro model, NHEK cells were used to test the efficacy of the solutions in a dose-response study to exclude any cytotoxic effects analyzing cell viability by MTT (<NUM>-(<NUM>,<NUM>-dimethylthiazol-<NUM>-yl)-<NUM>,<NUM>-diphenyl tetrazolium bromide) test. Since the combinations of substances have never been described in the literature, the range tested was based on TCA concentration reported in literature [<NPL>. In particular, <NUM>: <NUM> (<NUM>); <NUM>: <NUM> (<NUM>); <NUM>: <NUM> (<NUM>) and <NUM>: <NUM> (<NUM>) diluted with sterile physiological solution (<NUM>% NaCl) were tested. <NUM> different stimulation protocols were selected, as described below:.

Since the data highlighted the minimum effective dose and the importance of administering an adequate dose, in the subsequent experiments only the <NUM> concentration was tested using only the protocols A and C. Subsequently, tests were carried out to evaluate cell proliferation (Crystal Violet assay), migration (wound assay) and production of reactive oxygen species (ROS).

Migration and re-epithelization were investigated in an in vivo model using male C57BL/6JOlaHsd mice, excluding any toxic effects induced by the substances tested in protocol A and C. Before administration, the mice were depilated on the right side of the back spine. The depilated area was about <NUM>×<NUM>. The bedsore model was squeezed in the depilated area by intermittent mechanical extrusion to simulate the natural formation process of bedsores.

were used at <NUM>: <NUM> dilutions prepared in sterile physiological solution (<NUM>% NaCl) to treat skin wounds. At the end of each treatment, a planimetric analysis of the wounds was performed and wound areas were collected to make histological analysis as E/E (hematoxylin/eosin) and Masson staining.

Normal human epidermal keratinocyte cells (NHEK) purchased from Lonza (Basel, Switzerland) were cultured in EpiLife medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing <NUM> Ca2+ and HKGS (Gibco, Thermo Fisher Scientific) in a <NUM>% CO<NUM> incubator (Thermo Fisher Scientific) at <NUM>. The medium was replaced every two days, and the cells were used at <NUM>% to <NUM>% confluence [<NPL>]. Experiments were conducted at passages <NUM>-<NUM>.

After each stimulation, the NHEK cells were washed with sterile PBS <NUM>× and incubated with DMEM (Dulbecco s modified eagle medium) without red phenol and FBS (fetal bovine serum) containing <NUM>% MTT dye (MTT-Based In Vitro Toxicology Assay Kit; Sigma-Aldrich) for <NUM> at <NUM> and <NUM>% CO<NUM> [<NPL>. Cell viability was determined by measuring absorbance using a spectrometer (VICTORX4 multi-label plate reader) at <NUM> with correction at <NUM> and calculated by comparing the results to control cells (<NUM>% viable).

After each treatment the cells were fixed with <NUM>% glutaraldehyde (Sigma-Aldrich) for <NUM> at room temperature, washed, and stained with <NUM>µL <NUM>% aqueous crystal violet (Sigma- Aldrich) for <NUM> at room temperature. 100µL microliters of <NUM>% acetic acid were added to multi-well plates and mixed before reading the absorbance at <NUM> using a spectrometer (VICTORX4 multi-label plate reader). The estimated number of cells was calculated by comparing the results to the control cells (control T0), examined on the first treatment, and the variation of the untreated cells was also reported [<NPL>.

A scratch wound healing assay was performed as previously described [<NPL>. ] in confluent monolayer cells using a sterile p200 pipette tip. Afterwards, the cells were stimulated with different preparations in different protocols (A and C) and monitored for <NUM> days. After each time point, repopulation of the wounded areas was observed under a phase contrast microscope (Leitz, Germany). Using the ImageJ image-processing program, the size of the denuded area was determined at each time point from digital images taken at <NUM> different areas. The results are expressed as means ± SD (%) of migrated cells.

The rate of superoxide anion release was measured using a standard protocol based on reduction of cytochrome C. In both treated and untreated cells, <NUM>µL cytochrome C were added and in another sample <NUM>µL superoxide dismutase were also added for <NUM> in an incubator (all substances were from Sigma-Aldrich). The absorbance in culture supernatants was measured at <NUM> using a spectrometer (VICTORX4 multilabel plate reader) and O2 was expressed as the mean ± SD (%) of nanomoles per reduced cytochrome C per microgram of protein compared to the control [<NPL>].

The dose-response study was conducted evaluating the best concentration within the range found in the literature. This test allowed to determine the metabolism of the mitochondrion by analyzing the administration protocols. As shown in <FIG> the protocol A showed a dose-response effect for all substances with a peak around three days of stimulation; this effect can stabilize only with <NUM>-<NUM>-<NUM> and the main effect was observed with <NUM> (<NUM>:<NUM> dilution).

The same tests were also conducted with protocol B (<FIG>) in which the tested composition was administered one treatment/day for <NUM> days. Also, in this case the best results were obtained by <NUM> (p<<NUM>) for all tested solutions. Considering that the results are "lower" than those observed in protocol A, it is assumed that the daily stimulation is excessive but not toxic. The plateau phase tends to be slightly descending, demonstrating that protocol B is excessive and for this reason protocol B was replaced by protocol C (<NUM> treatment / alternate days carried out max <NUM> days) in subsequent experiments.

The proliferation assay was conducted using crystal violet on NHEK cells treated with the substances according to protocols A and C. As shown in <FIG>, <NUM>-<NUM>-<NUM> significantly increased cell proliferation compared to other compositions (p<<NUM>), particularly one more time compared to <NUM>-<NUM>-<NUM> and about <NUM>% to <NUM>-<NUM>-<NUM>. In addition, these effects are evident in both protocols, but the main effect was obtained by protocol C (p <<NUM> vs A). This is important because by increasing the number of cells it is possible that they can also migrate; in addition, these data suggested an activity of <NUM>-<NUM>-<NUM> higher than that of the individual components (p<<NUM>).

The effect of the different formulations on NHEK migration in wound healing were evaluated (<FIG>). Our results demonstrated an improvement in migration activity (p<<NUM>) in NHEK cells treated with <NUM>-<NUM>-<NUM> (both protocol A and C) compared to control and compared to <NUM>-<NUM>-<NUM> (about <NUM> times more) and <NUM>-<NUM>-<NUM> (about <NUM>%). This is a validated method to characterize various factors involved in migration. The data confirm what observed during proliferation analysis.

Since conflicting data are reported in literature on the ROS role on healing, further experiments were carried out to explore the role of ROS in this context. As shown in <FIG>, despite the presence of H<NUM>O<NUM>, ROS production does not increase above the physiological share of the tested substances; this is certainly important to ensure the effectiveness observed in previous tests. Obviously, the compound with hydrogen peroxide has a higher ROS production than the other substances in both protocols but <NUM>-<NUM>-<NUM> is able to remain below the physiological level of ROS compared to the control and compared to <NUM>-<NUM>-<NUM> (about <NUM>%) and compared to <NUM>-<NUM>-<NUM> (about <NUM>%), confirming once again the synergistic effect of TCA and hydrogen peroxide to neutralize the negative consequence of the two components administered individually. ROS production is dose dependent since protocol C gives higher results than protocol A but still physiological.

In order to validate the data obtained in vitro, in vivo tests were performed using <NUM> animals (C57BL / 6JOlaHsd mouse) of <NUM> weeks weighing <NUM>-<NUM>. The method allows to compare different preparations with a specific control by monitoring the wound healing area.

The experimental model of wound healing was performed in male mice treated for <NUM> days using <NUM>-<NUM>-<NUM>, <NUM>-<NUM>-<NUM> and <NUM>-<NUM>-<NUM>. As reported in <FIG>, in protocol A, <NUM>-<NUM>-<NUM> induced a greater closure area than control (p < <NUM>) and then compared to <NUM>-<NUM>-<NUM> and <NUM>-<NUM>-<NUM> (<NUM>% and <NUM>% respectively). This effect was also statistically significant (p < <NUM>) in comparison with untreated wounds. In protocol C the presence of <NUM>-<NUM>-<NUM> induced a faster closure in comparison to control (about <NUM>%), <NUM>-<NUM>-<NUM> (about <NUM>%) and <NUM>-<NUM>-<NUM> (about <NUM>%).

Claim 1:
Wound healing compositions in form of cream, ointment, liquid or gel comprising:
- a first component selected from trichloroacetic in a concentration by weight ranging from <NUM> to <NUM>%;
- a second component selected from <NUM>-<NUM>% aqueous hydrogen peroxide in a concentration by weight ranging from <NUM> to <NUM>% or trichloroperoxyacetic acid wherein:
- the relative molar ratio between trichloroacetic acid and hydrogen peroxide is from <NUM> to <NUM>; or
- the relative molecular ratio between trichloroperoxyacetic acid and trichloroacetic acid is from <NUM> to <NUM>;
said compositions having a pH value from <NUM> to <NUM>