Preparations for healing traumatic lesions of the skin and mucous membranes

Abstract

The present invention relates to preparations in the form of creams, ointments, liquids, or gels, and their use for promoting the healing of wounds and ulcers in mucous membranes and skin. The components of these preparations work together to effectively treat various surgical wounds, spontaneous ulcers, post-traumatic wounds, non-healing wounds, ulcers, and bedsores. The preparations of the present invention can promote the repair of wounds and ulcers by themselves, reduce inflammation and swelling, relieve pain and itching, and promote the epithelialization process in cases where repair does not occur despite treatment with antiseptics and fibrinolytic agents.

Description

Technical Field

[0001] The present invention relates to a preparation in the form of a cream, ointment, liquid or gel for use in the repair of surgical wounds of the skin and mucosa. Due to the synergistic effect of the components of the preparation, it enables surprising healing activity in the lesions after odontological and stomatological interventions such as tooth extraction, apicoectomy, maxillary sinus floor elevation, implant, gingivectomy, periodontal surgery, etc., and in the dermatological treatment of delayed wound healing, spontaneous ulcers, pain of various causes and necrotic pain (including pyogenic granuloma, chalazion, epulis, fistula and abscess).

Background Art

[0002] When the integrity of the skin and mucosal surfaces is disrupted, a wound is formed. Wound healing is a complex biological process that restores the integrity of tissues. Physiologically, wound healing can be roughly divided into four different stages: hemostasis, inflammation, proliferation and tissue remodeling. Immediately after injury, the hemostasis process begins: bleeding is controlled by platelet aggregation, followed by the formation of a fibrin thrombus to stop bleeding and provide a scaffold for cell attachment and proliferation. After 2 - 3 days, the inflammatory process progresses to the proliferative phase, and fibroblasts are attracted to the wound to synthesize granulation tissue. With this granulation tissue, white blood cells invade the wound site, and when the wound closes, the immature scar can progress to the final remodeling stage, which can last up to 1 year. The main function of fibroblasts in tissue regeneration is to maintain the physical integrity of connective tissue by producing and remodeling the extracellular matrix. Regardless of the cause of the wound, whether acute or chronic, the above repair process is the same, but acute wounds, such as surgical incisions, usually pass through these stages relatively quickly, but wounds with healing delays of up to 12 weeks from the initial injury, often as a result of long-term pathological inflammation, are defined as chronic wounds.

[0003] Several types of injuries, including those involving biofilm formation, microbial invasion, recurrent trauma, ischemia, edema, venous hypertension, and mechanical forces including pressure, can impair healing. These injuries reduce keratinocyte migration and delay epithelialization. In such slowly progressive cases, wound environment modification remains the standard treatment.

[0004] Effective wound environment adjustments include enzymatic debridement (J. Ramundo et al., J Wound Ostomy Continence Nurs Actions. May-Jun 2008;35(3):273-80; McCarty MS; Adv Wound Care (New Rochelle). 2013 Oct; 2(8): 438-447) and surgical debridement, stimulation of resident fibroblasts and growth factor release (L. Maddaluno et al., Development (2017) 144, 4047-4060), addition of exogenous growth factors to the wound (M. Yadav et al. Indian Journal of Fundamental and Applied Life Sciences (2012), 2 (2) April-June, 164-172), and biotechnology-based extracellular matrix (F. Urciolo et al., J. Clin. Med. 2019, 8, Any combination of the following may be involved: (2083), arrangement of collagen and alginate (BA Aderibigbe et al., Pharmaceutics, 2018, 10(2), 42), cultured keratinocyte suspension, and even bio-engineered dermal preparations (G. Schultz. Wound Repair and Regeneration, April 2003, 11 Suppl 1(s1):S1-28), and stem cell therapy is also promising (N. Kosaric et al., Expert Opinion on Biological Therapy, 19 (6), 2019).

[0005] Known treatment options remain moderately effective, often failing to promote the closure of unhealing wounds in susceptible populations such as the elderly and diabetic patients, or representing limited treatment options in cases of insect or spider bites (Hindawi Publishing Corporation, Case Reports in Emergency Medicine, Volume 2016, Article ID 7640789). Furthermore, some of these approaches, such as bioengineered skin grafts, while effective alternatives to autologous transplantation, induce skin cells in wound repair rather than guiding the regenerative process.

[0006] Similarly, surgical wounds of the oral mucosa following surgical incisions require a long recovery time, even without complications. While it usually takes up to five weeks to restore the oral cavity's original physiological function, anticipated complications can delay complete wound healing. Specifically, the normal healing response to tooth extraction results in significant bone loss and surrounding gum tissue collapse. A considerable percentage of extraction sites experience postoperative complications during normal healing.

[0007] In fact, after tooth extraction, a thrombus fills the socket, and after one week, the thrombus is replaced by granulation tissue; after three weeks, the granulation tissue is replaced by collagen, and bone formation begins at the base and around the socket. At five weeks, it is estimated that, on average, two-thirds of the socket is filled with bone. It has been found that it takes at least three weeks for the epithelium to completely cover the socket, and in some extraction sites, it can take up to five weeks to completely cover the socket (Amler MH et al. J Am Dent Assoc. 1960;61(7), 32-44; Amler MH Oral Surg Oral Med Oral Pathol, 1969, 27 (3), 309-318). Postoperative complications of normal healing according to tooth extraction may include dry socket, paresthesia, severe infection, pain, swelling, trismus, and bleeding. All of these anticipated complications significantly prolong the total healing time due to surgical incision of the oral mucosa.

[0008] Trichloroacetic acid can be used alone or in combination with other compounds as a "chemical peel" (Monheit GD; J. Dermatol. Surg. Oncol. (1989) 15 (9), 945-950). A 50% trichloroacetic acid (TCA) solution is commonly used as a moderate-depth chemical peel and is frequently used to treat fine wrinkles, photoirritation, hyperpigmentation, and even photo-related precancerous changes such as actinic keratosis.

[0009] Chloroacetic acid has been disclosed for the treatment of chronic wounds in combination with solvent-based surfactants and buffers for extracellular macromolecules (WO 2009152374). Preparations containing trichloroacetic acid and hydrogen peroxide in the pH range of 2.3 to 2.6 have also been proposed for the treatment of skin and mucous membrane lesions such as acne, sun damage, and freckles (EP 1979053). However, hydrogen peroxide, known as a mild disinfectant, can be used on the skin to prevent infection of minor cuts, abrasions, and burns, but it has some limitations due to its potentially adverse effects on tissue repair and should not be used for the treatment of deep wounds, animal, insect, or spider bites, or severe burns (Agency for Toxic Substances and Disease Registry, Buford Hwy NE Atlanta - Hydrogen Peroxide).

[0010] More recently, the biological effects of hydrogen peroxide in the wound healing process have been re-examined: at relatively high concentrations (i.e., 3%), hydrogen peroxide exhibits a strong oxidizing and pro-inflammatory ability to disinfect wound tissue (Roy S, et al. Mol Ther 2006;13:211-220); however, at lower concentrations (i.e., 1%), it shows good antibacterial effects and skin resistance (Capizzi R. et al. Br J Dermatol 2004;151:481-484). On the contrary, even though the oxidized form of trichloroacetic acid, namely trichloroperoxyacetic acid, had been confirmed to have disinfectant properties in diluted aqueous solutions against Bacillus subtilis, Escherichia coli, and Staphylococcus aureus in in vitro tests, its use in humans was never considered (Sita, F. et al. Journal of Hygiene, Epidemiology, Microbiology and Immunology (1968), 12(3), 370-3).

[0011] Therefore, innovative pharmacological treatments for surgical wounds of the skin and mucous membranes are needed to avoid the above-mentioned postoperative complications and enable optimal wound environment adjustment. [Overview of the project]

[0012] To solve the above problems, the present invention provides a formulation for repairing wounds of the skin and mucous membranes.

[0013] The wound healing composition of the present invention, hereinafter: - A first component selected from trichloroacetic acid or chloroacetic acid in a weight concentration ranging from 0.08 to 38%; - A second component selected from hydrogen peroxide solution or trichloroperoxyacetic acid in a weight concentration ranging from 0.01 to 5%. Includes.

[0014] The components of these formulations exhibit synergistic effects in the treatment of wounds on the skin and mucous membranes.

[0015] Trichloroacetic acid and hydrogen peroxide are preferably present in a relative molecular ratio of about 4.34 to 4.74. Trichloroperoxyacetic acid and trichloroacetic acid are preferably in a relative molecular ratio of about 3.6 to 4.0. The formulation of the present invention lacks the potential for negative effects of hydrogen peroxide on tissue. Alternatively, instead of trichloroacetic acid and hydrogen peroxide, chloroacetic acid and hydrogen peroxide in the same relative molecular ratio of 4.43 to 4.74 can be used together as components of the above formulation.

[0016] The formulations of the present invention are applied topically in the form of creams, ointments, liquids, gels, or similar dosage forms. These formulations are fast-acting, rapidly reducing swelling and pain, promoting wound healing, and shortening the recovery time of chronic wounds such as chronic ulcers and pressure ulcers. The use of these formulations is also effective in treating postoperative complications of normal healing in response to tooth extraction, including dry socket, paresthesia, severe infection, pain, swelling, trismus, and bleeding, as well as in treating delayed wound healing, spontaneous ulcers, pain of various causes, and necrotizing pain (including pyogenic granulomas, chalazions, epulis, fistulas, and abscesses). [Brief explanation of the drawing]

[0017] [Figure 1] Cell viability of NHEK cells treated with various concentrations of 14-03-01, 14-04-03, and 14-04-05 according to Protocol A. [Figure 2] Cell viability of NHEK cells treated with various concentrations of 14-03-01, 14-04-03, and 14-04-05 according to Protocol B. [Figure 3] Proliferation assay of NHEK cells treated with various concentrations of 14-03-01, 14-04-03, and 14-04-05 according to Protocol A (Panel A) and C (Panel B). [Figure 4] The involvement of Protocols A (left graph) and C (right graph) in NHEK wound closure on 14-03-01, 14-04-03, and 14-04-05. [Figure 5]ROS production by Protocol A (left) and C (right) in NHEK cells. [Figure 6] Graphs of postoperative wound healing measurement and relative wound closure.

[0018] Detailed Description of the Invention The preparation of the present invention is characterized in that the concentration of hydrogen peroxide solution (5 - 31% of 0.01 - 5%, preferably 29 - 31% hydrogen peroxide solution) is significantly lower than that disclosed in the prior art, particularly EP 1979053 (50 - 80% by weight).

[0019] The concentration of trichloroacetic acid is preferably in the range of 0.1 - 35% by weight, more preferably 0.15 - 15% by weight.

[0020] Alternatively, instead of trichloroacetic acid and hydrogen peroxide, chloroacetic acid and hydrogen peroxide with the same relative molecular ratio of 4.43 - 4.74 and a final concentration of 0.08 - 38% by weight can be used.

[0021] The preparation of the present invention can be diluted using a 0.9% w / v aqueous NaCl solution. The diluted preparation helps to ensure the viability of mucosal and skin cells and promotes the repair of surgical wounds, post-traumatic wounds, non-healing wounds, ulcers, and pressure ulcers. The pH value of this preparation is higher than 2.6 and is typically in the range of 2.6 - 3.6. Alternatively, trichloroperacetic acid can also be prepared in situ by adding an equimolar amount of hydrogen peroxide to trichloroacetic acid.

[0022] The liquid preparation of the present invention contains water in the range of 38 to 99% by weight, disodium EDTA salt in the range of 0.001 to 0.5% by weight, glycerol in the range of 0.025 to 15% by weight, aqueous ammonia (28 to 32%) in the range of 0.03 to 14% by weight, a thickening agent and a texturizing agent in the range of 0.005 to 5% by weight, and optionally sodium chloride at 0.8 to 0.9% w / v. Preferred thickening agents and texturizing agents include hydroxyethyl cellulose, guar gum, locust bean gum, xanthan gum, gelatin, and (hydroxyethyl acrylate / sodium acryloyldimethyltaurate) copolymer. More preferred thickening agents and texturizing agents include (hydroxyethyl acrylate / sodium acryloyldimethyltaurate) copolymer (Sepineo TM d.e.r.m). Alternatively, chloroacetic acid and hydrogen peroxide can be used instead of trichloroacetic acid and hydrogen peroxide.

[0023] The ointment preparation before dilution of the present invention also contains water in the range of 5 to 12% by weight, sodium lauryl sulfate in the range of 0.5 to 1.5% by weight, propylene glycol in the range of 10 to 一十五% by weight, stearyl alcohol in the range of 20 to 30% by weight, white petrolatum in the range of 20 to 30% by weight, aqueous ammonia (28 to 32%) in the range of 4 to 6% by weight, and optionally one or more preservatives at a concentration of 0.02 to 0.05%. Preferred preservatives include propyl paraben, methyl paraben, sodium benzoate, and ethylhexyl glycerin. In some cases, the preparation does not contain any preservatives. Alternatively, chloroacetic acid and hydrogen peroxide, or trichloroacetic acid and trichloroperacetic acid can be used instead of trichloroacetic acid and hydrogen peroxide. These preparations can optionally be administered after dilution with an isotonic 0.9% w / v NaCl aqueous solution.

[0024] The undiluted cream formulation of the present invention may contain, in addition to the above-mentioned combinations of trichloroacetic acid and hydrogen peroxide, or trichloroperoxyacetic acid and trichloroacetic acid, 20-25% by weight of water, 5-12% by weight of liquid paraffin, 3-7% by weight of propylene glycol, 8-12% by weight of glycerin, 10-14% by weight of aqueous ammonia (28-32%), and 1-2% by weight of polysorbate 60. Alternatively, chloroacetic acid and hydrogen peroxide, or trichloroacetic acid and trichloroperoxyacetic acid may be used instead of trichloroacetic acid and hydrogen peroxide. These formulations may, if applicable, be administered after dilution with an isotonic 0.9% w / v NaCl aqueous solution.

[0025] The gel formulations of the present invention, before dilution, may contain 25-35% by weight of ethanol, 11-15% by weight of propylene glycol, 10-14% of aqueous ammonia (28-32% w / v), 2-5% by weight of diethylene glycol monoethyl ether, and 1-4% of myristyl alcohol. Alternatively, chloroacetic acid and hydrogen peroxide, or trichloroacetic acid and trichloroperoxyacetic acid may be used instead of trichloroacetic acid and hydrogen peroxide. These formulations may, if applicable, be administered after dilution with an isotonic 0.9% w / v aqueous NaCl solution.

[0026] Experiment section

[0027] [Example 1] The ointment for repairing wounds of the skin and mucous membranes has the following composition:

[0028] [Table 1]

[0029] This concentrated ointment formulation can be diluted with a 0.9% w / v NaCl aqueous solution to obtain a final liquid formulation with a pH of ≥2.6.

[0030] [Example 2] The liquid for repairing wounds of the skin and mucous membranes has the following composition (Formulation 14-03-01: 1 / 400 dilution):

[0031] [Table 2]

[0032] This concentrated solution can be diluted up to 1:400 with a 0.9% w / v NaCl aqueous solution to obtain a final liquid formulation with a pH of ≥2.6.

[0033] [Example 2a] The fluid for repairing wounds of the skin and mucous membranes has the following composition:

[0034] [Table 3]

[0035] This concentrated solution can be diluted with a 0.9% w / v NaCl aqueous solution to obtain a final liquid formulation with a pH of ≥2.6. Alternatively, trichloroperoxyacetic acid can be prepared in situ by adding equimolar amounts of hydrogen peroxide to trichloroacetic acid.

[0036] [Example 3] The cream for repairing wounds of the skin and mucous membranes has the following composition:

[0037] [Table 4]

[0038] This concentrated formulation can be diluted with a 0.9% w / v NaCl aqueous solution to obtain a final liquid formulation with a pH of ≥2.6.

[0039] [Example 4] The gel for repairing wounds of the skin and mucous membranes has the following composition:

[0040] [Table 5]

[0041] This concentrated formulation can be diluted with a 0.9% w / v NaCl aqueous solution to obtain a final liquid formulation with a pH of ≥2.6.

[0042] [Comparative Example 1] The only difference from Example 2 (Formulation 14-03-01) is that the ingredients do not contain hydrogen peroxide.

[0043] [Table 6]

[0044] This concentrated formulation can be diluted 1:400 with a 0.9% w / v NaCl aqueous solution to obtain a final liquid formulation with a pH of ≥2.6.

[0045] [Comparative Example 2] [Table 7]

[0046] This concentrated solution can be diluted 1:400 with a 0.9% w / v NaCl aqueous solution to obtain a final liquid formulation with a pH of ≥2.6.

[0047] Next, liquid formulations 14-03-01 (TCA + H2O2), 14-04-03 (TCA only), and 14-04-05 (H2O2 only) were biologically evaluated in in vivo and in vitro studies. These studies confirmed the synergistic effect of trichloroacetic acid and hydrogen peroxide in promoting the healing of mucous membrane and skin wounds and ulcers, even after dilution up to 400 times with 0.9% NaCl aqueous solution to a pH of ≥2.6.

[0048] Experimental in vitro and in vivo trials Experimental protocol This trial was divided into two phases: the first phase involved verifying the effectiveness of the solution in an in vitro cell model using NHEK cells (normal human epidermal cells); the second phase involved testing the effectiveness of the solution in a mouse animal model that mimicked delayed wound healing in humans. The solutions tested in both phases included the following combinations: 14-03-01 (TCA + H2O2) 14-04-03 (TCA only) 14-04-05 (H2O2 only) These were included, and these effects were compared to an untreated control sample.

[0049] In the in vitro model, the potency of the solution was tested in a dose-response study using NHEK cells, and cell viability was analyzed by the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) test to rule out any cytotoxic effects. Since the combination of substances is not documented in the literature, the test range was based on TCA concentrations reported in the literature [Yang H, et al., Toxicol In Vitro. 2011 Dec;25(8):1638-43.]. Specifically, dilutions of 1:100 (20mM), 1:200 (10mM), 1:400 (5mM), and 1:800 (2.5mM) in sterile physiological solution (0.9% NaCl) were tested. Three different stimulation protocols were selected, as shown below: • Only one treatment, left on for a maximum of 6 days (total of 1 stimulation), Protocol A • Treatment once a day for a maximum of 6 days (5 stimulation sessions in total), Protocol B • Treatment every other day for a maximum of 6 days (total of 3 stimulations), Protocol C.

[0050] The data highlighted the importance of administering the minimum effective dose and appropriate dose; therefore, subsequent experiments tested only 5 mM concentrations using protocols A and C. Subsequently, tests were performed to evaluate cell proliferation (crystal violet assay), migration (wound assay), and reactive oxygen species (ROS) production.

[0051] Migration and re-epithelialization were investigated in an in vivo model using male C57BL / 6JOlaHsd mice, and toxic effects induced by the substances tested in protocols A and C were excluded. Before administration, the right side of the mouse spine was depilated. The depilated area was approximately 2 × 2 cm. The pressure ulcer model simulated the natural process of pressure ulcer formation by applying pressure to the depilated area through intermittent mechanical extrusion.

[0052] For more details, 14-03-01 (TCA + H2O2) 14-04-03 (TCA only) 14-04-05 (H2O2 only) Skin wounds were treated with a 1:400 dilution prepared in sterile physiological solution (0.9% NaCl). At the end of each treatment, wound area measurements were performed, and wound tissue was collected for histological analysis using E / E (hematoxylin / eosin) and Masson staining.

[0053] Method: In vitro model cell culture Normal human epidermal keratinocytes (NHEKs) purchased from Lonza (Basel, Switzerland) were treated with 60 μM Ca 2+ Cells were cultured in EpiLife medium (Gibco, Thermo Fisher Scientific) containing HKGS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in a 5% CO2 incubator (Thermo Fisher Scientific) at 37°C. The medium was changed every two days, and the cells were used at 70%–80% confluence [Jia, T et al., Molecules 2019, 24, 3156]. Experiments were conducted at passages 3–6.

[0054] Cell viability After each stimulus, NHEK cells were washed once with sterile PBS and incubated with red phenol-free DMEM (Dulbeccoo's Modified Eagle Medium) and FBS (fetal bovine serum) containing 1% MTT dye (MTT-Based In Vitro Toxicology Assay Kit; Sigma-Aldrich) at 37°C and 5% CO2 for 2 hours [Uberti, F et al., J Cardiovasc Pharmacol 57: 246-258.]. Cell viability was determined by measuring the absorbance at 570 nm corrected for 690 nm using a spectrometer (VICTORX4 multi-label plate reader), and the results were calculated by comparing them with control cells (100% viable).

[0055] Crystal Violet After each treatment, cells were fixed with 1% glutaraldehyde (Sigma-Aldrich) at room temperature for 15 minutes, washed, and stained with 100 μL of 0.1% crystal violet aqueous solution (Sigma-Aldrich) at room temperature for 20 minutes. 100 μL of 10% acetic acid was added to a multiwell plate, and after mixing, the absorbance at 595 nm was read using a spectrometer (VICTORX4 multilabel plate reader). The estimated number of cells was calculated by comparing the results with control cells (control T0) tested in the first treatment, and variability in untreated cells was also reported [Uberti F, Cells Tissues Organs. 2017;203(4):215-230. Epub 2016 Nov 25.].

[0056] In vitro wound assay Scratch healing assays were performed in confluent monolayer cells using sterile p200 pipette tips, as previously described [Uberti F, et al., Cells Tissues Organs. 2017;203(4):215-230. Epub 2016 Nov 25.]. Cells were then stimulated with different preparations according to different protocols (A and C) and monitored for 6 days. After each time point, regrowth of the wound area was observed using a phase-contrast microscope (Leitz, Germany). The size of the exposed area at each time point was determined from digital images taken in six different regions using the ImageJ image processing program. Results are expressed as the mean ± SD (%) of the number of migrating cells.

[0057] ROS production The release rate of superoxide anions was measured using a standard protocol based on cytochrome C reduction. 100 μL of cytochrome C was added to both treated and untreated cells, and 100 μL of superoxide dismutase was also added to a separate sample in an incubator for 30 minutes (all materials were obtained from Sigma-Aldrich). The absorbance of the culture supernatant was measured at 550 nm using a spectrometer (VICTORX4 multi-label plate reader), and O2 was expressed as the mean ± SD (%) of nanomoles per microgram of reduced cytochrome C per microgram of protein compared to the control [Uberti F, et al., Cells Tissues Organs. 2017;203(4):215-230. Epub 2016 Nov 25].

[0058] Results of in vitro trials Dose-response study of cell viability in NHEK cells. Dose-response studies were conducted to determine the optimal concentration within the range described in the literature. In these studies, mitochondrial metabolism could be determined by analyzing the administration protocol. As shown in Figure 1, protocol A showed a dose-response effect for all substances, peaking around day 3 of stimulation; this effect was stable only on 14-03-01, and the main effect was observed at 5 mM (1:400 dilution). The same test was also performed using Protocol B (Figure 2), in which the test composition was administered once daily for 6 days. In this example, the best results were obtained for all test solutions at 5 mM (p<0.05). Considering that the results were "lower" than those observed in Protocol A, the daily stimulation is considered excessive but not toxic. The plateau phase showed a slight downward trend, indicating that Protocol B was excessive, and therefore, in subsequent experiments, Protocol B was replaced with Protocol C (administered once every other day for a maximum of 6 days).

[0059] Growth analysis The proliferation assay was performed using crystal violet on NHEK cells treated with the substance according to protocols A and C. As shown in Figure 3, 14-03-01 significantly increased cell proliferation compared to the other compositions (p<0.05), specifically by 2 times compared to 14-04-03 and by approximately 33% compared to 14-04-05. Furthermore, while these effects were evident in both protocols, the main effect was obtained by protocol C (p<0.05 compared to A). This is important because increasing the number of cells may also allow them to migrate; furthermore, these data suggested that the activity of 14-03-01 was higher than that of the individual components (p<0.05).

[0060] Effects of 14-03-01, 14-04-03, and 14-04-05 on NHEK migration The effects of various formulations on NHEK migration during wound healing were evaluated (Figure 4). Our results demonstrated improved migratory activity of NHEK cells treated with 14-03-01 (both protocols A and C) compared to the control (p<0.05), and compared to 14-04-03 (approximately 3 times) and 14-04-05 (approximately 60%). This is an effective method for characterizing the various factors involved in migration. The data support observations made during proliferation analysis.

[0061] Effect of the formulation on ROS production Because conflicting data have been reported in the literature regarding the role of ROS in healing, further experiments were conducted to investigate the role of ROS in this regard. As shown in Figure 5, despite the presence of H2O2, ROS production does not increase beyond the physiological occupancy of the test substance; this is undoubtedly important to ensure the efficacy observed in previous studies. Clearly, compounds containing hydrogen peroxide have higher ROS production than the other substances in both protocols, but 14-03-01 remains below the physiological level of ROS compared to the control, and similarly compared to 14-04-03 (approximately 35%) and 14-04-05 (approximately 23%), reaffirming the synergistic effect of TCA and hydrogen peroxide neutralizing the negative results of the two components administered individually. Protocol C yields higher results than protocol A, but is still at a physiological level, indicating that ROS production is dose-dependent.

[0062] Methods and results of in vivo trials To validate the data obtained in vitro, an in vivo study was conducted using 60 eight-week-old animals (C57BL / 6JOlaHsd mice) weighing 20-25g. This method allows for the comparison of various formulations with a specific control by monitoring the wound healing area.

[0063] Evaluation in the field of wound healing The wound healing experimental model was performed on male mice treated for 8 days with 14-03-01, 14-04-03, and 14-04-05. As reported in Figure 6, in protocol A, 14-03-01 induced a larger closure area than the control (p<0.05), then compared to 14-04-03 and 14-04-05 (14% and 33%, respectively). This effect was also statistically significant compared to untreated wounds (p<0.05). In protocol C, the presence of 14-03-01 induced faster closure compared to the control (approximately 98%), 14-04-03 (approximately 2%), and 14-04-05 (approximately 4%).

[0064] These findings were confirmed by morphological analysis using E / E and Masson analysis, and formulation 14-03-01 was considered the better choice. In all treatments of protocol A, the granulation tissue still appeared to be very active, and healing was in the early stages. Specifically, 14-03-01 showed a very clear ability to repair wounds, and re-epithelialization was almost complete. In contrast, in protocol C, mice treated with 14-03-01 showed complete epithelialization of the wound compared to the control, 14-04-03, and 14-04-05, respectively. Epithelialization appeared without ulceration, with the presence of inactive granulation tissue, collagen reconstruction, and angiogenesis; therefore, restoration to the original state (restitutio ad integrum) can be assumed.

Claims

1. Compositions for wound healing in the form of creams, ointments, liquids, or gels, the following: - A first component, trichloroacetic acid, in a weight concentration ranging from 0.08% to 38%; - Hydrogen peroxide solution at a weight concentration in the range of 0.01 to 5%, with a weight concentration of 29 to 31% or Trichloroperoxyacetic acid A second component selected from Includes, Here, If the second component is hydrogen peroxide, the molar ratio of trichloroacetic acid to hydrogen peroxide in the composition is 4.34 to 4.74:1, or When the second component is trichloroperoxyacetic acid, the molar ratio of trichloroperoxyacetic acid to trichloroacetic acid in the composition is 3.6 to 4.0:1; A composition having a pH value of 2.6 to 3.

6.

2. The composition according to claim 1, in the form of a cream containing 20-25% by weight of water, 5-12% by weight of liquid paraffin, 3-7% by weight of propylene glycol, 8-12% by weight of glycerin, 10-14% by weight of aqueous ammonia (28-32% by weight), and 1-2% by weight of polysorbate 60.

3. The composition according to claim 1, in the form of an ointment containing 5 to 12% by weight of water, 0.5 to 1.5% by weight of sodium lauryl sulfate, 10 to 15% by weight of propylene glycol, 20 to 30% by weight of stearyl alcohol, 20 to 30% by weight of white petrolatum, and 4 to 6% by weight of aqueous ammonia (28 to 32%).

4. The composition according to claim 3, further comprising one or more preservatives in a concentration of 0.02 to 0.05%, wherein the preservatives are selected from the group consisting of propylparaben, methylparaben, sodium benzoate, and ethylhexylglycerin.

5. The composition according to claim 1, in the form of a liquid containing 38 to 99% by weight of water, 0.001 to 0.5% by weight of EDTA disodium salt, 0.025 to 15% by weight of glycerol, 0.03 to 14% by weight of aqueous ammonia (28 to 32%), and 0.005 to 5% of a thickening agent and a texturer.

6. The composition according to claim 1, in the form of a gel containing 25-35% by weight of ethanol, 11-15% by weight of propylene glycol, 10-14% by weight of aqueous ammonia (28-32% w / v), 2-5% by weight of diethylene glycol monoethyl ether, and 1-4% by weight of myristyl alcohol.

7. The composition according to claim 1, diluted with a 0.9% NaCl aqueous solution.

8. The composition according to claim 1, wherein trichloroperoxyacetic acid is prepared in situ by adding hydrogen peroxide to trichloroacetic acid.

9. The composition according to any one of claims 1 to 8, wherein wound healing includes the treatment of unhealed wounds, ulcers and pressure ulcers, treatment of postoperative complications of tooth extraction including dry socket, paresthesia, severe infection, pain, swelling, trismus and bleeding, treatment of delayed wound healing, spontaneous ulcers, pain of various causes and necrotizing pain (including suppurative granuloma, chalazion, epulis, fistula and abscess).

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