Pharmaceutical Composition for Topical Wound Treatment

Abstract

A pharmaceutical composition for topical wound treatment, comprising one or more nitrogen-containing heterocyclic compounds having five or six atoms and an imide group, one or more deoxyribonuclease enzymes having an active pH between 4.5 and 6.5, and one or more carboxylic acids; a kit and method for obtaining the pharmaceutical composition, and its use for wound treatment.

Description

[Technical field]

[0001] The present invention relates to topical pharmaceutical compositions for treating wounds. The present invention also relates to the field of topical pharmaceutical compositions and formulations useful in the treatment of chronic wounds. [Background technology]

[0002] Chronic wounds, understood as those that do not heal in a series of ordered stages and predictable times, such as venous ulcers, diabetic foot ulcers, pressure sores, decubitus ulcers, ischemic ulcers, and atypical lesions, are non-healing injuries (1). They affect approximately 2% of the world's population (2) and represent a major economic problem for the health care system (3). Chronic wounds are maintained in a chronic inflammatory state that evades healing (4), but there are multiple contributing factors to their failure to heal (5). Chronic wounds have been actively studied by scientists seeking to provide therapeutic solutions, resulting in many products. However, to date, there is no comprehensive and effective treatment for these types of wounds.

[0003] There are many products on the market for chronic wounds. Doctors usually use a combination of treatments for wound infection, debridement and wound care (6). These types of products do not guarantee healing, and doctors are often forced to use skin substitutes (7), negative pressure wound treatments (8), growth factors (9) and even hyperbaric therapy (10). However, they have limited therapeutic efficacy because none of them can simultaneously meet all the treatment requirements (11).

[0004] Among the products that may be mentioned in wound infections, 1) silver has a broad spectrum of activity and is available and soluble in many forms. However, it is necessary to continuously release silver in high enough concentrations to maintain efficacy. For example, silver nitrate must be administered 12 times a day to work properly (12). A recent survey also found no convincing evidence that silver sulfadiazine has an overall effect on wound healing, despite its widespread use among practitioners (13). 2) Iodine-containing compounds (i.e., cadexomer iodine in a starch lattice formed into microbeads) have been used in wound healing for many years, but there are concerns about the toxicity of iodine-containing compounds, especially in large wounds (14, 15). 3) Topical preparations of antibiotics have also been developed to apply to the wound site. However, recent surveys of the routine administration of antibiotic ointments have shown that they do not have better outcomes and often result in patient discomfort, as well as the possibility of antibiotic resistance and contact dermatitis (16-18).

[0005] Most pathogens in chronic wounds form biofilms that adhere to each other, forming a dense mass that surrounds a polymer matrix, allowing them to avoid destruction by antibiotics and the immune system. This creates a physical barrier to wound healing, and can prolong the normal resolution of the inflammatory phase, making biofilms a major challenge in wound healing. However, there is no anti-biofilm product with bacterial broadness for chronic wounds. In fact, to be effective in chronic wounds, a product should simultaneously exhibit bacteriostatic, bactericidal, biofilm-inhibiting and biofilm-disintegrating properties.

[0006] Debridement (19) involves the removal of non-viable tissue material to expose healthy, well-perfused tissue that is capable of growing and congesting the wound bed through epithelial cell migration, rather than maintaining necrotic debris that serves only as fuel for infection and impedes wound healing. Surgical debridement is an important component of wound care, but it has all the disadvantages that surgical procedures have, such as infection, pain, expense, and patient discomfort. Autolytic debridement refers to the autoactivity of endogenous enzymes involved in fibrin degradation that occurs in moist wound environments and is found in some types of wound dressings. However, it cannot function as an adequate substitute for surgical debridement, since it does not remove any devitalized tissue. Thus, there is no product that allows for precise debridement without invasive procedures and patient discomfort.

[0007] Wound care dressings (20) are very important in the management of chronic wounds considering the morbidity associated with them. However, all wound care dressings are not active products (cannot promote the healing process) and serve only to protect the wound while the healing process is proceeding. Also, none of them can be used on any type of chronic wound due to the following problems: 1) A limited amount of exudate is retained on the wound allowing for autolytic debridement which acts to further promote normal wound healing. Traditional dry gauze wound dressings (i.e. Curity, Vaseline gauze, Xeroform) may slow down this process while causing further injury if removed. 2) Low adhesion dressings and semi-permeable films (i.e. Bioclusive, Blisterfilm, Cutifilm, Flexigrid, OpSite, Tegaderm) are the basic types of wound dressings that aim to inhibit the penetration of liquids and microorganisms, but allow air and water vapor to pass through. Therefore, they can only be used on non-exuding wounds, which are among the most common. 3) Hydrocolloids (i.e., Aquacel, Comfeel, DuoDERM, Granuflex, Tegasorb) can absorb a certain amount of exudate to maintain a moist environment. However, they are impermeable and therefore should not be used on exuding wounds, as they prevent air exchange. They also require long times to change. 4) Hydrogels (i.e., Carrasyn, Curagel, Nu-Gel, Purilon, Restore, SAF-gel, XCell) can be used additionally to help promote moisturization in otherwise dry wounds. They are easily removed and changed, but can cause overhydration, which can slow healing. 5) Alginate dressings (i.e., Algisite, Kaltostat, Sorbsan, Tegagen) are nonwoven fibers derived from seaweed that are generally indicated for highly exuding wounds, as they can absorb large amounts of fluid. Therefore, adverse effects can be seen in dry wounds that are dressed with alginate dressings, for example, they can have a hemostatic effect (21). 6) Hydrofiber (i.e. Aquacel Hydrofiber), which has similar properties and adverse effects as alginate dressings.7) Foams (i.e. 3M Adhesive Foam, Allevyn, Lyofoam, Tielle) minimize trauma during dressing changes, but have limited absorptive capacity and can only be used on moderately exudative wounds. 8) There are combinations of traditional wound dressings (Kendall AMD) such as foams or hydrogels containing antimicrobial compounds such as silver, betaine, chitin or polyhexamethylene biguanide. These materials are not suitable for widespread application to wound healing, but may be suitable for chronic leg ulcers where infection can be a problem, especially with the formation of biofilms. However, they cannot bring about biofilm disruption, which is the true therapeutic requirement, although they can inhibit biofilm formation. 9) Collagen products: have been used for difficult-to-heal wounds and chronic ulcers. They are thought to help promote an environment that attracts cell types essential for wound healing while depleting negative effectors such as free radicals and proteases (22). However, this collagen is not a direct substitute for new collagen production in wound tissue (as it can be derived from a number of sources, including bovine and porcine collagen), and they are not useful for infected wounds.

[0008] Skin substitutes (7) generally consist of a combination of biomaterials and materials that allow their substitution on the wound. Overall, these dressings are quite difficult to adopt widely, since they are extremely expensive. Also, they have a high risk of infection and antigenicity. Several skin substitutes are commercially available. 1) Many skin substitutes are centered on mesh materials coated with porcine collagen or polypeptides (Biobrane and TransCyte), the latter of which also contain neofibroblasts or porcine xenografts (EZ Derm). However, they are only cost-effective for facial burns and not for general use. 2) Dermagraft is a material developed using fibroblasts derived from neoforeskin tissue, extracellular matrix and a bioabsorbable polyglactin mesh. However, it showed a high risk of rejection and hypersensitivity (thought to be due to traces of bovine serum in the formulation). 3) Apligraft, an allogeneic bilayered cultured skin equivalent that combines a dermal layer of cultured fibroblasts and bovine type I collagen with an epidermal layer of cultured keratinocytes. However, this is expensive, costing over US$1000 for a single application of Apligraft for a 7.5 cm diameter disc, and may only be cost-effective for refractory, chronic, and non-healing wounds, somewhat limiting the overall availability of these products. 4) Omnigraft, an acellular bilayered matrix composed of collagen, glycosaminoglycans, and chondroitin-6-sulfate with a layer of silicone for barrier function. However, Omnigraft should not be used in patients with known sensitivity to bovine collagen or chondroitin materials, and should never be used on clinically diagnosed infected wounds.

[0009] Negative Pressure Wound Dressing (VAC) by maintaining a moist environment, optimizing blood flow, removing exudate, and applying pressure to promote wound closure (8). These devices can alleviate many of the deficiencies that can occur in chronic wounds. However, negative pressure dressings can be dangerous if placed in direct contact with exposed blood vessels, bone, or nerves. They are also contraindicated if osteomyelitis is present or if the patient has an allergy or sensitivity to acrylic adhesives.

[0010] Growth factors (9). Chronic wounds contain so many perturbations in growth factors and cytokines that it would be beneficial to address some of these issues. However, in an environment where so many factors are scarce and disordered, simply replacing one is not enough to repair the chronic wound phenotype. In fact, the only such treatment shown to improve healing in a double-blind randomized controlled trial was platelet-derived growth factor (PDGF) (regranex becaplermin), and the results were rather poor.

[0011] Although wound oxygenation is one of the most important therapeutic targets for chronic wounds, there are no products available that increase angiogenesis by increasing vascular endothelial growth factor (VEGF) in situ.

[0012] Hyperbaric oxygen (10) has been used in wound healing based on the principle that it can promote fibroblast proliferation, enhance immune function, stimulate angiogenesis, etc. However, these ideals have not always been proven in practice, leading to some degree of controversy over its use. Importantly, this treatment is administered to patients in a hyperbaric oxygen chamber, where localized delivery of oxygen has not been proven effective and can produce serious side effects, including myopia, oxygen toxicity in the brain resulting in seizures, and pneumothorax.

[0013] It is possible to find several invention patents, such as Argentine Patent No. 093779, in which a specific approach is taught, some of whose inventors are the same as the inventors of the present invention. This application teaches a Lactobacillus enzyme supernatant proposed as being effective in the treatment of chronic wounds (23-25). In this document, said supernatant is called "LAPS". The technical problem with this supernatant is that it is a fermentation supernatant, and therefore contains an uncertain and variable composition depending on the manufacturing conditions. There is also evidence that it has limited stability over time, its therapeutic function not exceeding 60 days.

[0014] EP 0848951 discloses the use of N-acetylcysteine ​​(NAC) for preparing a composition for treating or preventing chronic wounds selected from the group consisting of diabetic foot ulcers and bedsores, the composition being in the form of an ointment for topical administration to chronic ulcers, the ointment comprising 1) 0.01-1% w / w NAC, or a pharma- ceutically acceptable salt or derivative thereof, and 2) a carrier: a hydrogel containing cellulose (i.e., hydroxyethylcellulose), a hydrogel of polyacrylic acid (i.e., carbopol), or a cream / ointment used in pharmaceutical formulations (i.e., cetomacrogol). The carrier may contain alginate as a thickening or stimulating agent, a preservative (i.e., benzyl alcohol), a pH-controlling buffer (i.e., Na2HPO4 / NaH2PO4), an agent for adjusting osmolality (i.e., NaCl), and a stabilizer (i.e., EDTA). The claimed composition for treating chronic wounds based on NAC exhibits a healing-promoting effect by inhibiting MMPs. However, in their procedure, NAC demonstrated a concentration-dependent inhibition of intercellular adhesion molecule-1 (ICAM-1) expression, which is not desirable for chronic wound treatment, since it has been shown that the absence of ICAM-1 delays wound healing (26).

[0015] US Patent No. 9,211,305 (B2) teaches a composition of glycosaminoglycans for the treatment of diabetic foot ulcers, and the patent relates to low molecular weight heparin (LMWH) and very low molecular weight heparin (VLMWH) in the manufacture of a medicinal product for the treatment of chronic ulcers, particularly diabetic foot ulcers, more particularly chronic ulcers, particularly diabetic foot ulcers and pressure ulcers. In particular, the patent claims a method for treating chronic wounds, but the treatment does not provide antimicrobial properties (bacteriostatic, bactericidal and antibiofilm), nor antioxidant or angiogenic properties. Therefore, it must be used in combination with other products.

[0016] The antimicrobial peptides of U.S. Patent No. 10,285,938 are a relatively new discovery in the immune system pathway. Recent designs of synthetically produced antimicrobial peptides have demonstrated increased potency and efficacy / resistance, enhanced specificity, and reduced relative toxicity. One such peptide, XYLENTRA®, has shown significant promise from significant in vitro testing against multiple pathogens. Furthermore, extensive animal testing has demonstrated that XYLENTRA® is an antimicrobial peptide against multiple pathogens. XYLENTRA® peptides are also solute resistant. The peptide XYLENTRA® shows significant antibacterial activity against the test organisms Staphylococcus aureus MTCC96 and Pseudomonas aeruginosa MTCC741. Substantial reductions in the levels of microbial populations have been observed in animals treated with the peptides using the protocols detailed in that application. Specifically, a wound treatment method using said antimicrobial peptides is claimed. However, this method does not envisage healing-promoting properties such as anti-oxidation, MMP inhibition and angiogenesis. Also, with regard to their antimicrobial properties, they have not demonstrated an effect on biofilms (inhibition and / or destruction). Therefore, it must be used in combination with other products.

[0017] WO 2019 / 193333 relates to a pharmaceutical composition comprising triclosan (2,4,4-trichlorohydroxydiphenyl ether) and a thickening agent for use in the treatment of chronic wounds, in particular diabetic foot ulcers. In particular, it relates to a topical composition comprising antimicrobial properties related to triclosan. Active pretreatment ingredients: Castor oil, Jojoba oil, Aloe vera 10:1, Active antimicrobial ingredients: Triclosan, Emulsifiers: Stearic acid, GMSSE, Cetyl palmitate, Silicone fluid 200 100 CS, Heavy liquid paraffin, Monopropylene glycol. Gelling agents: Carbopol 980 5%, Triethanolamine, Optional preservatives: Nipastat. However, this composition does not envisage healing-promoting properties such as antioxidant, MMP inhibition and angiogenesis etc. Also, with regard to their antimicrobial properties, they have not demonstrated an effect on biofilms (inhibition and / or disruption). Therefore, it must be used in combination with other products.

[0018] Many authors are convinced that targeting oxidative stress is an important factor in the treatment of impaired wound healing (27-29). In this regard, there are two types of antioxidants that can be applied in vivo: antioxidant enzymes (e.g., SOD, catalase, peroxidase, and glutathione peroxidase) and nonenzymatic antioxidants (e.g., vitamin C, nitric oxide, and metal-binding proteins, etc.) (27). However, most clinical trials with antioxidants have been limited to oral administration. For example, the effect of orally supplemented ascorbic acid on healing has been studied (28, 29). On the other hand, topical application of antioxidants from plant extracts or enzymatic antioxidants in hydrogels has been proposed for the treatment of chronic wounds (27). The only reported clinical trial of topical application of nonenzymatic antioxidants to chronic wounds was carried out with a cream containing 5% DMSO, a potent reactive oxygen species (ROS) scavenger, and this treatment failed to reduce the occurrence of ulcers (31). Also, a detrimental effect on the development of heel and ankle lesions was observed. The authors proposed several hypotheses to explain this unexpected result, in particular mentioning the possible prooxidative activity of DMSO (31). At physiological concentrations, DMSO and ascorbic acid are potent free radical scavengers in plasma and protect cells from oxidative damage caused by ROS (32).

[0019] Ajwee DM et al. (2021) teach that ethosuximide and phenobarbital promote wound healing by enhancing collagenization (33). They applied ethosuximide (Etho) to excision wounds in an albino rat model. They demonstrate that an ointment containing 10% w / w (80 mg / mL) ethosuximide in soft paraffin significantly promotes wound healing by enhancing collagenization. They applied 150 mg of ointment per day. That means 15 mg of Etho per day. Considering that albino rats weigh 140-180 g and that the standard blood volume for these animals is 50 mL per kilogram, these rats had an approximate blood volume of 10 mL. If 100% of the Etho applied per day was transferred to the bloodstream, the treatment Etho on day 1 would have a plasma concentration of 15 mg / 10 mL. That means 1.5mg / mL or 1500ug / mL. In humans, the acceptable therapeutic level is 40-100μg / mL (see Zarontin prospect FDA). Above this concentration, liver toxicity, leukopenia, thrombocytopenia, sedation and nausea are seen. Also, this composition did not show antimicrobial properties (bacteriostatic, bactericidal and antibiofilm). Therefore, it must be combined with other products for wound treatment.

[0020] In their study, Goldberg SR et al. (2020) suggested that there are five key points at which a wound becomes chronic (34): 1) Ischemia. Ischemia results in tissue injury, necrosis, and the development of open wounds that can rapidly become colonized by bacteria. 2) Infection. Infection sets the stage for chronic and uncontrolled inflammation. Most chronic wounds are polymicrobial in nature, containing primarily Staphylococcus and Pseudomonas species. 3) Biofilm formation. The proinflammatory response is sustained by the formation of biofilms that protect and defend the bacteria and the site of the inflamed ulcer. Biofilms stimulate the host immune response while directly resisting antimicrobial therapy and stimulating chronic inflammation. 4) Chronic inflammation and tissue damage. When host inflammatory cells attempt to remove damaged tissue, reactive oxygen species and proteases such as MMPs are released, causing further tissue damage. Overproduction of MMPs causes damage to the extracellular matrix, inducing the pathology underlying chronic non-healing wounds. Overproduction of MMPs also destroys important growth factors such as platelet-derived growth factor and transforming growth factor-b, which are necessary for wound healing. 5) Decreased mitogenic activity. Due to the disruption of the inflammatory environment, the remaining connective tissue cells have reduced mitogenic activity and become senescent.

[0021] Goldberg et al. suggest that this vicious cycle of inflammation and tissue destruction will continue until aggressive clinical techniques are used to remove bacteria, as well as damaged and necrotic tissue, and reduce inflammation. They also suggest that, given the current state of technology, the best approach to control patients' wounds is the DIME Assisted Products and Services Wound Care Guidelines (35). This guideline consists of a comprehensive approach to assessing wounds and recognizing all the products and services that are needed to be used simultaneously to enhance treatment effectiveness. As seen in this guideline, there are no products on the market that have the ability to simultaneously meet the five key points suggested by Goldberg (35).

[0022] Therefore, current treatments usually use a combination of treatments for wound infection, debridement and wound care. These kinds of products do not guarantee healing, and doctors often have to repeat the use of skin substitutes, negative pressure wounds, growth factors and hyperbaric treatments. However, none of them can meet all the above treatment requirements at the same time, and therefore still only show relative therapeutic efficacy.

[0023] Experts suggest that treatment will be more effective if it simultaneously includes all of the following therapeutic requirements (36): a) control of infection and elimination of biofilms (37, 38); b) control of the amount and composition of exudate (39), c) reconstitution of specific growth factors (9); d) Control of inflammation (4) and its deleterious effects, such as the accumulation of matrix metalloproteinases (40) and oxidative stress (29); and e) Pain management (41). Summary of the Invention [Means for solving the problem]

[0024] The present invention solves the problems presented by the current art by providing a composition for treating chronic wounds that simultaneously provides angiogenesis, broad spectrum bacterial bacteriostasis, bactericidal activity, biofilm inhibition and disruption (including S. aureus and P. aeruginosa, the bacteria most commonly isolated in chronic wounds), inhibition of matrix metalloproteinases (MMPs) (due to its acidifying and chelating properties), collagen and alpha-actin formation in the wound bed, and pro-healing properties.

[0025] The present invention also provides compositions for treating chronic wounds that unexpectedly exhibit functional synergy resulting from the combination of its components.

[0026] The present invention provides compositions and formulations containing pharmacologically used components which, in combination, constitute a solution to the problem of therapeutic treatment of chronic wounds. The present invention solves the problems posed by the current art.

[0027] The present invention aims to provide a composition for treating wounds.

[0028] It is also an object of the present invention to provide a composition for treating chronic wounds.

[0029] The present invention also aims to provide a composition for the treatment of chronic wounds, including venous ulcers, arterial ulcers, pressure ulcers (bed sores), diabetic foot ulcers, and mechanical or post-operative wounds that have become chronic due to superinfection with biofilm-forming bacteria.

[0030] It is also an object of the present invention to provide a formulation for in situ preparation for local delivery.

[0031] It is also an object of the present invention to provide an in situ prepared gel formulation for the treatment of wounds.

[0032] The present invention also aims to provide a method for making the composition.

[0033] The present invention also aims to provide a device that enables the use of an in situ prepared gel formulation for the treatment of wounds.

[0034] The present invention also aims to provide a method for treating mammals having venous ulcers, arterial ulcers, pressure ulcers (bed sores), diabetic foot ulcers, and mechanical or post-operative wounds that have become chronic due to superinfection with biofilm-forming bacteria. [Brief description of the drawings]

[0035] [Figure 1] FIG. 1 illustrates a device containing a formulation of the present invention, showing three containers separated by two collapsible membranes that allow for separation of each composition. When the membranes are ruptured, the compositions of the containers are mixed. [Diagram 2] Figure 1 shows the antipathogenic properties tested against P. aeruginosa, i.e. bacteriostatic activity, inhibition of biofilm (BF) formation and percentage of disruption of preformed biofilm, caused by compositions M0 and M13 and by the individual molecules at the concentrations present in the mentioned compositions. Etho = ethosuximide, AA = ascorbic acid, LA = lactic acid, PS80 = polysorbate 80, D1 = DNAse 1, MS = mixed solvent, LAPS = Lactobacillus plantarum supernatant, M = mixture. [Diagram 3]Figure 1 shows the bactericidal effect expressed on viable bacteria (CFU / mL) of Pseudomonas aeruginosa after 24 hours of treatment with compositions M0 and M13 and the individual molecules at the concentrations present in the mentioned compositions. Etho = ethosuximide, AA = ascorbic acid, LA = lactic acid, PS80 = polysorbate 80, D1 = DNAse 1, LAPS = Lactobacillus plantarum supernatant, M = mixture, MS = mixed solvent. [Figure 4] Figure 1 shows the different antipathogenic effects caused by the individual molecules at the concentrations present in composition M15 and the mentioned compositions, namely bacteriostatic activity against Pseudomonas aeruginosa, inhibition of biofilm (BF) formation and percentage of disruption of preformed biofilm. Etho = ethosuximide, AA = ascorbic acid, LA = lactic acid, PS80 = polysorbate 80, D1 = DNAse1, LAPS = Lactobacillus plantarum supernatant, M = mixture, MS = mixed solvent. [Diagram 5] Figure 1 shows the bactericidal effect expressed on viable bacteria (CFU / mL) of Pseudomonas aeruginosa after 24 hours of treatment with composition M15 and the individual molecules at the concentrations present in the mentioned compositions. Etho = ethosuximide, AA = ascorbic acid, LA = lactic acid, PS80 = polysorbate 80, D1 = DNAse 1, LAPS = Lactobacillus plantarum supernatant, M = mixture, MS = mixed solvent. [Figure 6]Figure 1 shows the different antipathogenic effects caused by the individual molecules at the concentrations present in composition M16 and the mentioned compositions, namely bacteriostatic activity against Pseudomonas aeruginosa, inhibition of biofilm (BF) formation and percentage of disruption of preformed biofilm. Etho = ethosuximide, AA = ascorbic acid, LA = lactic acid, PS80 = polysorbate 80, D1 = DNAse1, LAPS = Lactobacillus plantarum supernatant, M = mixture, MS = mixed solvent. [Figure 7] Figure 1 shows the bactericidal effect expressed on viable bacteria (CFU / mL) of Pseudomonas aeruginosa after 24 hours of treatment with composition M16 and the individual molecules at the concentrations present in the mentioned compositions. Etho = ethosuximide, AA = ascorbic acid, LA = lactic acid, PS80 = polysorbate 80, D1 = DNAse 1, LAPS = Lactobacillus plantarum supernatant, M = mixture, MS = mixed solvent. [Figure 8] Figure 1 shows the antipathogenic properties tested against Staphylococcus aureus (S. aureus), i.e. bacteriostatic activity, inhibition of biofilm (BF) formation and percentage of disruption of preformed biofilm, caused by compositions M0 and M13 and by the individual molecules at the concentrations present in the mentioned compositions. Etho = ethosuximide, AA = ascorbic acid, LA = lactic acid, PS80 = polysorbate 80, D1 = DNAse 1, LAPS = Lactobacillus plantarum supernatant, M = mixture, MS = mixed solvent. [Figure 9]Figure 1 shows the bactericidal effect expressed on viable bacteria (CFU / mL) of S. aureus after 24 hours of treatment with compositions M0 and M13 and with the individual molecules at the concentrations present in the mentioned compositions. Etho = ethosuximide, AA = ascorbic acid, LA = lactic acid, PS80 = polysorbate 80, D1 = DNAse 1, LAPS = Lactobacillus plantarum supernatant, M = mixture, MS = mixed solvent. [Figure 10] Figure 1 shows the different antipathogenic effects caused by the individual molecules at the concentrations present in composition M15 and the mentioned compositions, namely bacteriostatic activity against S. aureus, inhibition of biofilm (BF) formation and percentage of disruption of preformed biofilm. Etho = ethosuximide, AA = ascorbic acid, LA = lactic acid, PS80 = polysorbate 80, D1 = DNAse1, LAPS = Lactobacillus plantarum supernatant, M = mixture, MS = mixed solvent. [Figure 11] Figure 1 shows the bactericidal effect expressed on viable bacteria (CFU / mL) of S. aureus after 24 hours of treatment with composition M15 and the individual molecules at the concentrations present in the mentioned compositions. Etho = ethosuximide, AA = ascorbic acid, LA = lactic acid, PS80 = polysorbate 80, D1 = DNAse 1, LAPS = Lactobacillus plantarum supernatant, M = mixture, MS = mixed solvent. [Figure 12] Figure 1 shows the different antipathogenic effects caused by the individual molecules at the concentrations present in composition M16 and the mentioned compositions, namely bacteriostatic activity against S. aureus, inhibition of biofilm (BF) formation and percentage of disintegration of preformed biofilm. Etho = ethosuximide, AA = ascorbic acid, LA = lactic acid, PS80 = polysorbate 80, D1 = DNAse1, LAPS = Lactobacillus plantarum supernatant, M = mixture, MS = mixed solvent. [Figure 13] Figure 1 shows the bactericidal effect expressed on viable bacteria (CFU / mL) of S. aureus after 24 hours of treatment with composition M16 and the individual molecules at the concentrations present in the mentioned compositions. Etho = ethosuximide, AA = ascorbic acid, LA = lactic acid, PS80 = polysorbate 80, D1 = DNAse 1, LAPS = Lactobacillus plantarum supernatant, M = mixture, MS = mixed solvent. [Figure 14] Figure 1 shows the percentage of antioxidant activity against Trolox (%AAR) of MS, LAPS, AA (0.5mg / mL and 2.5mg / mL), M15 and M16. MS = mixed solvent, AA = ascorbic acid, LAPS = Lactobacillus plantarum supernatant, M = mixture. [Figure 15] Figure 1 shows the percentage of calcium and zinc chelation at pH 7.0 for mixed solvent (MS), solutions of EDTA in mixed solvent (0.25, 0.50 and 0.75 mg / mL), M0, M13, M15 and M16. MS = mixed solvent. [Figure 16] FIG. 1 shows the DNAse activity halo (mm) measured on DNAse agar at different times for different test samples to assess the maintenance of enzyme activity. [Figure 17] A, Halo (mm) of DNAse activity measured on DNAse agar for LAPS, 0.97 μg / mL dornase alfa, M0 and M13 (each containing 0.97 μg / mL dornase alfa), M15 and M16 (each containing 3.90 μg / mL dornase alfa). LAPS = Lactobacillus plantarum supernatant, M = mixture. [Figure 18] Figure 1 shows the percentage of increased angiogenesis in chicken chorioallantoic membrane. Etho = ethosuximide, LAPS = Lactobacillus plantarum supernatant, M = mixture. [Figure 19] FIG. 1 shows the effect of ethosuximide on VEGF levels normalized to GAPDH levels in macrophages J774 and keratinocytes HaCaT treated for 24 hours. Gene expression is changed on a Log2 scale. The Y-axis is the Log2 fold change in gene expression in each ethosuximide treatment relative to the control group. GAPDH served as a housekeeping gene to correct for cDNA input. [Figure 20] FIG. 1 shows the percentage of wound closure 24 hours after treatment application in different treatment animal groups. [Figure 21] FIG. 1 shows the immunohistochemistry technique on tissue sections from treated wounds to detect alpha-smooth muscle actin (α-SMA) using a monoclonal antibody (mouse anti-α-SMA - clone αsm-1) and revealing reaction with the streptavidin peroxidase system. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Short Description In one aspect of the present invention, provided herein is a pharmaceutical composition for topical wound treatment comprising at least one nitrogen-containing heterocyclic compound having 5 or 6 atoms and an imide group, one or more deoxyribonuclease enzymes having an active pH between 4.5 and 6.5, and one or more carboxylic acids. The nitrogen-containing heterocyclic compound having 5 or 6 atoms and an imido group includes a nitrogen-containing heterocyclic compound having 5 or 6 atoms and an imido group selected from the group consisting of ethosuximide, barbituric acid, phenobarbital, 5,5-diethylbarbituric acid, 5-ethyl-5-(1-methylbutyl)barbituric acid, pentobarbitone, pentobarbital, nembutal, 5-ethyl-5-(1-methylpropyl)barbituric acid, butobarbital, butisol, 5-allyl-5-(1-methylbutyl)-barbituric acid, secobarbital, seconal, phenytoin, hydantoin, and mixtures thereof, and preferably, the nitrogen-containing heterocyclic compound having 5 or 6 atoms and an imido group is ethosuximide. The deoxyribonuclease enzyme comprises a deoxyribonuclease enzyme selected from the group consisting of DNase, rh-dornase alpha, bovine pancreatic DNase I, DNase II, prokaryotic or eukaryotic DNase II, DNase II alpha, DNase II beta, porcine spleen DNase II, and mixtures thereof, preferably the deoxyribonuclease enzyme is recombinant human dornase-alpha.The carboxylic acid comprises a carboxylic acid selected from the group consisting of citric acid, lactic acid, acetic acid, formic acid, malic acid, tartaric acid, salicylic acid, oxalic acid, benzoic acid, propionic acid, and combinations thereof, preferably the carboxylic acid is lactic acid.

[0037] In another embodiment of the invention, the pharmaceutical composition further comprises one or more tensioactive agents selected from the group consisting of polysorbate 20, polysorbate 80, polysorbate 60, sorbitan tristearate, sorbitan monostearate, octoxynol-9, nonoxynol-9, and mixtures thereof, preferably the tensioactive agent is polysorbate 80.

[0038] In another embodiment of the invention, the pharmaceutical composition further comprises one or more complexing acids selected from the group consisting of ethylene glycol tetraacetic acid, ethylenediaminetetraacetic acid, dimercaptosuccinic acid, 2,3-dimercapto-1-propanesulfonic acid, lipoic acid (1,2-dithiolane-3-valeric acid), oxalic acid, and mixtures thereof, preferably the complexing acid is ethylenediaminetetraacetic acid.

[0039] In another embodiment of the invention, the pharmaceutical composition further comprises one or more hydrophilic reducing acids selected from the group consisting of uric acid, ascorbic acid, lipoic acid, and mixtures thereof, preferably the hydrophilic reducing acid is ascorbic acid.

[0040] In another embodiment of the invention, the pharmaceutical composition further comprises one or more gelling agents selected from the group consisting of cellulose, nanocrystalline cellulose, bacterial cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, microcrystalline cellulose, carbomethyl cellulose, carbopol, and mixtures thereof, preferably, the gelling agent is hydroxyethyl cellulose.

[0041] In another embodiment of the invention, the pharmaceutical composition comprises 0.2M acetic acid / 0.2M sodium acetate: ratio 2.78-0.66% (v / v), pH=4.2-4.8; 0.10M acetic acid / 0.01M sodium acetate: ratio 1.05-10.05% (v / v), pH=5.6; 0.05M potassium phosphate monobasic, pH=4.5; 0.36M potassium phosphate monobasic / 0.07M sodium phosphate disodium: ratio 4.92-0.98% (w / v), pH=5.7; 0.36M potassium phosphate monobasic / 0.10M sodium phosphate disodium: ratio 4.92-1.49% (w / v). , pH = 6.0; citric acid 0.31 M / disodium phosphate 0.20 M: ratio 2.99-1.42% (w / v), pH = 5.8; citric acid 0.10 M / sodium citrate 0.03 M: ratio 1.92-0.77% (w / v), pH = 5.8; Sörensen phosphate buffer: sodium phosphate monobasic 0.2 M / disodium phosphate 2.3 M: ratio 1.2-16.33% (w / v), pH = 5.8; Hanks' balanced salt solution (HBSS): sodium chloride 0.14 M (0.800%), potassium chloride 5 mM (0.040%), calcium chloride 1 mM (0.01 4%), magnesium sulfate heptahydrate 0.4 mM (0.010%), magnesium chloride hexahydrate 0.5 mM (0.010%), disodium phosphate dihydrate 0.3 mM (0.006%), potassium phosphate monobasic 0.4 mM (0.006%), glucose 6 mM (0.100%), sodium bicarbonate 4 mM (0.035%): pH = 5.7; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 1 M (pH = 6.5); 2-(N-morpholino)ethanesulfonic acid sodium salt, 4-morpholineethanesulfonic acid (MES sodium salt), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid sodium salt (BES) 0.5M (pH=6.0); N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) 0.5M (pH=6.0); 2,2'-[(2-amino-2-oxoethyl)imino]diacetic acid (ADA) 0.2M (pH=6.0); piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) 0.5M (pH=6.0~6.8); 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) 0.5M (pH=6.0~6.8).The solution further comprises a solution selected from the group consisting of: 2M (pH=6.0); saline (0.9%) / MgCL2 (5 mM), pH 5.5; 1M NaHCO3 solution, and combinations thereof.

[0042] In another embodiment of the invention, the pharmaceutical composition comprises a pH between about 4.5 and about 6.8.

[0043] In another embodiment of the present invention, the pharmaceutical composition contains about 0.5 mg / mL to about 50 mg / mL, preferably about 0.5 mg / mL to about 30 mg / mL, and more preferably about 0.5 mg / mL to about 3 mg / mL of one or more nitrogen-containing heterocyclic compounds having 5 or 6 atoms and an imido group.

[0044] In another embodiment of the invention, the pharmaceutical composition comprises one or more deoxyribonuclease enzymes at about 0.5 μg / mL to about 4000 μg / mL, more preferably about 1 μg / mL to about 1000 μg / mL, more preferably about 1 μg / mL to about 4 μg / mL, more preferably about 0.97 μg / mL to about 3.9 μg / mL.

[0045] In another embodiment of the invention, the pharmaceutical composition comprises from about 1 mg / mL to about 15 mg / mL, more preferably from about 1 mg / mL to about 9 mg / mL, more preferably from about 1.6 mg / mL to about 9 mg / mL of one or more carboxylic acids.

[0046] In another embodiment of the invention, the pharmaceutical composition comprises about 5 mg / mL or less, more preferably about 3 mg / mL or less, of one or more tensioactive agents.

[0047] In another embodiment of the invention, the pharmaceutical composition comprises about 1 mg / mL or less, more preferably about 0.75 mg / mL or less, of one or more complexing acids.

[0048] In another embodiment of the invention, the pharmaceutical composition comprises no more than about 3 mg / mL, more preferably no more than about 2.5 mg / mL, of one or more hydrophilic reducing acids.

[0049] In another embodiment of the invention, the pharmaceutical composition comprises no more than about 25 mg / mL, more preferably no more than about 17 mg / mL, of one or more gelling agents.

[0050] In a particularly preferred embodiment of the invention, the pharmaceutical composition comprises ethosuximide, recombinant human dornase-alpha and lactic acid in solution.

[0051] In a particularly preferred embodiment of the invention, the pharmaceutical composition comprises ethosuximide, recombinant human dornase-alpha, lactic acid, a hydrophilic reducing acid, polysorbate 80, ascorbic acid, ethylenediaminetetraacetic acid, hydroxyethylcellulose, and a solution comprising saline 0.9% and MgCl2 5 mM at pH 5.5.

[0052] In a particularly preferred embodiment of the invention, the pharmaceutical composition comprises an aqueous solution of about 0.5 mg / mL to about 30 mg / mL ethosuximide, about 1 μg / mL to about 4 μg / mL recombinant human dornase alfa, about 1 mg / mL to about 9 mg / mL lactic acid, about 2.5 mg / mL or less ascorbic acid, about 3 mg / mL or less polysorbate 80, about 0.75 mg / mL or less ethylenediaminetetraacetic acid, and about 17 mg / mL or less hydroxyethylcellulose.

[0053] In a particularly preferred embodiment of the invention, the pharmaceutical composition comprises an aqueous solution of about 0.5 mg / mL to about 3 mg / mL ethosuximide, about 0.97 μg / mL to about 3.9 μg / mL recombinant human dornase alfa, about 1.6 mg / mL to about 8.7 mg / mL lactic acid, about 2.5 mg / mL or less ascorbic acid, about 3 mg / mL or less polysorbate 80, about 0.75 mg / mL or less ethylenediaminetetraacetic acid, and about 17 mg / mL or less hydroxyethylcellulose.

[0054] In a particularly preferred embodiment of the present invention, a pharmaceutical preparation kit for topical wound treatment comprises a first composition and a second composition which are mixed before use to obtain a composition of the present invention, the first composition being a solid and comprising a deoxyribonuclease enzyme and a gelling agent powder, the second composition being an aqueous liquid comprising at least one nitrogen-containing heterocyclic compound having 5 or 6 atoms and an imide group and at least one carboxylic acid, and the second composition having a pH between 4.5 and 6.8.

[0055] In a particularly preferred embodiment of the present invention, a pharmaceutical preparation kit for topical wound treatment comprises a first composition, a second composition and a third composition to be mixed before use, wherein the first composition is solid and comprises a gelling agent powder, the second composition is solid and comprises a deoxyribonuclease enzyme, the third composition is an aqueous liquid comprising at least one nitrogen-containing heterocyclic compound having 5 or 6 atoms and an imide group and at least one carboxylic acid, and the third composition has a pH between 4.5 and 6.8.

[0056] In another embodiment of the invention, the pharmaceutical formulation kit comprises a first composition, a second composition and a third composition, each contained in a sealed individual container.

[0057] Another object of the invention is a method for preparing said pharmaceutical formulation, comprising the steps of: a. contacting a first composition with a second composition; and b. Mixing and shaking to obtain a gel pharmaceutical composition The method includes:

[0058] Another object of the present invention is a method for preparing a composition of the present invention from a pharmaceutical formulation of the present invention comprising a first composition, a second composition and a third composition mixed before use, wherein the first composition is solid and comprises a gelling agent powder, the second composition is solid and comprises a deoxyribonuclease enzyme, the third composition is an aqueous liquid comprising at least one nitrogen-containing heterocyclic compound having 5 or 6 atoms and an imide group, and at least one carboxylic acid, the third composition has a pH between 4.5 and 6.8, and the method comprises: a. contacting a third composition with a second composition; b. mixing and shaking the third composition with the second composition; c. contacting a first composition with the mixture obtained in step b.; and d. mixing and shaking to obtain a gel pharmaceutical composition.

[0059] Another object of the present invention is a method for treating a wound in a mammal, comprising the step of applying to the wound at least once a day a pharmaceutical composition according to the invention.

[0060] In another embodiment of the invention, the wound is a wound selected from the group consisting of diabetic foot ulcers, venous ulcers, arterial ulcers, pressure ulcers, mechanical wounds characterized by superinfection with biofilm forming bacteria, post-operative wounds characterized by superinfection with biofilm forming bacteria, and combinations thereof.

[0061] Another embodiment of the invention is a device comprising the pharmaceutical formulation of the invention, a first container containing a first composition; a second container containing a second composition; Including, The device has a first container and a second container separated by a disruptable membrane capable of separating the first composition from the second composition.

[0062] Another embodiment of the invention is a device comprising the pharmaceutical formulation of the invention, a first container containing a first composition; a second container containing a second composition; a third container containing a third composition; Including, the first container and the second container are separated by a first disruptable membrane capable of separating the first composition from the second composition; The device has a second container and a third container separated by a second disruptable membrane capable of separating the second composition and the third composition.

[0063] Another embodiment of the invention is a method for producing a pharmaceutical composition of the invention, comprising the steps of: a. mixing a nitrogen-containing heterocyclic compound having five or six atoms and an imide group, a deoxyribonuclease enzyme, and a carboxylic acid in a solution, thereby forming a mixture; , b. adding an alkaline solution dropwise to the mixture until the pH of the mixture is between 4.50 and 6.50, thereby forming a pH-adjusted mixture; and c. Sterilizing the pH adjusted mixture. The method includes:

[0064] d. The further step of adding a gelling agent.

[0065] e. adding a complexing acid, a tensioactive agent and a hydrophilic reducing acid.

[0066] Detailed Description The present invention provides pharmaceutical compositions and formulations, and kits for the topical treatment of wounds that achieve many technical advantages, primarily therapeutic advantages.

[0067] Wounds amenable to treatment according to the present invention are chronic ulcers that are refractory to conventional healing treatments, such as venous ulcers, arterial ulcers, pressure ulcers (bed sores), diabetic foot ulcers, and mechanical or post-operative wounds that have become chronic due to superinfection with biofilm-forming bacteria.

[0068] As used herein, the term "nitrogen-containing heterocyclic compound having five or six atoms and an imido group" refers to any pharma- ceutically acceptable chemical compound having a chemical ring having five or six atoms, about one of which is "N" and is part of an imido group. The imido group is -CO-NH-CO-. Examples of nitrogen-containing heterocyclic compounds having 5 or 6 atoms and imide groups in some embodiments of the present disclosure include, but are not limited to, ethosuximide, barbituric acid, phenobarbital, 5,5-diethylbarbituric acid (barbital or veronal), 5-ethyl-5-(1-methylbutyl)barbituric acid (pentobarbitone, pentobarbital or nembutal), 5-ethyl-5-(1-methylpropyl)barbituric acid (butabarbital, butizol), 5-allyl-5-(1-methylbutyl)-barbituric acid (secobarbital or seconal), phenytoin, hydantoin, and combinations thereof. Although ethosuximide is used in the examples of the present invention, any 5 or 6 pharma- ceutically acceptable nitrogen-containing heterocyclic compounds having imide groups listed above are considered equivalent.

[0069] The term "carboxylic acid" as used herein refers to any carboxylic acid that is pharma- ceutically acceptable. Examples of carboxylic acids in some embodiments of the present disclosure include, but are not limited to, citric acid, lactic acid, acetic acid, formic acid, malic acid, tartaric acid, salicylic acid, oxalic acid, benzoic acid, propionic acid, and combinations thereof. Although lactic acid is used in the present examples, any pharma- ceutically acceptable carboxylic acid listed above is considered to be equivalent.

[0070] The term "DNase enzyme" as used herein refers to any pharma- ceutically acceptable DNase enzyme with an active pH between 4.5 and 6.5. Examples of DNase enzymes in some embodiments of the present disclosure include, but are not limited to, DNase I of any origin, such as prokaryotic DNase I or eukaryotic DNase I. For example, recombinant human DNase or rh-Dornase alpha and bovine pancreatic DNase I, and DNase II of any origin, such as prokaryotic DNase II or eukaryotic DNase II. For example, DNase II alpha, DNase II beta and porcine spleen DNase II, and combinations thereof. Although rh-Dornase alpha is used in the examples of the present invention, any pharma-ceutically acceptable DNase enzyme listed above is considered to be equivalent.

[0071] The term "hydrophilic reducing acid" as used herein refers to any pharmaceutically acceptable hydrophilic reducing acid. Examples of hydrophilic reducing acids in some embodiments of the present disclosure include, but are not limited to, uric acid, ascorbic acid, lipoic acid, and combinations thereof. Although ascorbic acid is used in the examples of the present invention, any pharmaceutically acceptable hydrophilic reducing acid listed above is considered equivalent.

[0072] The term "complexing acid" as used herein refers to any pharma- ceutically acceptable complexing acid. Examples of complexing acids in some embodiments of the present disclosure include, but are not limited to, ethylene glycol tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), dimercaptosuccinic acid (DMSA), 2,3-dimercapto-1-propanesulfonic acid (DMPS), lipoic acid (1,2-dithiolane-3-valeric acid), oxalic acid, and combinations thereof. Although EDTA is used in the present example, any pharma-ceutically acceptable complexing acid listed above is considered equivalent.

[0073] The term "tensioactive agent" as used herein refers to any pharma- ceutically acceptable surfactant. Examples of tensioactive agents in some embodiments of the present disclosure include, but are not limited to, polysorbate 20, polysorbate 80, polysorbate 60, sorbitan tristearate, sorbitan monostearate, octoxynol-9, nonoxynol-9, which is castor oil ethoxylated with 40 moles of ethylene oxide, lauric alcohol ethoxylated with 7-10 moles, cremophor corifol, lipocoroxo 650, solutol HS15, emulgin B1, PH, Lanette 20PH, polysorbate, polysorbate 20, polysorbate 60, polysorbate 80, caprylic alcohol, decyl alcohol, lauryl alcohol, isotridecyl alcohol, myristyl alcohol, ethyl alcohol, palmoleyl, stearyl, oleyl, elaidyl, petroselinyl, linoleyl alcohol, linolenic, elaeostearyl alcohol, eicosyl, arachyl, gadoleyl, behenyl alcohol, erucyl alcohol and brassidyl, and combinations thereof. Polysorbate 80 was used in the examples of the present invention, but any of the pharma- ceutically acceptable tensioactive agents listed above are considered equivalents.

[0074] The term "gelling agent" as used herein refers to any pharma- ceutically acceptable gel-forming agent that forms a weakly cohesive internal structure when dissolved in a liquid phase as a colloidal mixture. Examples of pharma-ceutically acceptable gelling agents in some embodiments of the present disclosure include, but are not limited to, cellulose, nanocrystalline cellulose, bacterial cellulose, hydroxypropyl cellulose, microcrystalline cellulose, carbopol, cellulose derivatives, ethyl cellulose, hydroxyethyl cellulose, guar gum, gum arabic, hydroxyethyl methyl cellulose, hydroxyethyl propyl cellulose, carboxymethyl cellulose, xanthan gum, chitosan, alginates, gelatin, sodium starch glycolate, croscarmellose sodium, alginic acid, pectin, and combinations thereof. Hydroxyethyl cellulose was used in the examples of the present invention, but any pharma-ceutically acceptable gelling agent listed above is considered equivalent.

[0075] The present invention is a pharmaceutical composition for topical wound treatment comprising at least three components in solution, the concentrations of said components being in mg / mL, meaning mg of component per mL of said solution, and in % w / v, meaning % by weight of component per unit volume of said solution.

[0076] The terms "solution" or "MS" or "mixed solvent" as used interchangeably herein refer to any pharma- ceutically acceptable aqueous solvent. Examples of pharma-ceutically acceptable aqueous solvents of some embodiments of the present disclosure include, but are not limited to, acetic acid 0.2M / sodium acetate 0.2M: ratio 2.78-0.66% (v / v), pH=4.2-4.8; acetic acid 0.10M / sodium acetate 0.01M: ratio 1.05-10.05% (v / v), pH=5.6; potassium phosphate monobasic 0.05M, pH=4.5; potassium phosphate monobasic 0.36M / disodium phosphate disodium 0.07M: ratio 4.92-0.98% (w / v), pH=5.7; potassium phosphate monobasic 0.36M / disodium phosphate 0.10M: ratio 4.92-1.49% (w / v), pH = 6.0; citric acid 0.31M / disodium phosphate 0.20M: ratio 2.99-1.42% (w / v), pH = 5.8; citric acid 0.10M / sodium citrate 0.03M: ratio 1.92-0.77% (w / v), pH = 5.8; Sörensen phosphate buffer: monobasic sodium phosphate 0.2M / disodium phosphate 2.3M: ratio 1.2-16.33% (w / v), pH = 5.8; Hanks' balanced salt solution (HBSS): sodium chloride Calcium 0.14 M (0.800%), potassium chloride 5 mM (0.040%), calcium chloride 1 mM (0.014%), magnesium sulfate heptahydrate 0.4 mM (0.010%), magnesium chloride hexahydrate 0.5 mM (0.010%), disodium phosphate dihydrate 0.3 mM (0.006%), potassium phosphate monobasic 0.4 mM (0.006%), glucose 6 mM (0.100%), sodium bicarbonate 4 mM (0.035%): pH = 5.7; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 1M (pH=6.5); 2-(N-morpholino)ethanesulfonic acid sodium salt, 4-morpholineethanesulfonic acid (MES sodium salt) 0.5M (pH=5.5-6.7); N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid sodium salt (BES) 0.5M (pH=6.0); N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) 0.5M (pH=6.0); 2,2'-[(2-amino-2-oxoethyl)imino]diacetic acid (ADA) 0.2M (pH=6.0); piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) 0.5M (pH=6.0-6.8); 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) 0.2M (pH=6.0); saline (0.9%) / MgCL2 (5mM), pH 5.5; 1M NaHCO3 solution, and combinations thereof. Saline (0.9%) / MgCL2 (5mM), pH 5.5 was used in the examples of the present invention, but any pharma- ceutically acceptable solution or MS listed above is considered equivalent. The solution, or mixed solvent (MS), used in the examples of the present invention is saline (0.9%) and 5mM MgCl2, pH 5.5.

[0077] The compositions of the present invention are germicidal and may be formulated with a pharma- ceutically acceptable preservative selected from the group consisting of benzyl alcohol, benzoic acid, methyl hydroxybenzoate, propyl hydroxybenzoate, benzoalkonium chloride, chlorocresol, phenylmercuric nitrate, chlorobutanol, sodium dehydroacetate, thimerosal, sodium benzoate, sorbic acid, and combinations thereof.

[0078] The compositions of the present invention may be formulated with a pharma- ceutically acceptable anesthetic selected from the group consisting of lidocaine, pramoxine, benzocaine, and the like, and combinations thereof.

[0079] Among the technical advantages achieved by the present invention, the following advantages can be detailed:

[0080] Bacteriostatic and bactericidal effect: the compositions of the present invention are capable of eliminating 99% of the planktonic strains isolated from chronic wounds, whereas existing products on the market only have a small range of activity. This effect is not achieved by any of the current state of the art products, so currently two or more products must be used.

[0081] Inhibitory effect on bacterial biofilm formation: Whereas existing inhibitor products only inhibit one strain at a time, the composition of the present invention inhibits biofilm formation of 99% of strains isolated from chronic wounds.

[0082] Disruption effect of pre-formed bacterial biofilms: Whereas commercially available bacterial biofilm disruption products provide for the disruption of only a single species of biofilm at a time, the composition of the present invention provides for the disruption of 95% of biofilms of most isolated strains in chronic wounds, including mixed biofilms (formed by more than one species as in vivo), for which no comparable product is commercially available.

[0083] Debridement effect: The composition of the present invention removes the fibrin / biofilm / necrotic tissue layer from the wound surface

[0084] Inhibitory effect on wound expansion: This effect is achieved by the inhibition of MMPs and free radicals, which the composition of the present invention provides. It does so through a pharmaceutical form that absorbs exudate. There are no products on the market for such purposes.

[0085] Oxygenation effect: The oxygenation power of the present invention is achieved by inducing the expression of vascular endothelial growth factor (VEGF), which stimulates angiogenesis. Eliminating aerobic pathogenic flora also increases pO2 in the wound.

[0086] Collagenization Effect: The present invention stimulates the formation and deposition of collagen in the ulcer bed.

[0087] The compositions of the present invention have been demonstrated to be active for at least six months, thus demonstrating long-term stability compared to current technology.

[0088] A device for containing the formulation of the present invention is another object of the present invention. In one embodiment, the device comprises three containers: a first container containing a first composition, a second container containing a second composition, and a third container containing a third composition. The first composition is solid and comprises a gelling agent powder, the second composition is solid and comprises a deoxyribonuclease enzyme, and the third composition is an aqueous solution comprising at least one nitrogen-containing heterocyclic compound having 5 or 6 atoms and an imide group, and at least one carboxylic acid, and the third composition has a pH between 4.5 and 6.8. The third composition can further comprise one or more hydrophilic reducing acids, one or more tensioactive agents, and one or more complexing acids.

[0089] One embodiment of the present invention provides a three-container device, where the first and second containers are separated by a first collapsible membrane capable of separating the first and second compositions, and the second and third containers are separated by a second collapsible membrane capable of separating the second and third compositions.

[0090] The term "disintegrable membrane" as used herein refers to any membrane or film made of a rigid or flexible material that can be disrupted, broken, ruptured, torn or removed to separate the containers. These disintegrable membranes are made of any material known in the art, such as polymer, aluminum or silicon. When the disintegrable membrane separating adjacent containers is broken, the compositions in the containers come into contact.

[0091] The term "container" as used herein refers to any container that contains a composition that is isolated from the atmosphere and other compositions. The container shares a collapsible membrane with another container, and when the membrane breaks, the compositions contained in the containers come into contact. The container of the present invention can be an independent, separate container, or part of a single device having two or three containers that share a collapsible membrane between them, as seen in FIG. 1.

[0092] The device of the present invention is a cartridge or a dispenser.

[0093] One embodiment provides a dispenser device with a nozzle, which may include a removable cap.

[0094] An exemplary device is shown in Figure 1. The multi-component device is a cylindrical cartridge of rigid material. Materials disclosed herein that are useful for making the device include barrier-resistant polypropylene, aluminum, and glass to prevent exposure to moisture and oxygen. Two collapsible membranes divide the exemplary device into three containers. The collapsible membranes can be broken by pressure or any system known in the art for breaking films.

[0095] Another object of the present invention is a method for preparing the pharmaceutical composition of the present invention in situ prior to its use in wound treatment, said method comprising the steps of: a. disrupting the second disruptive membrane to contact the third composition with the second composition; b. mixing and shaking the third composition with the second composition; c. breaking the first collapsed membrane and contacting the first composition with the mixture obtained in step b.; and d. Mixing and shaking to obtain the gel pharmaceutical composition of the present invention. Includes.

[0096] In step a., a perfect mixture is possible. The enzyme of the second composition in the second container is distributed throughout its volume when it is shaken with the third liquid aqueous composition in the third container. This mixture according to step a. is mixed in step c. with the gelling agent of the first composition in the first container and shaken to obtain the homogenous gel composition of the present invention.

[0097] One embodiment provides a device for containing the formulation of the present invention. The device includes two containers, a first container containing a first composition and a second container containing a second composition. The first composition is solid and includes a gelling agent powder and a deoxyribonuclease enzyme, the second composition is an aqueous solution containing at least one nitrogen-containing heterocyclic compound having 5 or 6 atoms and an imide group and at least one carboxylic acid, and the third composition has a pH between 4.5 and 6.8. The third composition can further include one or more hydrophilic reducing acids, one or more tensioactive agents, and one or more complexing acids.

[0098] The formulations of the present invention that are prepared in situ prior to application exhibit a shelf life similar to that of the enzymes used.

[0099] The stability of the formulation of the present invention contained in the device of the present invention with two or three containers over time is about 2 years. This stability is the stability of the enzyme used, since it is in the form of a lyophilized powder in a separate container or compartment, without contact with a solution. Therefore, the formulation of the present invention in the device of the present invention shows a long-term stability compared to the current technology. And when the formulation is prepared, the first composition is contacted with the second composition, and (if the device provides three containers) with the third composition, and all the compositions are mixed by shaking, the prepared gel shows a stability of about 6 months or more. EXAMPLES

[0100] [Example 1] Component solution preparation Solutions of the different components of the composition of the present invention were prepared.

[0101] Mixed solvent (MS): Saline solution (0.9%) / MgCL2 (5mM). pH 5.5 A 1 liter solution is prepared by weighing out 9.0000 g of sodium chloride and 1.0106 g of magnesium chloride hexahydrate (MgCL2.6H2O) and dissolving them in distilled water.

[0102] Alkaline solution: 1M NaHC03 solution. In a volumetric flask, 100 mL of solution is prepared by dissolving 8.4 g of sodium bicarbonate in distilled water. It is important to completely dissolve the sodium bicarbonate in the solvent and beaker before dispensing up to the mark in the flask to avoid oversaturation. The final pH of the alkaline solution is approximately 8.6.

[0103] Lactic acid solution (LA) in mixed solvent. LA is diluted in the mentioned mixed solvent and then mixed for a few minutes. A final pH=5.5 is achieved with the alkaline solution. The concentrations prepared were LA 1.63mg / mL, LA 6.50mg / mL, LA 8.67mg / mL.

[0104] Polysorbate 80 solution (PS80) in mixed solvents. PS80 is diluted and then mixed for several minutes until homogenization is achieved. Final pH=5.5 is achieved using an alkaline solution. The concentrations prepared were PS80 2.0mg / mL, PS80 3.0mg / mL.

[0105] EDTA solution in mixed solvent: EDTA is dissolved in the mentioned mixed solvent and then mixed for a few minutes. A final pH=5.5 is achieved using an alkaline solution. The concentrations prepared were EDTA 0.25mg / mL, EDTA 0.50mg / mL, EDTA 0.75mg / mL.

[0106] Ascorbic acid solution (AA) in mixed solvents. AA is dissolved in the mentioned solvent mixture and then mixed for a few minutes. A final pH=5.50 is achieved using an alkaline solution. The concentrations prepared were AA 0.5mg / mL, AA 1.0mg / mL, AA 2.50mg / mL.

[0107] Ethosuximide solution (Etho) in mixed solvent. Etho is dissolved in the mixed solvent and then mixed for a few minutes. A final pH=5.5 is achieved using an alkaline solution. The concentrations prepared were Etho 0.50mg / mL, Etho 3.00mg / mL, Etho 30.00mg / mL.

[0108] Rh-Dornase alfa solution (D1) in mixed solvent. D1 is dissolved in the mixed solvent and then mixed for a few minutes. A final pH=5.5 is achieved with the alkaline solution. The concentrations prepared were: D1 250μg / mL, D1 125μg / mL, D1 62.5μg / mL, D1 31.2μg / mL, D1 15.6μg / mL, D1 7.8μg / mL, D1 3.9μg / mL, D1 1.9μg / mL, D1 0.97μg / mL, D1 0.5μg / mL.

[0109] Control Lactobacillus plantarum supernatant for 24 hours (LAPS 24h): L. plantarum ATCC10241 was grown for 12 hours at 37°C in de Man Rogosa Sharpe (MRS) (Britania. Argentina). The supernatant (LAPS) was obtained by centrifugation at 8000 rpm for 15 minutes and filtered through a 0.22 μm Millipore filter. The preparation was then stored for 24 hours at 2-8°C. Hydroxyethylcellulose 1.7% was added to obtain the pharmaceutical form as a gel.

[0110] Lactobacillus plantarum supernatant for 6 months (LAPS 6m) and control: LAPS 24h preparations were stored at 2–8 °C for 6 months before use.

[0111] [Example 2] Composition of the invention. Preparation of mixture No. 0 (MO) in the laboratory The composition of the present invention comprises: a. 6.50mg / mL lactic acid (85%~90% purity) b. 3.00mg / mL ethosuximide c. Consists of DNase (rh-Dornase alfa) 0.97 μg / mL

[0112] Solution: A mixture is formulated (a+b+c) In 50 mL of the mixed solvent, 150 mg of ethosuximide, 318 μL of lactic acid and 50 μg of rh-dornase alfa enzyme are added.

[0113] Preparation Mode 1. 150 mg of ethosuximide is weighed out on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland) on a watch glass. 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. 4. Next, 318 μL of lactic acid is added to the preparation. 5. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 6. The solution is brought to a final pH of 5.5 and 50 mL of the solution is placed in a 100 mL beaker and a pre-calibrated Lutron PH-206 pH meter electrode (Lutron Electronics Co. Inc., Pennsylvania, USA) is immersed in it. 7. The mass is placed on a stirrer at low speed and the alkaline solution is added dropwise until a pH of 5.5 is achieved, taking note of the foaming produced by the agglomeration. 8. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min. 9. After the sterilization process is complete, 50 mL of the preparation is placed in a conical tube and 50 μg of rh-Dornase alfa enzyme (Roche, Basel, Switzerland) is added under aseptic conditions to achieve a final concentration of 0.97 μg / mL. This step is performed in a biological safety cabinet. 10. The tube is sealed and mixed by vortexing for 1 minute. 11. The preparation is protected from light using aluminum foil and then stored at 4°C to 25°C.

[0114] [Example 3] Composition of the invention. Preparation of mixture No. 13 (M13) in the laboratory The composition of the present invention comprises: a. 3.00mg / mL Polysorbate 80 b. 1.00 mg / mL ascorbic acid c. 6.50mg / mL lactic acid (85%~90% purity) d. 0.50mg / mL EDTA e. 3.00mg / mL ethosuximide f. Contains 0.97μg / mL DNase

[0115] Solution: A mixture is formulated (a+b+c+d+e+f) In 50 mL of the mixed solvent, 50 mg of ascorbic acid, 25 mg of EDTA, 150 mg of ethosuximide, 50 μg of rh-dornase alfa, 141 μL of polysorbate 80 and 318 μL of lactic acid are added.

[0116] Preparation mode: 1. 50 mg of ascorbic acid and 25 mg of EDTA are weighed on a watch glass on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland). 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. During stirring, 141 μL of polysorbate 80 is added with a 200 μL tip, with the tip cut off. 4. Next, 318 μL of lactic acid is added to the preparation. 5. 150 mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. The mixture is brought to a final pH of 5.5 and 50 mL of the mixture is placed in a 100 mL beaker and a pre-calibrated Lutron PH-206 pH meter electrode (Lutron Electronics Co. Inc., Pennsylvania, USA) is immersed in it. 8. The mixture from step 7 is placed in a stirrer at low speed and the alkaline solution is added dropwise until a pH of 5.5 is achieved, taking care to avoid foaming produced by the agglomeration. 9. The mixture from step 8 is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 minutes. 10. After the sterilization process is complete, 50 mL of the mixture is placed in a 50 mL conical tube and 50 μg of rh-Dornase alfa enzyme (Roche, Basel, Switzerland) is added under aseptic conditions to achieve a final concentration of 0.97 μg / mL. This step is performed in a biological safety cabinet. 11. The tube is sealed and mixed by vortexing for 1 minute. 12. The composition of the present invention in step 11 is protected from light using aluminum foil and then stored at 4°C to 25°C.

[0117] [Example 4] Composition of the invention. Preparation of Mixture No. 15 (M15) in the laboratory The composition of the present invention comprises: a. 2.00mg / mL Polysorbate 80 b. 0.50mg / mL ascorbic acid c. 1.63mg / mL lactic acid (85%~90% purity) d. 0.25mg / mL EDTA e. 0.50mg / mL ethosuximide f. Contains 3.90μg / mL DNase

[0118] The mixture is formulated (a+b+c+d+e+f) In 50 mL of the mixture solvent, 25.00 mg of ascorbic acid, 12.50 mg of EDTA, 25 mg of ethosuximide, 200 μg of rh-dornase alfa, 94 μL of polysorbate 80 and 80 μL of lactic acid are added.

[0119] Preparation mode: 1. 25 mg of ascorbic acid and 12.50 mg of EDTA are weighed on a watch glass on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland). 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. While stirring, 94 μL of polysorbate 80 is added with a 200 μL tip cut off. 4. Next, 80 μL of lactic acid is added to the preparation. 5. 25 mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. The mixture is brought to a final pH of 5.5 and 50 mL of the mixture is placed in a 100 mL beaker and a pre-calibrated Lutron PH-206 pH meter electrode (Lutron Electronics Co. Inc., Pennsylvania, USA) is immersed in it. 8. The mixture from step 7 is placed in a stirrer at low speed and the alkaline solution is added dropwise until a pH of 5.5 is achieved, taking care to avoid foaming produced by the agglomeration. 9. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min. 10. 9. After the sterilization process is finished, 50 mL of the prepared solution is placed in a 50 mL conical tube and 200 μg of rh-Dornase alfa enzyme (Roche, Basel, Switzerland) is added under aseptic conditions to achieve a final concentration of 3.90 μg / mL. This step is performed in a biological safety cabinet. 11. The tube is sealed and mixed by vortexing for 1 minute. 12. The composition of the present invention in step 11 is protected from light using aluminum foil and then stored at 4°C to 25°C.

[0120] [Example 5] Compositions of the Invention Preparation of Mixture No. 16 (M16) in the Laboratory The composition of the present invention comprises: a. 3.00mg / mL Polysorbate 80 b. 2.50mg / mL ascorbic acid c. 8.67mg / mL lactic acid (85%~90% purity) d. 0.75mg / mL EDTA e. 30.00mg / mL ethosuximide f. Contains 3.90μg / mL DNase

[0121] The mixture is formulated (a+b+c+d+e+f) In 50 mL of the mixed solvent, 125 mg of ascorbic acid, 37.5 mg of EDTA, 1500 mg of ethosuximide, 200 μg of rh-dornase alfa, 141 μL of polysorbate 80 and 424 μL of lactic acid are added.

[0122] Preparation mode: 1. 125 mg of ascorbic acid and 37.5 mg of EDTA are weighed on a watch glass on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland). 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. During stirring, 141 μL of polysorbate 80 is added with a 200 μL tip, with the tip cut off. 4. Next, 424 μL of lactic acid is added to the preparation. 5. 1500mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. A Lutron PH-206 pH meter electrode is calibrated (Lutron Electronics Co. Inc., Pennsylvania, USA) to measure the pH of the preparation. 8. The solution is brought to a final pH of 5.5, 50 mL of the solution is placed in a 100 mL beaker on a slow stirrer with a pH meter and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its condensation. 9. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min. 10. After the sterilization process is complete, 50 mL of the prepared solution is placed in a 50 mL conical tube and 200 μg of rh-Dornase alfa enzyme (Roche, Basel, Switzerland) is added under aseptic conditions to achieve a final concentration of 3.90 μg / mL. This step is performed in a biological safety cabinet. 11. The tube is sealed and mixed by vortexing for 1 minute. 12. The composition of the present invention in step 11 is protected from light using aluminum foil and then stored at 4°C to 25°C.

[0123] [Example 6] Composition of the invention. Preparation of gel of mixture no. 0 (MO) in the laboratory The composition of the present invention comprises: a. 6.50mg / mL lactic acid (85%~90% purity) b. 3.00mg / mL ethosuximide c. DNase 0.97μg / mL d. Consists of 17 mg / mL hydroxyethyl cellulose

[0124] Solution: A mixture is formulated (a+b+c) In 50 mL of the mixed solvent, 150.0 mg of ethosuximide, 318 μL of lactic acid and 50 μg of rh-dornase alfa enzyme are added.

[0125] Preparation Mode 1. 150.0 mg of ethosuximide is weighed out on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland) on a watch glass. 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. 4. Next, 318 μL of lactic acid is added to the preparation. 5. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 6. The solution is brought to a final pH of 5.5 and 50 mL of the solution is placed in a 100 mL beaker and a pre-calibrated Lutron PH-206 pH meter electrode (Lutron Electronics Co. Inc., Pennsylvania, USA) is immersed in it. 7. The mass is placed on a stirrer at low speed and the alkaline solution is added dropwise until a pH of 5.5 is achieved, taking note of the foaming produced by the agglomeration. 8. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min. 9. After the sterilization process is complete, 50 mL of the preparation is placed in a conical tube and 50 μg of rh-Dornase alfa enzyme (Roche, Basel, Switzerland) is added under aseptic conditions to achieve a final concentration of 0.97 μg / mL. This step is performed in a biological safety cabinet. 10. The tube is sealed and mixed by vortexing for 1 minute.

[0126] Gel: A mixture is formulated (solution + g) 11. Add 0.85 g of hydroxyethyl cellulose to the prepared mixture. 12. Mix gently until homogenous. 13. The preparation is protected from light using aluminum foil and then stored covered at 4-25°C. It should be left for between 6 and 8 hours until the air bubbles disappear.

[0127] [Example 7] Composition of the invention. Preparation of gel of mixture no. 13 (M13) in the laboratory The composition of the present invention comprises: a. 3.00mg / mL Polysorbate 80 b. 1.00 mg / mL ascorbic acid c. 6.50mg / mL lactic acid (85%~90% purity) d. 0.50mg / mL EDTA e. 3.00mg / mL ethosuximide f. 0.97μg / mL DNase g. Contains 17mg / mL hydroxyethylcellulose

[0128] Solution: A mixture is formulated (a+b+c+d+e+f) In 50 mL of the mixed solvent, 50 mg of ascorbic acid, 25 mg of EDTA, 150 mg of ethosuximide, 50 μg of rh-dornase alfa, 141 μL of polysorbate 80 and 318 μL of lactic acid are added.

[0129] Preparation mode: 1. 50 mg of ascorbic acid and 25 mg of EDTA are weighed on a watch glass on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland). 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. During stirring, 141 μL of polysorbate 80 is added with a 200 μL tip, with the tip cut off. 4. Next, 318 μL of lactic acid is added to the preparation. 5. 150 mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. The mixture is brought to a final pH of 5.5 and 50 mL of the mixture is placed in a 100 mL beaker and a pre-calibrated Lutron PH-206 pH meter electrode (Lutron Electronics Co. Inc., Pennsylvania, USA) is immersed in it. 8. The mixture from step 7 is placed in a stirrer at low speed and the alkaline solution is added dropwise until a pH of 5.5 is achieved, taking care to avoid foaming produced by the agglomeration. 9. The mixture from step 8 is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 minutes. 10. After the sterilization process is complete, 50 mL of the mixture is placed in a 50 mL conical tube and 50 μg of rh-Dornase alfa enzyme (Roche, Basel, Switzerland) is added under aseptic conditions to achieve a final concentration of 0.97 μg / mL. This step is performed in a biological safety cabinet. 11. The tube is sealed and mixed by vortexing for 1 minute.

[0130] Obtaining the gel: the mixture is formulated (solution + g) 12. Add 0.85 of hydroxyethyl cellulose to the prepared mixture. 13. Mix gently until homogenous. 14. The preparation is protected from light using aluminum foil and then stored covered at 4-25°C. It should be left for between 6 and 8 hours until the air bubbles disappear.

[0131] [Example 8] Composition of the invention. Preparation of gel of mixture number 15 (M15) in the laboratory The composition of the present invention comprises: a. 2.00mg / mL Polysorbate 80 b. 0.50mg / mL ascorbic acid c. 1.63mg / mL lactic acid (85%~90% purity) d. 0.25mg / mL EDTA e. 0.50mg / mL ethosuximide f. 3.90μg / mL DNase g. Contains 17mg / mL hydroxyethylcellulose

[0132] The mixture is formulated (a+b+c+d+e+f) In 50 mL of the mixture solvent, 25.00 mg of ascorbic acid, 12.50 mg of EDTA, 25 mg of ethosuximide, 200 μg of rh-dornase alfa, 94 μL of polysorbate 80 and 80 μL of lactic acid are added.

[0133] Preparation mode: 1. 25 mg of ascorbic acid and 12.5 mg of EDTA are weighed on a watch glass on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland). 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. While stirring, 94 μL of polysorbate 80 is added with a 200 μL tip cut off. 4. Next, 80 μL of lactic acid is added to the preparation. 5. 25 mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. The mixture is brought to a final pH of 5.5 and 50 mL of the mixture is placed in a 100 mL beaker and a pre-calibrated Lutron PH-206 pH meter electrode (Lutron Electronics Co. Inc., Pennsylvania, USA) is immersed in it. 8. The mixture from step 7 is placed in a stirrer at low speed and the alkaline solution is added dropwise until a pH of 5.5 is achieved, taking care to avoid foaming produced by the agglomeration. 9. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min. 10. After the sterilization process is complete, 50 mL of the prepared solution is placed in a 50 mL conical tube and 200 μg of rh-Dornase alfa enzyme (Roche, Basel, Switzerland) is added under aseptic conditions to achieve a final concentration of 3.90 μg / mL. This step is performed in a biological safety cabinet. 11. The tube is sealed and mixed by vortexing for 1 minute.

[0134] Obtaining the gel: the mixture is formulated (solution + g) 12. Add 0.85 g of hydroxyethyl cellulose to the prepared mixture. 13. Mix gently until homogenous. 14. The preparation is protected from light using aluminum foil and then stored covered at 4-25°C. It should be left for between 6 and 8 hours until the air bubbles disappear.

[0135] [Example 9] Composition of the invention. Preparation of gel of mixture number 16 (M16) in the laboratory The composition of the present invention comprises: a. 3.00mg / mL Polysorbate 80 b. 2.50mg / mL ascorbic acid c. 8.67mg / mL lactic acid (85%~90% purity) d. 0.75mg / mL EDTA e. 30.00mg / mL ethosuximide f. 3.90μg / mL DNase g. Contains 17mg / mL hydroxyethylcellulose

[0136] The mixture is formulated (a+b+c+d+e+f) In 50 mL of the mixed solvent, 125 mg of ascorbic acid, 37.5 mg of EDTA, 1500 mg of ethosuximide, 200 μg of rh-dornase alfa, 141 μL of polysorbate 80 and 424 μL of lactic acid are added.

[0137] Preparation mode: 1. 125 mg of ascorbic acid and 37.5 mg of EDTA are weighed on a watch glass on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland). 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. During stirring, 141 μL of polysorbate 80 is added with a 200 μL tip, with the tip cut off. 4. Next, 424 μL of lactic acid is added to the preparation. 5. 1500mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. A Lutron PH-206 pH meter electrode is calibrated (Lutron Electronics Co. Inc., Pennsylvania, USA) to measure the pH of the preparation. 8. The solution is brought to a final pH of 5.5, 50 mL of the solution is placed in a 100 mL beaker on a slow stirrer with a pH meter and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its condensation. 9. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min. 10. After the sterilization process is complete, 50 mL of the prepared solution is placed in a 50 mL conical tube and 200 μg of rh-Dornase alfa enzyme (Roche, Basel, Switzerland) is added under aseptic conditions to achieve a final concentration of 3.90 μg / mL. This step is performed in a biological safety cabinet. 11. The tube is sealed and mixed by vortexing for 1 minute.

[0138] Obtaining the gel: the mixture is formulated (solution + g) 12. Add 0.85 g of hydroxyethyl cellulose to the prepared mixture. 13. Mix gently until homogenous. 14. The preparation is protected from light using aluminum foil and then stored covered at 4-25°C. It should be left for between 6 and 8 hours until the air bubbles disappear.

[0139] [Example 10] Compositions of the Invention Preparation of Mixture No. 0 (MO) in a preferred pharmaceutical formulation. The composition of the present invention comprises: a. 6.5mg / mL lactic acid (85%~90% purity) b. 3.00mg / mL ethosuximide c. 0.97μg / mL DNAse d. Consists of 17 mg / mL hydroxyethyl cellulose

[0140] Solution: A mixture is formulated (a+b) In 50 mL of the mixed solvent, 150.0 mg of ethosuximide and 318 μL of lactic acid are added.

[0141] Preparation Mode 1. 150.0 mg of ethosuximide is weighed out on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland) on a watch glass. 2. With the help of total mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. The beaker with the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 min until the solid particles are partially dissolved. 4. Next, 318 μL of lactic acid is added to the preparation. 5. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 6. A Lutron PH-206 pH meter electrode is calibrated (Lutron Electronics Co. Inc., Pennsylvania, USA) to measure the pH of the preparation. 7. The solution is brought to a final pH of 5.5 by placing 50 mL of the solution in a 100 mL beaker on a slow stirrer along with a calibrated pH meter, Lutron PH-206, and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its aggregation. 8. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min and stored tightly sealed at 4-25°C and protected from light sources.

[0142] Pharmaceutical formulation: A preferred pharmaceutical form of the present invention is a gel. The gel is obtained after a two-stage process. The application device contains three containers separated by two collapsible membranes. The formulation of the present invention contains: Vessel 1: This vessel is filled with 0.85 g of lyophilized hydroxyethylcellulose sufficiently to obtain a gel with the expected rheological properties. Container 2: Into this container is placed 50 μg of lyophilized rh-dornase alfa in sufficient quantity to give a final solution of 0.97 μg / mL. Container 3: In this container 50 mL of the preparation from step 8 is placed.

[0143] In the first mixing stage, membrane number 2 is broken allowing mixing of the contents of vessels 2 and 3. Mixing improves homogenization.

[0144] In the second mixing stage, membrane number 1 is broken to allow mixing of the homogenate with the contents of container 1, which contains the excipients and therefore at this stage the final pharmaceutical form of the gel is achieved. Mixing improves homogenization of all the components.

[0145] [Example 11] Compositions of the invention.Preparation of Mixture No. 13 (M13) in a preferred pharmaceutical formulation. The composition of the present invention comprises: a. 3.00mg / mL Polysorbate 80 b. 1.00 mg / mL ascorbic acid c. 6.50mg / mL lactic acid (85%~90% purity) d. 0.50mg / mL EDTA e. 3.00mg / mL ethosuximide f. 0.97μg / mL DNase g. Contains 17mg / mL hydroxyethylcellulose

[0146] Solution: A mixture is formulated (a+b+c+d+e) In 50 mL of the mixed solvent, 50 mg of ascorbic acid, 25 mg of EDTA, 150 mg of ethosuximide, 141 μL of polysorbate 80 and 318 μL of lactic acid are added.

[0147] Preparation mode: 1. 50 mg of ascorbic acid and 25 mg of EDTA are weighed on a watch glass. 2. The mixed solvent is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. While stirring, 141 μL of polysorbate 80 is added with a 200 μL tip cut off. 4. A volume of 318 μL of lactic acid is then added to the preparation. 5. 150 mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. The solution is brought to a final pH of 5.5 by placing 50 mL of the solution in a 100 mL beaker on a slow stirrer along with a calibrated pH meter, Lutron PH-206, and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its aggregation. 8. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min and stored tightly sealed at 4-25°C and protected from light sources.

[0148] Pharmaceutical formulation: A preferred pharmaceutical form of the present invention is a gel. The gel is obtained after a two-stage process. The application device contains three containers separated by two collapsible membranes. The formulation of the present invention contains: Vessel 1: This vessel is filled with 0.85 g of lyophilized hydroxyethylcellulose sufficiently to obtain a gel with the expected rheological properties. Container 2: 50 μg of lyophilized rh-dornase alfa is placed into this container in sufficient quantity to give a 0.97 μg / mL solution. Container 3: In this container 50 mL of the preparation from step 8 is placed.

[0149] In the first mixing stage, membrane number 2 is broken allowing mixing of the contents of vessels 2 and 3. Mixing improves homogenization.

[0150] In the second mixing stage, membrane number 1 is broken to allow mixing of the homogenate with the contents of container 1, which contains the excipients and therefore at this stage the final pharmaceutical form of the gel is achieved. Mixing improves homogenization of all the components.

[0151] [Example 12] Compositions of the Invention Preparation of Mixture No. 15 (M15) in a preferred pharmaceutical formulation. The composition of the present invention comprises: a. 2.00mg / mL Polysorbate 80 b. 0.50mg / mL ascorbic acid c. 1.63mg / mL lactic acid (85%~90% purity) d. 0.25mg / mL EDTA e. 0.50mg / mL ethosuximide f. 3.90μg / mL DNase g. Contains 17mg / mL hydroxyethylcellulose

[0152] Solution: A mixture is formulated (a+b+c+d+e) In 50 mL of the mixed solvent, 25 mg of ascorbic acid, 12 mg of EDTA, 25 mg of ethosuximide, 94 μL of polysorbate 80 and 80 μL of lactic acid are added.

[0153] Preparation mode: 1. 25 mg of ascorbic acid and 12.5 mg of EDTA are weighed on a watch glass. 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. While stirring, 94 μL of polysorbate 80 is added with a 200 μL tip cut off. 4. A volume of 80 μL of lactic acid is then added to the preparation. 5. 25 mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. The solution is brought to a final pH of 5.5, 50 mL of the solution is placed in a 100 mL beaker on a slow stirrer with a pH meter and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its condensation. 8. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min and stored tightly sealed at 4-25°C and protected from light sources.

[0154] Pharmaceutical formulation: A preferred pharmaceutical form of the present invention is a gel. The gel is obtained after a two-stage process. The application device contains three containers separated by two collapsible membranes. The formulation of the present invention contains: Vessel 1: This vessel is filled with 0.85 g of lyophilized hydroxyethylcellulose sufficiently to obtain a gel with the expected rheological properties. Container 2: Into this container, 200 μg of lyophilized rh-dornase alfa is placed in sufficient quantity to give a 3.90 μg / mL solution. Container 3: In this container 50 mL of the preparation from step 8 is placed.

[0155] In the first mixing stage, membrane number 2 is broken allowing mixing of the contents of vessels 2 and 3. Mixing improves homogenization.

[0156] In the second mixing stage, membrane number 1 is broken to allow mixing of the homogenate with the contents of container 1, which contains the excipients and therefore at this stage the final pharmaceutical form of the gel is achieved. Mixing improves homogenization of all the components.

[0157] [Example 13] Compositions of the Invention Preparation of Mixture No. 16 (M16) in a preferred pharmaceutical formulation. The composition of the present invention comprises: a. 3.00mg / mL Polysorbate 80 b. 2.50mg / mL ascorbic acid c. 8.67mg / mL lactic acid (85%~90% purity) d. 0.75mg / mL EDTA e. 30.00mg / mL ethosuximide f. 3.90μg / mL DNase g. Contains 17mg / mL hydroxyethylcellulose

[0158] Solution: A mixture is formulated (a+b+c+d+e) In 50 mL of the mixed solvent, 125 mg of ascorbic acid, 37.5 mg of EDTA, 1500 mg of ethosuximide, 141 μL of polysorbate 80 and 424 μL of lactic acid are added.

[0159] Preparation mode: 1. 125 mg of ascorbic acid and 37.5 mg of EDTA are weighed on a watch glass on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland). 2. With the help of a solvent mixture, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of mixed solvent to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 min until the solid particles are partially dissolved. While stirring, 141 μL of polysorbate 80 is added with a 200 μL tip cut off. 4. A volume of 424 μL of lactic acid is then added to the preparation. 5. 1500mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. The mixture from step 6 is brought to a final pH of 5.5 by placing 50 mL of the solution in a 100 mL beaker on a slow stirrer with a pH meter Lutron PH-206 electrode and adding alkaline solution dropwise until the pH level is achieved, taking note of the foaming produced by its aggregation. 8. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 minutes and after this step is stored tightly closed at 4-25°C and protected from light sources.

[0160] Pharmaceutical formulation: A preferred pharmaceutical form of the present invention is a gel. The gel is obtained after a two-stage process. The application device contains three containers separated by two collapsible membranes. The formulation of the present invention contains: Vessel 1: This vessel is filled with 0.85 g of lyophilized hydroxyethylcellulose sufficiently to obtain a gel with the expected rheological properties. Container 2: Into this container, 200 μg of lyophilized rh-dornase alfa is placed in sufficient quantity to give a 3.90 μg / mL solution. Container 3: In this container 50 mL of the preparation from step 8 is placed.

[0161] In the first mixing stage, membrane number 2 is broken allowing mixing of the contents of vessels 2 and 3. Mixing improves homogenization.

[0162] In the second mixing stage, membrane number 1 is broken to allow mixing of the homogenate with the contents of container 1, which contains the excipients and therefore at this stage the final pharmaceutical form of the gel is achieved. Mixing improves homogenization of all the components.

[0163] [Example 14] Compositions of the Invention Preparation of Mixture No. 0 (MO) in a preferred pharmaceutical formulation. The composition of the present invention comprises: a. 6.50mg / mL lactic acid (85%~90% purity) b. 3.00mg / mL ethosuximide c. 0.97μg / mL DNase d. Contains 17 mg / mL hydroxyethyl cellulose

[0164] Solution: A mixture is formulated (a+b) In 50 mL of the mixed solvent, 150 mg of ethosuximide and 318 μL of lactic acid are added.

[0165] Preparation mode: 1. 150.0 mg of ethosuximide is weighed out on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland) on a watch glass. 2. With the help of total mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. The beaker with the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 min until the solid particles are partially dissolved. 4. Next, 318 μL of lactic acid is added to the preparation. 5. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 6. The solution is brought to a final pH of 5.50 by placing 50 mL of the solution in a 100 mL beaker on a slow stirrer with a calibrated Lutron PH-206 pH meter electrode and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its aggregation. 7. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min and stored tightly sealed at 4-25°C and protected from light sources.

[0166] Pharmaceutical form: A preferred pharmaceutical form of the present invention is a gel. The gel is obtained after a one-step process. The application device comprises two containers separated by a disintegrable membrane. The formulation of the present invention comprises: Container 1: In this container 50 mL of the preparation from step 7 is placed. Container 2: Into this container, 50 μg of lyophilized rh-dornase alfa is placed in sufficient quantity to obtain a 0.97 μg / mL solution and 0.85 g of lyophilized hydroxyethylcellulose is placed in sufficient quantity to obtain a gel with the expected hydrodynamic properties.

[0167] In the mixing process, the disintegrable membrane is broken, allowing mixing of the contents of containers 1 and 2. Mixing allows achieving the final pharmaceutical form and improves homogenization of all ingredients.

[0168] [Example 15] Compositions of the Invention Preparation of Mixture No. 13 (M13) in a preferred pharmaceutical formulation. The composition of the present invention comprises: a. 3.00mg / mL Polysorbate 80 b. 1mg / mL ascorbic acid c. 6.50mg / mL lactic acid (85%~90% purity) d. 0.50mg / mL EDTA e. 3.00mg / mL ethosuximide f. 0.97μg / mL DNase g. Consists of 17mg / mL hydroxyethyl cellulose

[0169] Solution: A mixture is formulated (a+b+c+d+e) In 50 mL of the mixed solvent, 50 mg of ascorbic acid, 25 mg of EDTA, 150 mg of ethosuximide, 141 μL of polysorbate 80 and 318 μL of lactic acid are added.

[0170] Preparation mode: 1. 50 mg of ascorbic acid and 25 mg of EDTA are weighed on a watch glass on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland). 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. While stirring, 141 μL of polysorbate 80 is added with a 200 μL tip cut off. 4. A volume of 318 μL of lactic acid is then added to the preparation. 5. 150 mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. The solution is brought to a final pH of 5.5 by placing 50 mL of the solution in a 100 mL beaker on a slow stirrer along with a calibrated pH meter, Lutron PH-206, and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its aggregation. 8. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min and stored tightly sealed at 4-25°C and protected from light sources.

[0171] Pharmaceutical form: A preferred pharmaceutical form of the present invention is a gel. The gel is obtained after a one-step process. The application device comprises two containers separated by a disintegrable membrane. The formulation of the present invention comprises: Container 1: In this container 50 mL of the preparation from step 8 is placed. Container 2: Into this container, 50 μg of lyophilized rh-dornase alfa is placed in sufficient quantity to obtain a 0.97 μg / mL solution and 0.85 g of lyophilized hydroxyethylcellulose is placed in sufficient quantity to obtain a gel with the expected hydrodynamic properties.

[0172] In the mixing process, the disintegrable membrane is broken, allowing mixing of the contents of containers 1 and 2. Mixing allows achieving the final pharmaceutical form and improves homogenization of all ingredients.

[0173] [Example 16] Compositions of the Invention Preparation of Mixture No. 15 (M15) in a preferred pharmaceutical formulation. The composition of the present invention comprises: a. 2.00mg / mL Polysorbate 80 b. 0.50mg / mL ascorbic acid c. 1.63mg / mL lactic acid (85%~90% purity) d. 0.25mg / mL EDTA e. 0.50mg / mL ethosuximide f. 3.90μg / mL DNase g. Contains 17mg / mL hydroxyethylcellulose

[0174] Solution: A mixture is formulated (a+b+c+d+e) In 50 mL of the mixed solvent, 25 mg of ascorbic acid, 12.5 mg of EDTA, 25 mg of ethosuximide, 94 μL of polysorbate 80 and 80 μL of lactic acid are added.

[0175] Preparation mode: 1. 25 mg of ascorbic acid and 12.5 mg of EDTA are weighed on a watch glass on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland). 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. While stirring, 94 μL of polysorbate 80 is added with a 200 μL tip cut off. 4. A volume of 80 μL of lactic acid is then added to the preparation. 5. 25 mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. The solution is brought to a final pH of 5.5 by placing 50 mL of the solution in a 100 mL beaker on a slow stirrer along with a calibrated pH meter, Lutron PH-206, and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its aggregation. 8. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min and stored tightly sealed at 4-25°C and protected from light sources.

[0176] Pharmaceutical form: A preferred pharmaceutical form of the present invention is a gel. The gel is obtained after a one-step process. The application device comprises two containers separated by a disintegrable membrane. The formulation of the present invention comprises: Container 1: In this container 50 mL of the preparation from step 8 is placed. Container 2: Into this container, 200 μg of lyophilized rh-dornase alfa is placed in sufficient quantity to obtain a 3.90 μg / mL solution and 0.85 g of lyophilized hydroxyethylcellulose is placed in sufficient quantity to obtain a gel with the expected hydrodynamic properties.

[0177] In the mixing process, the disintegrable membrane is broken, allowing mixing of the contents of containers 1 and 2. Mixing allows achieving the final pharmaceutical form and improves homogenization of all ingredients.

[0178] [Example 17] Compositions of the Invention Preparation of Mixture No. 16 (M16) in a preferred pharmaceutical formulation. The composition of the present invention comprises: a. 3.00mg / mL Polysorbate 80 b. 2.50mg / mL ascorbic acid c. 8.67mg / mL lactic acid (85%~90% purity) d. 0.75mg / mL EDTA e. 30.00mg / mL ethosuximide f. 3.90mg / mL DNAse g. Consists of 17mg / mL hydroxyethyl cellulose

[0179] Solution: A mixture is formulated (a+b+c+d+e) In 50 mL of the mixed solvent, 125 mg of ascorbic acid, 37.5 mg of EDTA, 1500 mg of ethosuximide, 141 μL of polysorbate 80 and 424 μL of lactic acid are added.

[0180] Preparation mode: 1. 125 mg of ascorbic acid and 37.5 mg of EDTA are weighed on a watch glass on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland). 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. While stirring, 141 μL of polysorbate 80 is added with a 200 μL tip cut off. 4. A volume of 424 μL of lactic acid is then added to the preparation. 5. 1500mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. The solution is brought to a final pH of 5.5 by placing 50 mL of the solution in a 100 mL beaker on a slow stirrer along with a calibrated pH meter, Lutron PH-206, and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its aggregation. 8. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min and stored tightly sealed at 4-25°C and protected from light sources.

[0181] Pharmaceutical form: A preferred pharmaceutical form of the present invention is a gel. The gel is obtained after a one-step process. The application device comprises two containers separated by a disintegrable membrane. The formulation of the present invention comprises: Container 1: In this container 50 mL of the preparation from step 8 is placed. Container 2: Into this container, 200 μg of lyophilized rh-dornase alfa is placed in sufficient quantity to obtain a 3.90 μg / mL solution and 0.85 g of lyophilized hydroxyethylcellulose is placed in sufficient quantity to obtain a gel with the expected hydrodynamic properties.

[0182] In the mixing process, the disintegrable membrane is broken, allowing mixing of the contents of containers 1 and 2. Mixing allows achieving the final pharmaceutical form and improves homogenization of all ingredients.

[0183] [Example 18] Composition of the present invention. Preparation of Mixture No. 0 (MO) in the Laboratory The composition of the present invention comprises: a. 6.50mg / mL lactic acid (85%~90% purity) b. 3.00mg / mL ethosuximide c. Contains 0.97μg / mL DNAse

[0184] Solution: A mixture is formulated (a+b+c) In 50 mL of the mixed solvent, 150 mg of ethosuximide, 50 μg of rh-dornase alfa, and 318 μL of lactic acid are added.

[0185] Preparation mode: 1. 150.0 mg of ethosuximide is weighed out on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland) on a watch glass. 2. With the help of total mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. The beaker with the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 min until the solid particles are partially dissolved. 4. Next, 318 μL of lactic acid is added to the preparation. 5. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 6. The solution is brought to a final pH of 5.5 by placing 50 mL of the solution in a 100 mL beaker on a slow stirrer along with a calibrated pH meter, Lutron PH-206, and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its aggregation. 7. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min. 8. The 50 mL preparation from step 7 is placed in a conical tube (50 mL) and 0.05 mL of dornase alfa 1 mg / mL enzyme Pulmozyme (Roche, Basel, Switzerland) is added under sterile conditions to achieve a final concentration of 0.97 μg / mL. This step is performed in a biological safety cabinet. 9. Once sealed, the tube is mixed by vortexing for 10 seconds. 10. The preparation is protected from light using aluminum foil and then stored at 4°C to 25°C.

[0186] [Example 19] Composition of the present invention. Preparation of Mixture No. 13 (M13) in the Laboratory The composition of the present invention comprises: a. 3.00mg / mL Polysorbate 80 b. 1mg / mL ascorbic acid c. 6.50mg / mL lactic acid (85%~90% purity) d. 0.50mg / mL EDTA e. 3.00mg / mL ethosuximide f. Contains 0.97μg / mL DNase

[0187] Solution: A mixture is formulated (a+b+c+d+e+f) In 50 mL of the mixed solvent, 50 mg of ascorbic acid, 25 mg of EDTA, 150 mg of ethosuximide, 50 μg of rh-dornase alfa, 141 μL of polysorbate 80 and 318 μL of lactic acid are added.

[0188] Preparation mode: 1. 50 mg of ascorbic acid and 25 mg of EDTA are weighed on a watch glass on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland). 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. While stirring, 141 μL of polysorbate 80 is added with a 200 μL tip cut off. 4. A volume of 318 μL of lactic acid is then added to the preparation. 5. 150 mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. The solution is brought to a final pH of 5.5 by placing 50 mL of the solution in a 100 mL beaker on a slow stirrer along with a calibrated pH meter, Lutron PH-206, and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its aggregation. 8. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min. 9. The 50 mL preparation from step 8 is placed in a conical tube (50 mL) and 0.05 mL of dornase alfa 1 mg / mL enzyme Pulmozyme (Roche, Basel, Switzerland) is added under sterile conditions to achieve a final concentration of 0.97 μg / mL. This step is performed in a biological safety cabinet. 10. Once sealed, the tube is mixed by vortexing for 10 seconds. 11. The preparation is protected from light using aluminum foil and then stored at 4°C to 25°C.

[0189] [Example 20] Compositions of the Invention Preparation of Mixture No. 15 (M15) in the Laboratory The composition of the present invention comprises: a. 2.00mg / mL Polysorbate 80 b. 0.50mg / mL ascorbic acid c. 1.63mg / mL lactic acid (85%~90% purity) d. 0.25mg / mL EDTA e. 0.50mg / mL ethosuximide f. Consists of 3.90μg / mL DNase

[0190] Solution: A mixture is formulated (a+b+c+d+e+f) In 50 mL of the mixed solvent, 25 mg of ascorbic acid, 12.5 mg of EDTA, 25 mg of ethosuximide, 200 μg of rh-dornase alfa, 94 μL of polysorbate 80 and 80 μL of lactic acid are added.

[0191] Preparation mode: 1. 25 mg of ascorbic acid and 12.5 mg of EDTA are weighed on a watch glass on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland). 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. While stirring, 94 μL of polysorbate 80 is added with a 200 μL tip cut off. 4. A volume of 80 μL of lactic acid is then added to the preparation. 5. 25 mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. The solution is brought to a final pH of 5.5 by placing 50 mL of the solution in a 100 mL beaker on a slow stirrer along with a calibrated pH meter, Lutron PH-206, and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its aggregation. 8. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min. 9. The 50 mL preparation from step 8 is placed in a conical tube (50 mL) and 0.20 mL of dornase alfa 1 mg / mL enzyme Pulmozyme (Roche, Basel, Switzerland) is added under sterile conditions to achieve a final concentration of 3.90 μg / mL. This step is performed in a biological safety cabinet. 10. Once sealed, the tube is mixed by vortexing for 10 seconds. 11. The preparation is protected from light using aluminum foil and then stored at 4°C to 25°C.

[0192] [Example 21] Compositions of the Invention Preparation of Mixture No. 16 (M16) in the Laboratory The composition of the present invention comprises: a. 3.00mg / mL Polysorbate 80 b. 2.50mg / mL ascorbic acid c. 8.67mg / mL lactic acid (85%~90% purity) d. 0.75mg / mL EDTA e. 30.00mg / mL ethosuximide f. Contains 3.90μg / mL DNase

[0193] Solution: A mixture is formulated (a+b+c+d+e+f) In 50 mL of the mixed solvent, 125 mg of ascorbic acid, 37.5 mg of EDTA, 1500 mg of ethosuximide, 200 μg of rh-dornase alfa, 141 μL of polysorbate 80 and 424 μL of lactic acid are added.

[0194] Preparation mode: 1. 125 mg of ascorbic acid and 37.50 mg of EDTA are weighed on a watch glass on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland). 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. While stirring, 141 μL of polysorbate 80 is added with a 200 μL tip cut off. 4. A volume of 424 μL of lactic acid is then added to the preparation. 5. 1500mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. The solution is brought to a final pH of 5.5 by placing 50 mL of the solution in a 100 mL beaker on a slow stirrer along with a calibrated pH meter, Lutron PH-206, and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its aggregation. 8. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min. 9. The 50 mL preparation from step 8 is placed in a conical tube (50 mL) and 0.20 mL of dornase alfa 1 mg / mL enzyme Pulmozyme (Roche, Basel, Switzerland) is added under sterile conditions to achieve a final concentration of 3.90 μg / mL. This step is performed in a biological safety cabinet. 10. Once sealed, the tube is mixed by vortexing for 10 seconds. 11. The preparation is protected from light using aluminum foil and then stored at 4°C to 25°C.

[0195] [Example 22] Gel Composition of the Invention. Preparation of Mixture No. 0 (MO) in the Laboratory The composition of the present invention comprises: a. 6.50mg / mL lactic acid (85%~90% purity) b. 3.00mg / mL ethosuximide c. 0.97μg / mL DNase d. Contains 17 mg / mL hydroxyethyl cellulose

[0196] Solution: A mixture is formulated (a+b+c) In 50 mL of the mixed solvent, 150 mg of ethosuximide, 50 μg of rh-dornase alfa, and 318 μL of lactic acid are added.

[0197] Preparation mode: 1. 150.0 mg of ethosuximide is weighed out on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland) on a watch glass. 2. With the help of total mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. The beaker with the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 min until the solid particles are partially dissolved. 4. Next, 318 μL of lactic acid is added to the preparation. 5. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 6. The solution is brought to a final pH of 5.5 by placing 50 mL of the solution in a 100 mL beaker on a slow stirrer along with a calibrated pH meter, Lutron PH-206, and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its aggregation. 7. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min. 8. The 50 mL preparation from step 7 is placed in a conical tube (50 mL) and 0.05 mL of dornase alfa 1 mg / mL enzyme Pulmozyme (Roche, Basel, Switzerland) is added under sterile conditions to achieve a final concentration of 0.97 μg / mL. This step is performed in a biological safety cabinet. 9. Once sealed, the tube is mixed by vortexing for 10 seconds. Obtaining the gel: the mixture is formulated (solution + g) 10. Add 0.85 g of hydroxyethyl cellulose to the prepared mixture. 11. Mix gently until homogenous. 12. The preparation is protected from light using aluminum foil and then stored covered at 4-25°C. It should be left for between 6 and 8 hours until the air bubbles disappear.

[0198] [Example 23] Gel composition of the present invention. Preparation of Mixture No. 13 (M13) in the laboratory The composition of the present invention comprises: a. 3.00mg / mL Polysorbate 80 b. 1mg / mL ascorbic acid c. 6.50mg / mL lactic acid (85%~90% purity) d. 0.50mg / mL EDTA e. 3.00mg / mL ethosuximide f. 0.97μg / mL DNase g. Contains 17mg / mL hydroxyethylcellulose

[0199] Solution: A mixture is formulated (a+b+c+d+e+f) In 50 mL of the mixed solvent, 50 mg of ascorbic acid, 25 mg of EDTA, 150 mg of ethosuximide, 50 μg of rh-dornase alfa, 141 μL of polysorbate 80 and 318 μL of lactic acid are added.

[0200] Preparation mode: 1. 50 mg of ascorbic acid and 25 mg of EDTA are weighed on a watch glass on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland). 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. While stirring, 141 μL of polysorbate 80 is added with a 200 μL tip cut off. 4. A volume of 318 μL of lactic acid is then added to the preparation. 5. 150 mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. The solution is brought to a final pH of 5.5 by placing 50 mL of the solution in a 100 mL beaker on a slow stirrer along with a calibrated pH meter, Lutron PH-206, and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its aggregation. 8. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min. 9. The 50 mL preparation from step 8 is placed in a conical tube (50 mL) and 0.05 mL of dornase alfa 1 mg / mL enzyme Pulmozyme (Roche, Basel, Switzerland) is added under sterile conditions to achieve a final concentration of 1.00 μg / mL. This step is performed in a biological safety cabinet. 10. Once sealed, the tube is mixed by vortexing for 10 seconds. Obtaining the gel: the mixture is formulated (solution + g) 12. Add 0.85 g of hydroxyethyl cellulose to the prepared mixture. 13. Mix gently until homogenous. 14. The preparation is protected from light using aluminum foil and then stored covered at 4-25°C. It should be left for between 6 and 8 hours until the air bubbles disappear.

[0201] [Example 24] Gel Composition of the Present Invention Preparation of Mixture No. 15 (M15) in the Laboratory The composition of the present invention comprises: a. 2.00mg / mL Polysorbate 80 b. 0.50mg / mL ascorbic acid c. 1.63mg / mL lactic acid (85%~90% purity) d. 0.25mg / mL EDTA e. 0.50mg / mL ethosuximide f. 3.90μg / mL DNase g. Consists of 17mg / mL hydroxyethyl cellulose

[0202] Solution: A mixture is formulated (a+b+c+d+e+f) In 50 mL of the mixed solvent, 25 mg of ascorbic acid, 12.5 mg of EDTA, 25 mg of ethosuximide, 200 μg of rh-dornase alfa, 94 μL of polysorbate 80 and 80 μL of lactic acid are added.

[0203] Preparation mode: 1. 25 mg of ascorbic acid and 12.5 mg of EDTA are weighed on a watch glass on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland). 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. While stirring, 94 μL of polysorbate 80 is added with a 200 μL tip cut off. 4. A volume of 80 μL of lactic acid is then added to the preparation. 5. 25 mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. The solution is brought to a final pH of 5.5 by placing 50 mL of the solution in a 100 mL beaker on a slow stirrer along with a calibrated pH meter, Lutron PH-206, and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its aggregation. 8. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min. 9. The 50 mL preparation from step 8 is placed in a conical tube (50 mL) and 0.20 mL of dornase alfa 1 mg / mL enzyme Pulmozyme (Roche, Basel, Switzerland) is added under sterile conditions to achieve a final concentration of 3.90 μg / mL. This step is performed in a biological safety cabinet. 10. Once sealed, the tube is mixed by vortexing for 10 seconds. Obtaining the gel: the mixture is formulated (solution + g) 11. Add 0.85 g of hydroxyethyl cellulose to the prepared mixture. 12. Mix gently until homogenous. 13. The preparation is protected from light using aluminum foil and then stored covered at 4-25°C. It should be left for between 6 and 8 hours until the air bubbles disappear.

[0204] [Example 25] Gel Composition of the Present Invention Preparation of Mixture No. 16 (M16) in the Laboratory The composition of the present invention comprises: a. 3.00mg / mL Polysorbate 80 b. 2.50mg / mL ascorbic acid c. 8.67mg / mL lactic acid (85%~90% purity) d. 0.75mg / mL EDTA e. 30.00mg / mL ethosuximide f. 3.90μg / mL DNase g. Contains 17mg / mL hydroxyethylcellulose

[0205] Solution: A mixture is formulated (a+b+c+d+e+f) In 50 mL of the mixed solvent, 125 mg of ascorbic acid, 37.50 mg of EDTA, 1500 mg of ethosuximide, 200 μg of rh-dornase alfa, 141 μL of polysorbate 80 and 424 μL of lactic acid are added.

[0206] Preparation mode: 1. 125 mg of ascorbic acid and 37.50 mg of EDTA are weighed on a watch glass on a Radwag AS 220 R2 analytical balance (Radwag LLC, Radom, Poland). 2. With the help of mixed solvent, the substance is dissolved in a glass beaker without leaving any particles behind in the watch glass. 3. After adding the remaining 50 mL of the solvent mixture to the beaker, the preparation is placed on a NUMAK GL-3250A magnetic stirrer (Numak Technology LLC, Dubai, UAE) for 5 minutes until the solid particles are partially dissolved. While stirring, 141 μL of polysorbate 80 is added with a 200 μL tip cut off. 4. A volume of 424 μL of lactic acid is then added to the preparation. 5. 1500mg of ethosuximide is added. 6. Once these materials have been added, the mixture is allowed to stir for an additional 5 minutes. 7. The solution is brought to a final pH of 5.5 by placing 50 mL of the solution in a 100 mL beaker on a slow stirrer along with a calibrated pH meter, Lutron PH-206, and the alkaline solution is added dropwise until the pH level is achieved, taking note of the foaming produced by its aggregation. 8. The preparation is sterilized in an autoclave Arcano LS-B75L at 120°C and 1 atm overpressure for 10 min. 9. The 50 mL preparation from step 8 is placed in a conical tube (50 mL) and 0.20 mL of dornase alfa 1 mg / mL enzyme Pulmozyme (Roche, Basel, Switzerland) is added under sterile conditions to achieve a final concentration of 3.90 μg / mL. This step is performed in a biological safety cabinet. 10. Once sealed, the tube is mixed by vortexing for 10 seconds. Obtaining the gel: the mixture is formulated (solution + g) 11. Add 0.85 g of hydroxyethyl cellulose to the prepared mixture. 12. Mix gently until homogenous. 13. The preparation is protected from light using aluminum foil and then stored covered at 4-25°C. It should be left for between 6 and 8 hours until the air bubbles disappear.

[0207] EXAMPLES FOR TESTING WITH COMPOSITIONS OF THE PRESENT ART Various compositions of the present invention have been formulated and tested to demonstrate their technical effectiveness.

[0208] A summary of some compositions of the present invention that were used to evaluate the present invention can be seen in the table below:

[0209] [Table 1]

[0210] Solutions of the individual molecules were also tested (prepared as can be seen in Example 1) to demonstrate the synergistic properties of the compositions of the invention. The solutions tested were: 1. Lactic acid (LA) in mixed solvents: 1.63 mg / mL, 6.50 mg / mL, 8.67 mg / mL. Final pH of all solutions: 5.5 2. Polysorbate 80 (PS80) in mixed solvent: 2.0 mg / mL, 3.0 mg / mL. Final pH of the total solution: 5.5 3. EDTA in mixed solvents: 0.25mg / mL, 0.50mg / mL, 0.75mg / mL. Final pH of all solutions: 5.5 4. Ascorbic acid (AA) in mixed solvents: 0.5 mg / mL, 1.0 mg / mL, 2.5 mg / mL. Final pH of all solutions: 5.5 5. Ethosuximide (Etho) in mixed solvents: 0.50 mg / mL, 3.00 mg / mL, 30.00 mg / mL. Final pH of all solutions: 5.5 6. Dornase alfa (D1) in mixed solvents: 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.2 μg / mL, 15.6 μg / mL, 7.8 μg / mL, 3.9 μg / mL, 1.9 μg / mL, 0.97 μg / mL, 0.5 μg / mL. Final pH of all solutions: 5.5.

[0211] [Example 26] Anti-pathogenic effect Bacterial strains: 1) Mucoid Pseudomonas aeruginosa (internal code LEF037): A clinical isolate from a chronic venous ulcer of more than 6 months of development. 2) Methicillin-resistant Staphylococcus aureus (internal code LEF006), a clinical isolate from a chronic venous leg ulcer of >6 months duration.

[0212] Bacterial suspension: P. aeruginosa LEF037 was grown in Luria-Bertani (LB) culture medium at 37° C. for 12 hours. From this culture, a suspension (OD 600nm= 0.120) was prepared in LB. S. aureus LEF006 was grown in brain heart infusion (BHI) culture medium at 37°C for 12 hours. From this culture, a suspension (OD 600nm = 0.120) was prepared in BHI.

[0213] Controls: The following controls are used in the assays performed: a. Normal growth control for P. aeruginosa: Luria Bertani broth (LB), pH 7.2 a. Routine growth control for S. aureus: Brain Heart Infusion (BHI) medium (pH 7.2) c. Dilution effect control: Mixed solvent (MS) (pH 5.50). d. Control for freshly obtained LAPS effect: LAPS stored for 24 hours at 4° C. (pH 5.22) (LAPS 24h). e. LAPS activity maintenance control: LAPS (LAPS6m) stored at 4°C (pH 6.05) for 6 months.

[0214] Lactobacillus plantarum Supernatant (LAPS): LAPS control was prepared according to the details of Example 1. L. plantarum ATCC10241 was grown for 12 hours at 37°C in de Man Rogosa Sharpe (MRS) (Britania. Argentina). The supernatant (LAPS) was obtained by centrifugation at 8000 rpm for 15 minutes and filtered through a 0.22 μm Millipore filter. This supernatant corresponds to patent application No. AR093779 and is taken as the known product of best therapeutic effect for chronic wound treatment. This product raises some problems that the present invention intends to solve. On the one hand, it was observed from the tests raised below that its efficiency is lost over time. Regulatory problems are also raised, since it does not have an absolutely defined composition, since it depends on the fermentation process. The present invention achieves extended high stability over time, is composed of absolutely defined components, and is approved for human therapeutic use.

[0215] Solutions of individual molecules: All solutions were prepared in mixed solvents with a final pH of 5.5 according to Example 1. The solutions prepared were: 1. Lactic acid (LA) (1.63, 6.50 and 8.67 mg / mL); 2. Polysorbate 80 (PS80) (2.0 and 3.0 mg / mL); 3. EDTA (0.25, 0.50 and 0.75 mg / mL); 4. Ascorbic acid (AA) (0.5, 1.0 and 2.5 mg / mL); 5. Ethosuximide (Etho) (0.50, 3.00 and 30.00 mg / mL); 6. rh-Dornase alfa (D1) (0.97 and 3.9 μg / mL).

[0216] M0, M13, M15 and M16 (see Table 1). The compositions of the invention were worked up according to Examples 18 to 21.

[0217] Tests for the determination of the bacteriostatic effect: For the bacteriostatic assays, 96-well plates (Deltalab. Argentina) (200 μL capacity) were used. All assays were performed in triplicate using 100 μL of LB or BHI culture medium with 50 μL of bacterial suspension of P. aeruginosa or S. aureus, respectively. The following samples were then placed: a) 50 μL control (see Controls) b) 50 μL solutions of individual molecules (see solutions of individual molecules) c) A 50 μL mixture of molecules.

[0218] The microplate was then placed in a microplate reader (Multiskan Go. Thermo Scientific. Germany) using the following reading program: a) incubation at 37°C; b) slight agitation (3 seconds duration) every 58 minutes; c) absorbance reading cycle at 600 nm, once per hour for 24 hours. The initial optical density (OD) was 0.100 for all wells. Data was analyzed by calculating the mean in Microsoft Excel®. Results are shown in columns as percentage of bacteriostatic effect at 24 hours.

[0219] Tests for determination of bactericidal effect: From the tests for determination of bacteriostatic effect, 10 μL aliquots were taken from each well, serially diluted and one aliquot of each dilution was plated on LB or BHI agar depending on the bacterial strain. The initial number of colony forming units (CFU) was counted compared to the final number of CFU obtained 24 hours after treatment. The number of surviving bacteria was calculated (representing them in the columns according to the number of CFU / mL obtained after treatment).

[0220] Test for determining biofilm inhibition effect: After 24 hours of growth inhibition test, the contents of the wells were discarded and the empty wells were washed three times with sterile saline solution. The plate was dried at room temperature for 15 minutes (face down, on absorbent paper). To each well, 200 μL of 0.1% crystal violet was added and the plate was incubated at room temperature for 15 minutes with gentle and constant agitation on a vortex (Labnet) to stain the biofilm attached to the walls of the wells. After that, the contents of the wells were discarded, washed three times with sterile saline solution and dried at room temperature for 15 minutes (face down, on absorbent paper). Then, to dissolve the crystal violet attached to the biofilm, 200 μL of 96° ethanol was added to each well and the plate was incubated at room temperature for 10 minutes with gentle and constant agitation on a vortex (Labnet). The amount of crystal violet is directly proportional to the amount of biofilm formed. In this manner, a solution in each well was obtained, the color intensity of which is directly proportional to the amount of biofilm formed. Absorbance readings were taken accurately at 540 nm and 37° C. in a Multiskan Go microplate reader (Thermo Scientific). Data were processed in Microsoft Excel®, means were calculated and results are presented in columns as percentage inhibition of biofilm formation.

[0221] Test for determining biofilm disruption effect: In a 96-well plate (Deltalab), 150 μL of culture medium + 50 μL of bacterial suspension were placed. The plate was incubated for 24 hours at 37 °C in a humid chamber until biofilm formation. After that, the contents of the wells were carefully discarded and gently washed three times with sterile saline solution, avoiding the loss of biofilm attached to the wall. The plate was dried for 20 minutes at room temperature in a biological safety cabinet. Then, 150 μL of culture medium was added to each of the following: 50 μL of control, 50 μL of solution of individual molecules and 50 μL of mixture of molecules. All trials were performed in triplicate. The plate was incubated for 24 hours at 37 °C in a humid chamber. After that, the contents of the wells were discarded and washed three times with sterile saline. The plate was dried for 15 minutes at room temperature (face down on absorbent paper). The wells were stained with 200 μL / well of 0.1% crystal violet for 15 min at room temperature with gentle and constant agitation by vortexing (Labnet). The contents of the wells were discarded, washed three times with sterile saline, and the plates were dried (face down on absorbent paper) for 15 min at room temperature. Finally, 200 μL / well of 96° ethanol was added to dissolve the crystal violet attached to the biofilm. The plates were incubated for 10 min at room temperature with gentle and constant agitation by vortexing (Labnet). The absorbance of the resulting solution was measured at 540 nm (37° C.) in a Multiskan Go microplate reader (Thermo Scientific). The data were processed in Microsoft Excel®, the average was calculated, and the results are presented in columns as percentage of biofilm destruction.

[0222] Results and Discussion Figure 2: Percentage of different antipathogenic effects of bacteriostatic activity against Pseudomonas aeruginosa, inhibition of biofilm (BF) formation and disruption of preformed biofilms caused by compositions MO and M13 and by the individual molecules at the concentrations present in the compositions mentioned. The figure also shows these characteristics for LAPS 24h, LAPS 6 months and mixed solvent (MS).

[0223] Each molecule exhibited some activity for each property. Nevertheless, MO and M13 show synergistic ability for all activities. LAPS 24h had high bacteriostatic and inhibitory BF effects, but its disruptive effect on biofilm was lower than that of the composition. In addition, after 6 months, LAPS dramatically lost their antipathogenic effect.

[0224] FIG. 3 shows the bactericidal effect, expressed in viable bacteria (CFU / mL), of Pseudomonas aeruginosa 24 hours after treatment using compositions MO and M13 and the individual molecules at the concentrations present in the compositions mentioned.

[0225] As the figure shows, none of the individual molecules caused a bactericidal effect, since the levels of viable bacteria were similar to the control (no treatment). LAPS showed a significant bactericidal effect, which was lost over time. MO showed a significant bactericidal effect, while M13 caused a complete elimination of the bacteria.

[0226] FIG. 4: Percentage of different antipathogenic effects of bacteriostatic activity against Pseudomonas aeruginosa, inhibition of biofilm (BF) formation and disruption of preformed biofilms caused by individual molecules at concentrations present in composition M15 and the mentioned compositions. The figure also shows these properties for LAPS 24h, LAPS 6 months and mixed solvent (MS). Individual molecules exhibited some activity for each property. Nevertheless, M15 shows synergistic potential for all activities. LAPS 24h had high bacteriostatic and inhibitory BF effects, but its disruption effect on biofilms was lower than composition M15. In addition, after 6 months, LAPS dramatically lost their antipathogenic effect.

[0227] Figure 5 shows the bactericidal effect, expressed in viable bacteria (CFU / mL), of Pseudomonas aeruginosa after 24 hours of treatment using composition M15 and the different individual molecules at the concentrations present in the mentioned compositions. As the figure shows, none of the individual molecules caused a bactericidal effect, since the viable bacteria levels were similar to the control (no treatment). LAPS had an important bactericidal effect, which was lost over time. M15 caused a high clearance of bacteria.

[0228] Figure 6 shows the percentage of different antipathogenic effects of bacteriostatic activity against Pseudomonas aeruginosa, inhibition of biofilm (BF) formation and disruption of preformed biofilms caused by the individual molecules at the concentrations present in composition M16 and the mentioned compositions. The figure also shows these properties for 24h LAPS, 6-month LAPS and mixed solvent (MS). The individual molecules exhibited some activity for each property. Nevertheless, M16 shows a significant synergistic ability for all activities. LAPS 24h had a high bacteriostatic and inhibitory BF effect, but its disruption effect on biofilms was lower than the formulation. In addition, after 6 months LAPS dramatically lost their antipathogenic effect.

[0229] Figure 7 shows the bactericidal effect, expressed in viable bacteria (CFU / mL), of Pseudomonas aeruginosa after 24 hours of treatment using composition M16 and the different individual molecules at the concentrations present in the mentioned compositions. As the figure shows, some of the individual molecules caused a low bactericidal effect, since the viable bacteria level was similar to the control (no treatment). LAPS had an important bactericidal effect, which was lost over time, whereas M16 caused a complete elimination of the bacteria.

[0230] Figure 8 shows the percentage of different antipathogenic effects of bacteriostatic activity against Staphylococcus aureus, inhibition of biofilm (BF) formation and disruption of preformed biofilms caused by compositions MO and M13 and the individual molecules at the concentrations present in the compositions mentioned. The figure also shows these properties for 24 hours, 6 months LAPS and mixed solvent (MS).

[0231] Individual molecules exhibited some activity for each property. Nevertheless, M13 shows synergistic ability for all activities. MO exhibited synergistic effect for inhibition of biofilm (BF) formation and biofilm disruption. LAPS 24h had high bacteriostatic and inhibitory BF effect, but its disruptive effect on biofilm was lower than formulation. In addition, after 6 months, LAPS dramatically lost their anti-pathogenic effect.

[0232] FIG. 9 shows the bactericidal effect, expressed in viable bacteria (CFU / mL), of Staphylococcus aureus 24 hours after treatment using compositions MO and M13 and the different individual molecules at the concentrations present in the compositions mentioned.

[0233] As the figure shows, most of the individual molecules did not cause a bactericidal effect, since the viable bacterial levels were similar to the control (no treatment). LAPS had a significant bactericidal effect, which was lost over time. MO showed a significant bactericidal effect, but bacterial clearance by M13 was higher.

[0234] Figure 10 shows the percentage of different antipathogenic effects of bacteriostatic activity against Staphylococcus aureus (S. aureus), inhibition of biofilm (BF) formation and disruption of preformed biofilms caused by the individual molecules at the concentrations present in composition M15 and the compositions mentioned. The figure also shows these properties for 24 hours, 6 months LAPS and mixed solvent (MS).

[0235] Each molecule exhibited some activity for each property. Nevertheless, M15 shows synergistic ability for all activities. LAPS 24h had high bacteriostatic and inhibitory BF effects, but its disruptive effect on biofilm was lower than that of the formulation. In addition, after 6 months, LAPS dramatically lost their antipathogenic effect.

[0236] FIG. 11 shows the bactericidal effect, expressed in viable bacteria (CFU / mL), of Staphylococcus aureus 24 hours after treatment using composition M15 and the different individual molecules at the concentrations present in the compositions mentioned.

[0237] As the figure shows, most of the individual molecules did not cause a bactericidal effect, since the viable bacteria levels were similar to the control (no treatment). LAPS had some bactericidal effect, but this was lost over time. Finally, M15 caused a high clearance of bacteria.

[0238] Figure 12 shows the percentage of different antipathogenic effects of bacteriostatic activity against Staphylococcus aureus, inhibition of biofilm (BF) formation and disruption of preformed biofilms caused by the concentrations present in the mentioned formulations of composition M16 and the individual molecules. The figure also shows these properties for 24 hours, 6 months LAPS and mixed solvent (MS).

[0239] Individual molecules exhibited some activity for each property. Nevertheless, M16 exhibited significant synergistic potential for all activities. LAPS 24h had high bacteriostatic and inhibitory BF effects, but its disruptive effect on biofilms was lower than that of the formulation. In addition, after 6 months, LAPS dramatically lost their antipathogenic effect.

[0240] FIG. 13 shows the bactericidal effect, expressed in viable bacteria (CFU / mL), of Staphylococcus aureus 24 hours after treatment using composition M16 and the different individual molecules at the concentrations present in the compositions mentioned.

[0241] As the figure shows, some of the individual molecules caused a low bactericidal effect, since the viable bacterial levels were similar to the control (no treatment). LAPS had a significant bactericidal effect, but this was lost over time, whereas M16 caused the greatest elimination of bacteria.

[0242] The ability to disrupt biofilms is one of the most desired properties for chronic wound treatments, since patients visit the doctor when biofilms have already formed in the wound. It is known that traditional antimicrobial agents are incapable of causing biofilm disruption, especially with relative efficacy in vivo, due to the following: Each species has a different chemical composition in its biofilm matrix. · The infection is polymicrobial resulting in the formation of a mixed biofilm. · Existing treatments are directed towards specific chemical compositions.

[0243] That is why existing treatments always leave mixed biofilms behind, which become new sources of infection and make the infection chronic. A good disruption treatment should attack mixed and different species biofilms and cause close to 100% disruption.

[0244] conclusion Surprisingly, the components of the present invention exhibit synergistic bacteriostatic, bactericidal, biofilm inhibiting and disrupting properties in the bacteria most commonly identified in chronic wounds (P. aeruginosa and S. aureus). The present invention also exhibits superior efficacy against LAPS in the properties studied in P. aeruginosa and S. aureus.

[0245] [Example 27] Antioxidant Capacity of the Compositions of the Invention The method for determining antioxidant activity is based on the ability of 2,2-diphenyl-1-picrylhydrazyl (DPPH) to capture free radicals (proton donating substances are antioxidants). DPPH * The radical accepts a proton from the antioxidant to become DPPH. In this manner, the antioxidant effect is * Proportional to the decrease. * has its maximum absorbance at 517 nm and changes from purple to yellow when DPPH is formed. Therefore, the antioxidant effect could be measured after the absorbance was dropped to 517 nm by spectrophotometry. Then, the inhibition coefficient 50 (IC50) is calculated according to the initial and final absorbance values ​​of the sample. IC50 is the inhibition coefficient of 50% of DPPH. * It represents the minimum concentration of antioxidant required to reduce the oxidative stress. To obtain the IC50, two concentration-increasing curves were performed: the first one using the tested sample and the second one using the reference standard (Trolox). Finally, the percentage of relative antioxidant activity (%AAR) was calculated according to the recommendations of Sharma et al. (42).

[0246] D.P.H.P. * Preparation of the radical stock solution (SS) and working solution (WS): A solution of approximately 100 ppm DPPH in anhydrous methanol is prepared. The solution must be left in the dark at room temperature for at least 30 minutes. WS is then prepared in anhydrous methanol from SS until an absorbance of 0.800 ± 0.100 is obtained in a microplate (wavelength 517 nm).

[0247] Preparation of standard Trolox solution: Prepare SS = 500 ppm. To obtain a volume of 10 mL, 5 mg of Trolox should be dissolved in 10 mL of anhydrous methanol. Six WS were prepared at concentrations of 25, 50, 100, 150, 200 and 250 ppm.

[0248] Samples: 1) mixed solvent (MS), 2) solutions containing 0.5 and 2.5 mg / mL ascorbic acid in mixed solvent, respectively, prepared as shown in Example 1, 3) M15 (a composition of the invention made according to Example 20), 4) M16 (a composition of the invention made according to Example 21), and 5) LAPS (prepared according to the details of Example 1).

[0249] Spectrophotometry: Absorbance (Abs) readings were taken in triplicate on a spectrophotometer at 517 nm using a microplate suitable for UV-Vis. a. Place the following into wells: sample blank (12.5 μL of sample or WS 25 ppm); Sample (12.5 μL of sample or WS 25 to 250 ppm); Initial DPPH value (12.5 μL of anhydrous methanol) b. Achieve zero absorbance in absolute methanol. c. Add 250 μL of absolute methanol to the sample blank wells. d. 250 μL of DPPH * WS is added to DPPH baseline and sample wells. e. Record the absorbance at 517 nm (initial DPPHAbs). f. Set aside for 30 minutes (dark and at room temperature). g. Record absorbance at 517 nm (final DPPHAbs). h. Record the absorbance reading (Sample Blank Abs). i. Calculation of capture percentages for samples and Trolox

[0250] Curves with the reference standard (Trolox) and the analyzed samples: Two curves were fitted plotting the % capture on the vertical axis (y) and the natural logarithm (Ln) of the concentration of Trolox or the analyzed samples (ppm of TEAC or sample, respectively) on the horizontal axis (x). The value 50 was entered on the (y) axis of each graph and the IC50 was calculated using the formula obtained by linear regression of the graphs. The %AAR was then calculated using the respective IC50 values.

[0251] result In Figure 14 it can be seen that the mixed solvents do not exhibit antioxidant activity. LAPS does not have ascorbic acid, but the values ​​shown for its %AAR are due to its phenolic content. Surprisingly, compositions M15 and M16 had better antioxidant activity than their equivalent ascorbic acid concentration solutions (AA 0.5 and 2.5 mg / mL).

[0252] conclusion The compositions of the present invention enhance the antioxidant properties of ascorbic acid.

[0253] [Example 28] Chelating Ability of the Composition The following solutions were prepared in mixed solvent (MS) at pH 7.0. All solutions were supplemented with ZnCl2 (1 ppm), CaCl2 (100 ppm) and MgCl2 (25 ppm). The concentrations of Zn, Ca and Mg used here are close to those typically found in exudates from chronic wounds. The pH selected for the reaction (7.0) would result from the combination of the composition of the present invention (pH 5.5) and exudates from chronic wounds (pH 8.5). The solutions prepared were: EDTA (0.25, 0.50 and 0.75 mg / mL) as shown in Example 1; M0, M13, M15 and M16 were worked up according to Examples 18 to 21 and LAPS was deproteinized: For the LAPS 24h samples obtained according to Example 1, the deproteinization process was carried out using the reagents and instructions of the commercially available kit used.

[0254] Measurement of calcium (unchelated) remaining in the prepared solutions: A commercially available calcium determination kit (Wiener Lab) was used, which is a colorimetric method based on the reaction of calcium with o-cresolphthalein complexone (o-CPC) at alkaline pH, which produces a magenta complex that can be measured photometrically at 570 nm.

[0255] Measurement of zinc (unchelated) remaining in the prepared solutions: A commercial zinc determination kit (Randox Lab) was used, which is a colorimetric method based on the reaction of zinc with dimethylglyoxime and salicylaldoxime at alkaline pH, resulting in a colored complex that can be measured photometrically at 560 nm.

[0256] For the determination of zinc in LAPS, a previous deproteinization step was necessary. Therefore, equal volumes of LAPS and deproteinization reagent (trichloroacetic acid 370 mmol / L) were mixed in a conical tube, homogenized, and centrifuged for 10 min at 10,000 x g. The supernatant was used for the test.

[0257] result FIG. 15 shows that even with a combination of zinc, calcium and magnesium, EDTA was able to completely chelate zinc at all concentrations tested. Even with a combination of zinc, calcium and magnesium, EDTA was also able to completely chelate calcium at concentrations of 0.50, 0.75 and 1.00 mg / mL. However, EDTA 0.25 mg / mL was not able to chelate all available calcium. M0 does not have EDTA, yet it still shows chelating activity for both calcium and zinc. M15, containing 0.25 mg / mL EDTA, lost calcium chelating capacity (but not zinc chelating capacity) compared to its equivalent concentration of EDTA alone. In contrast, M13 and M16, containing 0.50 mg / mL and 0.75 mg / mL EDTA, respectively, retained maximum chelating capacity over their EDTA equivalents alone. All mixtures showed superior chelating capacity compared to LAPS, which also did not have zinc chelating capacity.

[0258] conclusion The composition has a chelating ability independent of EDTA. Surprisingly, the components of the composition improve the chelating ability of EDTA.

[0259] [Example 29] Increase and maintenance of DNAse activity from the composition over time DNAse agar is a culture medium containing highly polymerized DNA and agar as a solidifying agent. DNA is a substrate for the deoxyribonuclease enzyme (DNAse), which hydrolyzes DNA. This medium makes it possible to detect DNAse activity in different samples and microorganisms. The presence of the enzyme could be visualized by adding 1N hydrochloric acid. Hydrolyzed deoxyribonucleic acid is transparent, while polymerized deoxyribonucleic acid produces a precipitate, resulting in turbidity in the culture medium.

[0260] Plate preparation: DNAse agar plates were prepared according to the manufacturer's recommendations (Britania, CABA, Argentina). A volume of 20 mL of molten medium was used to achieve an agar height of 2 mm in the plate. Wells of 5 mm diameter were made in the agar using a sterile punch. The seeding volume in each well was therefore πxr 2 xh = πx (2.5 mm) 2 x2mm=40mm=40μL.

[0261] Calibration curve: rh-Dornase alfa solution (Roche. Basel, Switzerland) was used diluted in the mixed solvent at the following concentrations: C1 (0.1 μg / mL); C2 (0.97 μg / mL); C3 (2.0 μg / mL); 4 (3.90 μg / mL); C5 (8.0 μg / mL); C6 (10.0 μg / mL); C7 (100.0 μg / mL) gamma C8 (1000 μg / mL).

[0262] Samples analyzed: LAPS, M0, M13, M15, M16 (all gelled with hydroxyethylcellulose) were stored at 25° C. and 4° C. DNAse activity was measured once a week for 6 months. LAPS was prepared as shown in Example 1 and compositions of the invention were worked up according to Examples 22 to 25.

[0263] Plating onto DNAse agar plates: points of the calibration curve, different gelling samples were plated in duplicate.

[0264] Incubation: 24 hours, 32°C (normal human skin temperature)

[0265] Presentation: The plates were flooded by adding enough HCl (1M) to cover the entire surface of the plate. The plates were then left to stand and observed for up to 5 minutes after adding HCl. DNase activity was measured, loss of transparency around the planted zone was analyzed, and the halo diameter in millimeters was recorded.

[0266] result FIG. 16 shows that DNAse activity in the compositions of the present invention was retained for up to 6 months, while LAPS lost this enzymatic activity in less than 2 months.

[0267] As shown in Figure 17, the compositions of the present invention have higher enzymatic activity than their equivalent DNAse-only solutions. For example, MO and M13 with a DNAse concentration of 0.97μg / mL showed halos of 13-15mm, whereas DNAse concentration of 0.97μg / mL alone (C2) showed halos of 8-10mm. In addition, M15 and M16 with a DNAse concentration of 3.90μg / mL showed halos of 17-20mm, whereas DNAse concentration of 3.90μg / mL alone (C4) showed halos of 13-15mm. This demonstrates the amplification of DNAse activity for dornase alfa in the compositions of the present invention.

[0268] conclusion The components of the composition improve the DNAse activity of dornase alfa and make it possible to extend the enzymatic activity over time.

[0269] [Example 30] Angiogenic Ability of the Composition Angiogenesis is a complex biological process that involves the generation of new blood vessels from pre-existing vasculature. The process involves the cooperation of several different cell types, particularly proliferation, survival, and tissue invasion by activated endothelial cells. Most investigations of angiogenesis include at least one in vivo study. The chick chorioallantoic membrane assay (CAM) is appropriate for a variety of applications. In this assay, the angiogenic properties of different concentrations of ethosuximide and lactate and mixtures were tested.

[0270] material and method Eggs of a commercial strain with weights between 30 and 90 g were used. By using an ovoscope, the viability of the chorioallantoic membrane was determined to select only fertilized and viable eggs.

[0271] Procedure: Before placing the eggs in the incubator, they were disinfected with povidone-iodine and on days 6 to 9 of incubation, the embryonated eggs were observed with an ovoscope to determine viability and control age. After that, the air cell was identified and the eggshell was carefully broken and trimmed to facilitate exposure of the inner shell membrane. The membrane was moistened with 2 mL of NaCl (0.9%) at 37°C. The saline solution was then discarded and the shell membrane was carefully removed with forceps to access the underlying chorioallantoic membrane. Eggs with damaged chorioallantoic membrane (presence of bleeding or any other injury) were discarded.

[0272] Next, 300 μL of the following solutions were slowly placed onto the chorioallantoic membrane: ethosuximide 3.0 mg / mL; ethosuximide 0.5 mg / mL; M0; M13; M15; LAPS; negative control (PBS). Each solution was evaluated in triplicate.

[0273] Ethosuximide solution and LAPS were obtained as shown in Example 1. Meanwhile, the compositions of the present invention were prepared according to Examples 18 to 21.

[0274] The eggs were covered with clear film and photographs were taken 5 minutes, 24, 48 and 72 hours after application.

[0275] Angiogenesis test: Digital image analysis was performed using the software Image J Plus. CAM area (cm 2 The length of angiogenesis (cm) per 100 mm was assessed before addition of the solution (basal) and at 24 and 48 hours. The percentage of increase in angiogenesis was then calculated.

[0276] result FIG. 18 shows the kinetics of angiogenesis in the presence of LAPS and different solutions of ethosuximide, M0, M13 and M15. As can be seen, LAPS did not show angiogenic effects. Ethosuximide showed angiogenic properties, but it was better at 0.5 mg / mL than at 3.0 mg / mL. Mixtures M0, M13 and M15 showed increased angiogenic effects compared to solutions with the same concentration of ethosuximide.

[0277] conclusion Surprisingly, the compositions of the present invention enhance the angiogenic potential of ethosuximide.

[0278] [Example 31] Regulation of vascular endothelial growth factor (VEGF) expression by ethosuximide. material and method Cell lines were grown and maintained using minimum essential medium (MEM), fetal bovine serum (FBS), and L-glutamine according to the American Type Culture Collection (ATCC, Rockville, MD, USA) protocol.

[0279] To assess VEGF gene expression, the following cell lines were used: a. Mouse macrophage cell line J774 (ATCC), stimulated with 25 ng / mL lipopolysaccharide (LPS) from E. coli (L-8274, Sigma). b. Adult human skin keratinocyte cell line, HaCaT (ATCC). Cells were plated with different concentrations of ethosuximide for 24 hours to assess their effect on gene expression.

[0280] Monolayers of J774 macrophage cell line and HaCaT keratinocytes were used until confluent cells were obtained and stimulated with ethosuximide 0.5 and 3.0 mg / mL in a final volume of 300 μL / well in 12-well plates as shown in Example 1. For the J774 macrophage line, all treatments were supplemented with LPS (25 ng / mL), and MEM 1X with LPS (25 ng / mL) (control+LPS) was used as a control. HaCaT keratinocytes were treated with multiple concentrations of ethosuximide in MEM with 10% FBS, and 1X MEM was used as a control.

[0281] The plates were incubated for 24 hours at 37° C. in a humidified atmosphere with 5% CO2. Each experiment was performed in triplicate plates for each cell line.

[0282] VEGF and GAPDH mRNA levels were measured in triplicate for each sample using StepOnePlus Real-Time PCR Systems (Applied Biosystems) and Power Up SYBR Green Master Mix (ROCHE) kits. Primer sequences for VEGF and GAPDH were: 5'-ACCTCCACCATGCCAAGT-3' (sense) and 5'-TTGGTCTGCATTCACATCTG-3' (antisense) for VEGF and 5'-CGACCACTTTGTCAAGCTCA-3' (sense) and 5'-CCCTGTTGCTGTAGCCAAAT-3' (antisense) for GAPDH. Mean values ​​for VEGF were normalized to the mean GAPDH value for each sample. Group means were determined and normalized to the MEM group for each experiment.

[0283] Results and Discussion As a result, VEGF expression levels were induced in both macrophages and keratinocytes. The level of induction of VEGF expression occurs at 0.5 mg / mL of ethosuximide in macrophages (FIG. 19) and at 3.0 mg / mL in keratinocytes (FIG. 19).

[0284] During the proliferative phase of the normal healing process, angiogenesis occurs, allowing the repair of new blood vessels in the injured area under the control of angiogenic growth factors such as VEGF (vascular endothelial growth factor) (43, 44). In chronic wounds, VEGF tends to show reduced or absent levels (45), a plausible cause of poor vascularization of wounds, due to its low activity (46). VEGF stimulates macrophage engulfment of apoptotic cells via VEGFR-1 (46, 47). When this occurs during healing, increased expression of VEGF in macrophages helps resolve inflammation (46) and supports their passage towards the proliferative phase of healing.

[0285] Keratinocytes are also one of the main sources of VEGF during wound healing (46, 48). VEGF produced by epithelial keratinocytes acts by regulating endothelial cells in the underlying blood vessels within the dermis (46). Thus, increased expression of VEGF in keratinocytes stimulates angiogenesis.

[0286] Conclusion: There is an increase in the expression of VEGF in macrophages and keratinocytes caused by ethosuximide.

[0287] [Example 32] Wound healing effect of the composition (49) Animals: 42 male rats (8-10 weeks old) of Wistar / Cmedc strain were used, divided into 7 experimental groups of 6 animals each (groups A, B, C, D, E, F and G).

[0288] Excision wound model (50): The operation was started by anesthetizing the animal and preparing the surgical field. For this, the shaved dorsal skin was washed and disinfected with povidone-iodine and 70° ethanol solution. Then, using a sterile 5 mm diameter biopsy punch, the skin and flesh were incised in a panicle shape to create two wounds on the dorsal thorax (wound 1 corresponding to the left side of the animal and wound 2 corresponding to the right side of the animal). After that, adhesive cyanoacrylate (Tegaderm® 3M) was placed on each silicone ring and glued to the skin, separating the inside of the ring with the incision. Once the adhesive had fixed the skin and silicone ring, five simple sutures were placed on the skin, penetrating the silicone ring on its exterior, and their position was secured using polypropylene sutures. After the operation, once the animal had recovered, an Elizabethan collar was placed to prevent the animal from removing the silicone ring.

[0289] Assessment of bacterial colonization in the wound bed: To estimate the total unintended bacterial load, one animal from each experimental group was swabbed on wound 1. The resulting samples were placed in tubes containing 10 mL of sterile 1XPBS and the total bacterial load was determined by serial dilution and plated on agar (APC) for plate counting. After 24 hours of incubation at 37°C, CFU / wound were calculated.

[0290] Infection with Staphylococcus aureus ATCC 29213 to mimic chronic wound conditions: both wounds were inoculated locally in all experimental groups of animals. The inoculum was 10 μL of a bacterial suspension of S. aureus ATCC 29213 in sterile 1X PBS (1.5x10 8 The bacterial concentration in each wound was adjusted to the McFarland 0.5 scale (equivalent to 1.5x10 CFU / mL). As a result, the final bacterial concentration obtained in each wound was 1.5x10 6 The total CFU was

[0291] Treatment: Both wounds of all animals in each group were topically treated with the following hydroxyethylcellulose gelling compositions: Group A (M0), Group B (M13), Group C (M15), Group D (M16), Group E (LAPS), Group F (mixed solvent) and Group G (control without treatment). The first administration was administered 48 hours after infection, followed by daily applications until the end of the trial. Before application of the corresponding test substance, the animal's wounds were cleaned using a swab and sterile physiological solution. Until day 7, 20 μL of each test substance composition was administered to each wound, and from day 8, the dose was reduced to 10 μL.

[0292] The compositions of the invention (M0, M13, M15 and M16) were finished according to Examples 22-25.

[0293] Observation and monitoring of animals: Animals were evaluated daily for clinical signs, behavior and weight changes. In addition, wounds were photographed every 3 to 5 days using an Olympus Stylus SZ-15 digital camera and their areas were quantified using the Image ProPlus 6.0 program.

[0294] Necropsy: Animals 1, 3, and 5 from all experimental groups were sacrificed when the wounds of the untreated control group achieved 40% healing (around day 6 after surgery). Animals 2, 4, and 6 from all experimental groups were sacrificed when the wounds of the untreated control group achieved approximately 80% healing (around day 10 after surgery). At the endpoint, animals from the control groups were euthanized.

[0295] Histopathology: In skin samples from animals 2, 4 and 6 of each group, epithelialization and collagen formation were determined by digital analysis of histological images stained with Masson's trichrome.

[0296] Immunohistochemistry: Immunohistochemistry technique was performed on tissue sections to detect alpha smooth muscle actin (α-SMA) using a monoclonal antibody (mouse anti-α-SMA-Clone asm-1 Novocastra, NCL-SMA), antigenic retrieval was performed in a microwave using citrate buffer pH 6, and the reaction was revealed with a streptavidin peroxidase system.

[0297] result Figure 20 shows the percentage of wound closure at 24 hours of treatment applied to different groups of treated animals. Compositions (MO, M13, M15 and M16) were tested 24 hours after infection. The percentage of wound area closure was measured by analyzing the photos using the software Image ProPlus 6.0. Animals treated with M0 and M13 (with ethosuximide 3.00 mg / mL), M15 (with ethosuximide 0.5 mg / mL) and M16 (with ethosuximide 30.0 mg / mL) showed a higher percentage of healing compared to untreated controls.

[0298] [Table 2]

[0299] The presence of collagen was evaluated with Masson's trichrome in animals of all experimental groups (untreated control, MS, LAPS, M0, M13, M15 and M16). The histopathological classification used to estimate collagenization is the Abramov scoring system adapted from Nisbet et al., (2010), where the amount of collagen is classified as 0=absence of collagen, 1=little, 2=moderate and 3=large amount. Both wounds of each animal were studied with 5 high power fields (400X) examining the epidermis through the dermis (Nisbet et al., 2010. Effect of three types of honey on cutaneous wound healing. Wounds 22:11;275-283).

[0300] Ajwee et al. (2012). Ethosuximide and phenobarbital promote wound healing via enhancing collagenization. Chemical Biology & Drug Design, 79(1), 137-142 applied ethosuximide (Etho) to excision wounds in an albino rat model. They demonstrated that an ointment containing ethosuximide 10% w / w (80 mg / mL) in soft paraffin significantly promoted wound healing by enhancing collagenization. The inventors of this patent also applied ethosuximide in combination with other ingredients (M0, M13, M15 and M16) to an excision wound model of Wistar rats infected with Staphylococcus aureus (S. aureus). The composition of the present invention makes it possible to obtain similar or better collagenization results in infected wounds with minute amounts of ethosuximide (0.5 to 30 mg / mL). The results from both assays can be seen in the table below. The data in the Ajwee et al. column was extracted from his paper and the 10% w / w ethosuximide concentration was converted to 80 mg / mL by using the soft paraffin density value.

[0301] [Table 3]

[0302] Alpha smooth muscle actin (α-SMA) expression was studied as a specific marker of blood vessels and myofibroblast muscle layer associated with the healing process. Myofibroblasts are specialized contractile fibroblasts essential for wound closure and tissue contracture. α-SMA expression is seen as brown staining in the photograph (Figure 21). Higher amount and intensity of brown color means higher α-SMA expression. At 7 days after surgery (first end point of the trial), the untreated control (Figure 21G) and the group treated with mixed solvent (MS) gel only (Figure 21F) showed α-SMA expression in blood vessels. LAPS slightly increased α-SMA expression in myofibroblasts (Figure 21E). On the other hand, the composition-treated wounds showed α-SMA expression directly proportional to the concentration of its components. For example, MO and M15 showed moderate expression (Figure 21C), M13 showed high expression (Figure 21B), and M16 showed very high expression (Figure 21D).

[0303] Conclusion: The inventive composition accelerates the healing process, during which it stimulates collagen and alpha-smooth muscle actin production, while achieving a bactericidal effect.

[0304] References

[0305] [Table 4-1]

[0306] [Table 4-2]

[0307] [Table 4-3]

[0308] [Table 4-4]

Claims

1. Ethosuximide, DNase I, Lactic acid and 1. A pharmaceutical composition for topical wound treatment comprising:

2. 2. The pharmaceutical composition of claim 1, wherein the DNase I is selected from the group consisting of recombinant human dornase alfa, bovine pancreatic DNase I, and mixtures thereof.

3. about 0.5 mg / mL to about 50 mg / mL of ethosuximide; about 0.5 μg / mL to about 4000 μg / mL DNase I; About 1 mg / mL to about 15 mg / mL lactic acid; The pharmaceutical composition according to claim 1 or 2, comprising:

4. 4. The pharmaceutical composition of any one of claims 1 to 3, further comprising one or more tensioactive agents selected from the group consisting of polysorbate 20, polysorbate 80, polysorbate 60, sorbitan tristearate, sorbitan monostearate, octoxynol-9, nonoxynol-9, and mixtures thereof.

5. 5. The pharmaceutical composition of claim 4, comprising about 5 mg / mL or less of said tensioactive agent.

6. 6. The pharmaceutical composition of any one of claims 1 to 5, further comprising one or more complexing acids selected from the group consisting of ethylene glycol tetraacetic acid, ethylenediaminetetraacetic acid, dimercaptosuccinic acid, 2,3-dimercapto-1-propanesulfonic acid, lipoic acid (1,2-dithiolane-3-valeric acid), oxalic acid, and mixtures thereof.

7. 7. The pharmaceutical composition of claim 6, comprising about 1 mg / mL or less of said complexing acid.

8. 8. The pharmaceutical composition of any one of claims 1 to 7, further comprising one or more hydrophilic reducing acids selected from the group consisting of uric acid, ascorbic acid, lipoic acid, and mixtures thereof.

9. 9. The pharmaceutical composition of claim 8, comprising no more than about 3 mg / mL of said hydrophilic reducing acid.

10. 10. The pharmaceutical composition of any one of claims 1 to 9, further comprising one or more gelling agents selected from the group consisting of cellulose, nanocrystalline cellulose, bacterial cellulose, hydroxyethyl cellulose, hydroxypropyl cellulos, microcrystalline cellulose, carbomethyl cellulose, carbopol, and mixtures thereof.

11. 11. The pharmaceutical composition of claim 10, comprising about 25 mg / mL or less of one of said gelling agents.

12. 0.2M acetic acid / 0.2M sodium acetate: 2.78-0.66% (v / v), pH=4.2-4.8; 0.10M acetic acid / 0.01M sodium acetate: 1.05-10.05% (v / v), pH=5.6; 0.05M potassium phosphate monobasic, pH=4.5; 0.36M potassium phosphate monobasic / 0.07M sodium phosphate disodium: 4.92-0.98% (w / v), pH=5.7; 0.36M potassium phosphate monobasic / 0.10M sodium phosphate disodium: 4.92-1.49% (w / v), pH=6.0; 0.31M citric acid / phosphate disodium 0.20M: ratio 2.99-1.42% (w / v), pH = 5.8; citric acid 0.10M / sodium citrate 0.03M: ratio 1.92-0.77% (w / v), pH = 5.8; Sorensen phosphate buffer: sodium phosphate monobasic 0.2M / sodium phosphate disodium 2.3M: ratio 1.2-16.33% (w / v), pH = 5.8; Hanks' balanced salt solution (HBSS): sodium chloride 0.14M (0.800%), potassium chloride 5mM (0.040%), calcium chloride 1mM (0.014%), magnesium sulfate heptahydrate 0. 4 mM (0.010%), magnesium chloride hexahydrate 0.5 mM (0.010%), disodium phosphate dihydrate 0.3 mM (0.006%), potassium phosphate monobasic 0.4 mM (0.006%), glucose 6 mM (0.100%), sodium bicarbonate 4 mM (0.035%): pH = 5.7; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 1 M (pH = 6.5); 2-(N-morpholino)ethanesulfonic acid sodium salt, 4-morpholineethanesulfonic acid (MES sodium salt) 0.5 M (pH = 5.5- 6.7); N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid sodium salt (BES) 0.5M (pH=6.0); N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) 0.5M (pH=6.0); 2,2'-[(2-amino-2-oxoethyl)imino]diacetic acid (ADA) 0.2M (pH=6.0); piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) 0.5M (pH=6.0-6.8); 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) 0.2M (pH=6.0);The pharmaceutical composition of any one of claims 1 to 11, further comprising a solution selected from the group consisting of saline (0.9%) / MgCl2 (5 mM), pH 5.5; 1M NaHCO3 solution, and combinations thereof.

13. The pharmaceutical composition of claim 1, wherein the DNase I comprises recombinant human dornase alfa.

14. one or more hydrophilic reducing acids; one or more tensioactive agents; one or more complexing acids; one or more gelling agents; Solution and 14. The pharmaceutical composition of claim 13, further comprising:

15. The method of claim 1, wherein the DNase I comprises recombinant human dornase alfa, The pharmaceutical composition comprises: Ascorbic acid, Polysorbate 80, Ethylenediaminetetraacetic acid, Hydroxyethyl cellulose, Saline 0.9% and MgCl ２ A solution containing 5 mM of pH 5.5 2. The pharmaceutical composition of claim 1, further comprising:

16. The pharmaceutical composition according to any one of claims 1 to 15, having a pH between 4.5 and 6.

8.

17. about 0.5 mg / mL to about 30 mg / mL of ethosuximide; about 1 μg / mL to about 4 μg / mL of recombinant human dornase alfa, and About 1 mg / mL to about 9 mg / mL lactic acid and about 2.5 mg / mL or less of ascorbic acid; about 3 mg / mL or less of polysorbate 80; about 0.75 mg / mL or less of ethylenediaminetetraacetic acid; Hydroxyethylcellulose, about 17 mg / mL or less; 2. The pharmaceutical composition of claim 1, further comprising:

18. about 0.5 mg / mL to about 3 mg / mL of ethosuximide; about 0.97 μg / mL to about 3.9 μg / mL of recombinant human dornase alfa; About 1.6 mg / mL to about 8.7 mg / mL lactic acid; Including, about 2.5 mg / mL or less of ascorbic acid; about 3 mg / mL or less of polysorbate 80; about 0.75 mg / mL or less of ethylenediaminetetraacetic acid; Hydroxyethylcellulose, about 17 mg / mL or less; 2. The pharmaceutical composition of claim 1, further comprising:

19. 1. A pharmaceutical preparation kit for topical wound treatment comprising a first composition and a second composition, which are mixed before use to obtain a pharmaceutical composition, a. the first composition is solid and comprises DNase I and a gelling agent powder; b. the second composition is an aqueous liquid comprising ethosuximide and lactic acid; c. The pharmaceutical formulation kit, wherein the second composition has a pH between 4.5 and 6.

8.

20. A pharmaceutical preparation kit for topical wound treatment comprising a first composition, a second composition and a third composition, which are mixed before use to obtain a pharmaceutical composition, a. the first composition is solid and comprises a gelling agent powder; b. the second composition is a solid and comprises DNase I; c. the third composition is an aqueous liquid comprising ethosuximide and lactic acid; d. The pharmaceutical formulation kit, wherein said third composition has a pH between 4.5 and 6.

8.

21. 20. The pharmaceutical formulation kit of claim 19, wherein each of the first composition and the second composition is contained in a sealed individual container.

22. 21. The pharmaceutical formulation kit of claim 20, wherein each of the first composition, the second composition and the third composition is contained in a sealed individual container.

23. 20. A method for preparing the pharmaceutical composition of claim 19, comprising: a. contacting the first composition with the second composition; and b. Mixing and shaking to obtain said pharmaceutical composition. The method includes:

24. 11. A method for preparing the pharmaceutical composition of claim 10 from a pharmaceutical formulation comprising a first composition, a second composition and a third composition mixed prior to use, wherein the first composition is solid and comprises a gelling agent powder, the second composition is solid and comprises DNase I, the third composition is an aqueous liquid comprising ethosuximide and lactic acid, and the third composition has a pH between 4.5 and 6.8, the method comprising: a. contacting the third composition with the second composition; b. mixing and shaking the third composition and the second composition to form a mixture (b); c. contacting the first composition with the mixture (b) to form a mixture (c); d. Mixing and shaking to obtain said pharmaceutical composition. The method includes:

25. A pharmaceutical composition according to any one of claims 1 to 18, applied to a wound at least once daily.

26. The pharmaceutical preparation kit of any one of claims 19 to 22, wherein the pharmaceutical composition is applied to the wound at least once a day.

27. 26. The pharmaceutical composition of claim 25, wherein the wound is selected from the group consisting of diabetic foot ulcers, venous ulcers, arterial ulcers, pressure ulcers, mechanical wounds characterized by superinfection with biofilm forming bacteria, post-operative wounds characterized by superinfection with biofilm forming bacteria, and combinations thereof.

28. 27. The pharmaceutical preparation kit of claim 26, wherein the wound is selected from the group consisting of diabetic foot ulcers, venous ulcers, arterial ulcers, pressure ulcers, mechanical wounds characterized by superinfection with biofilm forming bacteria, post-operative wounds characterized by superinfection with biofilm forming bacteria, and combinations thereof.

29. A device comprising the pharmaceutical formulation kit of claim 19, a first container containing a first composition; a second container containing a second composition; Including, A device, wherein the first container and the second container are separated by a disruptable membrane capable of separating the first composition and the second composition.

30. A device comprising the pharmaceutical formulation kit of claim 20, a first container containing a first composition; a second container containing a second composition; a third container containing a third composition; and Including, the first container and the second container are separated by a first disruptable membrane capable of separating the first composition from the second composition; The device, wherein the second container and the third container are separated by a second disruptable membrane capable of separating the second composition and the third composition.

31. A method for producing the pharmaceutical composition of claim 1, comprising the steps of: a. mixing ethosuximide, DNase I, and lactic acid in a solution, thereby forming a mixture; b. adding an alkaline solution dropwise to the mixture until the pH of the mixture is between 4.50 and 6.50, thereby forming a pH-adjusted mixture; and c. Sterilizing the pH adjusted mixture. The method includes:

32. d) The method of claim 31, further comprising the step of adding a gelling agent.

33. The method of claim 31, further comprising the steps of: e) adding a complexing acid, a tensioactive agent, and a hydrophilic reducing acid.

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