CHITOSAN GELS (A) CONTAINING METAL NANOPARTICLES OF COPPER, SILVER AND ANTIBIOTICS (CIPROFLOXACIN, CEFOTAXIME, GENTAMICIN AND CLOXACILLIN)

Abstract

The present invention describes chitosan gel (poly-beta-glucosamine) having bacteriocidal and fungicidal properties in a mixture with nanoparticles of copper and/or silver, and/or antibiotics (ciprofloxacin, cloxacillin, gentamicin and cefotamine, and a mixture of ciprofloxacin and cloxacillin, gentamicin and cefotaxim), and a process of obtaining and using the described gel.

Claims

1. Method for preparing a biodegradable and biocompatible gel, CHARACTERIZED in that said method includes the following steps: i) preparation of chitosan gels: a chitosan polymer with a deacetylation degree higher than 90% and a molar mass between 25,000 and 500,000 g/mol is used, which is obtained by dissolution at room temperature (15-25° C.) in weak organic acids (acetic acid, formic acid, lactic acid, among others) until a concentration between 1 and 5% by mass is achieved; ii) preparation of chitosan mixed with antibiotics: one or more antibiotics are added to the chitosan matrix obtained in (i) by the method of dispersion and agitation in a gaseous nitrogen atmosphere, wherein the antibiotics are at a concentration of 0.1 to 5% by mass and in a ratio of 1:1, if combined; iii) preparation of nanoparticles: co-deposition reaction: this method involves the physical co-deposition of metal vapors (copper and silver) with organic vapors (2-ethoxyethanol) in liquid nitrogen, in a reactor of metal atoms; the metal to be evaporated is introduced into an alumina crucible; the magnet and the solid chitosan are introduced into the bottom of the reactor; the flask is connected to the solvent to be used (2-ethoxyethanol) and the whole system is evacuated until a high vacuum of 10.sup.−5 bar is reached. The reactor is then immersed in a 5000 ml Dewar with liquid nitrogen. The power source is turned on up to 40A, which evaporates both the solvent and then the metal at its respective boiling temperature, penetrating the solvent into the reactor in vaporous state. The power source is turned off, the metal and the solvent are co-deposited in a process called nucleation for a period of approximately 60 minutes, finally forming a frozen “metal-organic” matrix on the internal walls of the reactor; iv) preparation of chitosan doped with nanoparticles The chitosan doped with the metallic nanoparticles is obtained by defrosting the “metal-organic” matrix found on the internal walls of the reactor. The matrix is shaken therein for 12 hours with a magnetic stirrer, and it rests for a period of approximately 2 hours, thus obtaining the “metal-organic-polymer” matrix from which it is extracted by connecting a receiving flask to the vacuum line. Once the matrix is removed, the solvent is evaporated, which acts as a support for the nanoparticle/chitosan mixture until solid chitosan particles are obtained. These particles are doped with metallic nanoparticles, wherein said particles are in a concentration of 1 to 5% by mass with respect to the chitosan polymer.

2. The method for preparing a biodegradable and biocompatible gel, according to claim 1, CHARACTERIZED in that the antibiotics correspond to ciprofloxacin and/or cloxacillin alone or in a mixture thereof and/or cefotaxime and/or gentamicin alone or in a mixture thereof.

3. The method for preparing a biodegradable and biocompatible gel, according to claim 1, CHARACTERIZED in that the nanoparticles correspond to copper and/or silver alone or in a mixture thereof.

4. A biodegradable and biocompatible gel, CHARACTERIZED in that said gel comprises a chitosan polymeric matrix in a concentration of 1 to 5% by mass, containing metallic nanoparticles in a range from 10 to 100 nm, in a concentration of 1 to 5% by mass and/or containing antibiotics in a concentration of 0.1 to 5% by mass and additives between 1 to 5% by mass.

5. Gel according to claim 4, CHARACTERIZED in that the chitosan corresponds to poly-beta-D-glucosamine and the molar mass of the polymeric matrix ranges between 25,000 and 500,000 g/mol with a degree of purity greater than 95% by mass.

6. Gel according to claim 4, CHARACTERIZED in that the metallic ions correspond to copper and/or silver alone or in a mixture thereof.

7. Gel according to claim 4, CHARACTERIZED in that the antibiotics correspond to ciprofloxacin and/or cloxacillin alone or in a mixture thereof and/or cefotaxime and/or gentamicin alone or in a mixture thereof.

8. Gel according to claim 4, CHARACTERIZED in that the combined antibiotics are in a 1:1 ratio.

9. Gel according to claim 4, CHARACTERIZED in that the additives correspond to carbopol, hydroquinone, guar gum, xanthan gum and/or gum arabic.

10. Gel according to claim 4, CHARACTERIZED in that it is useful for treating bacterial skin infections whether they are neuropathic ulcers or diabetic foot.

11. Gel according to claim 4, CHARACTERIZED in that it is useful for the restoration of epidermal and dermal tissues, neovascularization and angiogenesis.

12. Gel according to claim 4, CHARACTERIZED in that it is useful as a matrix for fibroblast cell growth in epidermal and dermal tissues.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0072] The present application describes the preparation of topical use gels containing a chitosan matrix and nanoparticles charged with metallic nanoparticles, metallic ions and/or antibiotics; these components provide the gels with tissue regenerating and antimicrobial properties, which are useful for use on skin suffering from lesions and/or bacterial infections.

[0073] Method of Production

[0074] Step 1—Elaboration and Characterization of Gel Formulations Synthesis of Gels

[0075] i) Preparation of chitosan gels

[0076] The formulations will be prepared according to classic techniques of pharmaceutical technology for the preparation of semi-solid pharmaceutical forms.

[0077] A chitosan polymer with a deacetylation degree higher than 90% and a molar mass between 25,000 and 500,000 g/mol is used, which is obtained by dissolution at room temperature (15-25° C.) in weak organic acids (acetic acid, formic acid, lactic acid, among others) until a concentration between 1 and 5% by mass is achieved.

[0078] ii) Preparation of chitosan mixed with antibiotics

[0079] One or more antibiotics are added to a chitosan matrix in a concentration of 0.1 to 5% by mass and in a ratio of 1:1 if there is a combination of antibiotics, by means of the method of dispersion and agitation in gaseous nitrogen atmosphere.

[0080] iii) Preparation of chitosan gel doped with metallic nanoparticles [0081] a) Co-deposition reaction: this method involves the physical co-deposition of metal vapors with organic vapors (2-ethoxyethanol) in liquid nitrogen, in a reactor of metal atoms; the metal to be evaporated is introduced into an alumina crucible; the magnet and the solid chitosan are introduced into the bottom of the reactor; the flask is connected to the solvent to be used (2-ethoxyethanol) and the whole system is evacuated until a high vacuum of 10.sup.−5 bar is reached. The reactor is then immersed in a 5000 ml Dewar with liquid nitrogen. The power source is turned on up to 40A, which evaporates both the solvent and then the metal at its respective boiling temperature, penetrating the solvent into the reactor in vaporous state. The power source is turned off, the metal and the solvent are co-deposited in a process called nucleation for a period of approximately 60 minutes, finally forming a frozen “metal-organic” matrix on the internal walls of the reactor (Chemical Liquid Deposition). [0082] b) Preparation of chitosan doped with nanoparticles: the chitosan doped with the metallic nanoparticles is obtained by defrosting the “metal-organic” matrix found in the internal walls of the reactor. The matrix is shaken for 12 hours with a magnetic stirrer, and it rests for a period of approximately 2 hours, thus obtaining the “metal-organic-polymer” matrix from which it is extracted by connecting a receiving flask to the vacuum line. Once the matrix is removed, the solvent is evaporated, which acts as a support for the nanoparticle/chitosan mixture until solid chitosan particles are obtained. These particles are doped with metallic nanoparticles. (Solvated Metal Atom Dispersion, SMAD). The concentration of nanoparticles ranges between 0.1 and 5% by mass.

[0083] iv) Characterization of chitosan doped with nanoparticles: Ag and Cu nanoparticles supported in chitosan are characterized by Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) equipped with an energy dispersive X-ray analyzer coupled to the microscope, thermogravimetric analysis (TGA) and Fourier Transform Spectroscopy (FTIR).

[0084] v) Quality Control—the antimicrobial activity of metallic nanoparticles and/or antibiotics supported in chitosan is analyzed through susceptibility tests to pathogenic microorganisms Escherichia coli, Staphylococcus aureus, Salmonella typhymorium, S. epidermidis and Candida albicans through dilution in broth and disc diffusion.

[0085] The following are examples of the preparation, application and use of antimicrobial gels, which allow a better description of the present invention, but they do not limit in any way the scope thereof.

EXAMPLES

[0086] 1. Determination of the antibacterial activity of chitosan gels with antibiotics

[0087] Chitosan gels were added with antibiotics: gentamicin, ciprofloxacin, cloxacillin and cefotaxime and two mixtures thereof. For each antibiotic two concentrations were tested—a low concentration (LC=1 mg/ml) and a high concentration (HC=10 mg/ml). Furthermore, gels of copper sulfate (2.64 mg/ml), copper oxychloride (1.5 mg/ml) and copper nanoparticles were added. The antibacterial activity of the chitos a gels with antibiotic and/or copper salts is determined by measuring the inhibition halos using an agar diffusion method. For this purpose, the microorganisms to be tested were cultured on tryptictic soy agar +5% of lamb blood for 18-24 hours at 35° C. From these cultures, a suspension adjusted to 0.5 Mc Farland was prepared in sterile distilled water and plates containing 20 ml of Mueller-Hinton agar were inoculated with a swab. Once the plates were inoculated, 6 mm diameter wells were prepared in the agar (5 per plate) with the help of a glass rod (Pasteur pipette). Approximately 30 g of antibiotic gel is placed on the wells. The plates were incubated at 35° C. for 24 hours and the inhibition halo was measured.

[0088] Six bacterial strains were used to carry out this test: Pseudomonas aeruginosa (ATCC 27853), Pseudomonas sp. (multi-resistant strain of clinical origin) Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 29212) and two strains of Staphylococcus aureus (ATCC 29213 y ATCC 25923).

TABLE-US-00001 TABLE 1 Inhibition haloes (mm) of chitosan gels added with antibiotics or copper salts on different bacterial species Pseudomonas sp. E. coli S. aureus E. faecal S. aureus Antimicrobial in ATCC 27853 ATCC ATCC is ATCC ATCC chitosan gel P. aeruginosa Clinic* 25922 25923 29212 29213 Gentamicin/lactic acid/LC 35 35 35 28 31 30 Gentamicin/lactic acid/HC 31 29 40 37 30 36 Cefotaxime/lactic acid/LC 18 18 37 32 26 33 Cefotaxime/acetic acid/LC 15 10 30 27 27 28 Cefotaxime/lactic acid/HC 41 40 45 31 34 30 Cloxacillin/lactic acid/LC 6 6  6 6 6 6 Cloxacillin/lactic acid/HC 6 6 n.d. 8 6 6 Ciprofloxacin/lactic acid/HC 40 40 40 32 32 30 Ciprofloxacin/lactic acid/LC 40 40 44 30 34 30 Gentamicin-cephotaxime/acetic 32 37 37 26 30 25 acid/LC Gentamicin-cephotaxime/lactic 38 40 41 35 37 35 acid/LC Cloxacilin-cyprophloxacin/lactic 40 40 44 30 34 30 acid/HC Copper Nanoparticle/lactic 6 6 20 8 6 6 acid Copper sulfate/acetic acid 9 9 15 6 6 6 Copper oxychloride/acetic 14 13 20 11 14 15 acid *multiresistant strain

[0089] In general, it is observed that ciprofloxacin is the antibiotic that shows more activity against the tested microorganisms, having inhibition halos that ranged between 30 and 44 mm and between 30 and 46 mm for the antibiotic at low and high concentrations, respectively. Furthermore, the best activity occurred against Gram-negative bacteria. These results are consistent with the corresponding information in the literature. However, it shall be noted that there are no important differences in the diameters of the halos when comparing the antibiotic at low and high concentrations for the same microorganism.

[0090] 2. Tests on humans

[0091] A descriptive, longitudinal and prospective therapeutic intervention study was conducted.

[0092] Thirty patients, who met the eligibility criteria, were included, configuring the sample for the current study. All patients started the study on the same day and remained in the study until the completion thereof. Conventional anamnesis, physical examination, nursing, clinical laboratory, anatomopathological and microbiological techniques were used for blood extraction and collection of biomaterial for biopsy and culture. Patients were treated with the combination of chitosan-silver gel and ciprofloxacin-cloxacillin gel, three times a week up to two weeks, and they were monitored for 21 days after the last therapeutic application. The application procedure was characterized by photographic record, topical application of the combined therapeutic product in the proportion of 1 mm on the whole surface of the lesion and placement of sterile dressing after full absorption of the therapeutic product. Photographic records were used to evaluate exposure to diagnosis and treatment. The outcome was evaluated by categorizing the clinical improvement of patients as a function of time. The researchers evaluated their consistency with the collected photographs to identify possible interpretation and typing errors. These inconsistencies were discussed in a group and corrected according to the data established in the photographs. From the digitalization of the photographic images, the tracking over time was framed and the information obtained was used to design a model that allowed establishing the risk that a patient has of clinical improvement. For the clinical classification of skin ulcers a technique validated in Chile in previous studies was used, which develops the following parameters, where Type 1 expresses the lowest degree of pathological affectation, whereas Type 4 shows the highest one.

TABLE-US-00002 1 2 3 4 ASPECT Erythematous Reddened Yellow Necrotic Greater extension 0-1 cm >1-3 cm >3-6 cm >6 cm Depth 0   >1 cm  1-3 cm >3 cm Exudate amount Absent Rare Moderate Abundant Exudate quality No exudate Serous Turbid Purulent Sphacellated or necrotic tissue Absent <25% 25%-50% >50% Granulatory tissue 100-75% 50%-<75%  25%-<50% <25% Edema Absent + ++ +++ Pain 0-1  2-3   4-6 7-10 Surrounding skin Healthy Peeled Erythematous Macerated

[0093] The process of application of the therapeutic product was always preceded by asepsis actions concerning the region involved in the lesion to be treated. The study was developed as expressed by the following algorithm:

TABLE-US-00003 Days Activities 0 21 42 Clinical Evaluation and X Selection Consultation Distribution of patients X according to age, sex and morbid history. Photographic record of the X X X skin ulcers and of the alterations in the solution of continuity of the skin. Classification of skin ulcers X X based on the assessment of macroscopic morphological parameters Application set by protocol of therapeutic gel I and therapeutic gel II (described in annex: product data sheet) Obtaining the analytical X X parameters Obtaining the anatomopathological X X X and microbiological parameters

[0094] Description and operationalization of variables

TABLE-US-00004 Operational Dimension Variable Classification definition Scale Indicator Sociodemographic Age Qualitative In exact years a) 18-37 years old Absolute and morbid Ordinal completed according b) 38-55 years old frequency history Polytomous to date of birth at the c) ≥56 years old and percentage time of the study. Sex Qualitative It was considered a) Male Absolute Nominal according to the b) Female frequency Dichotomous condition of the and percentage individual determined by the characteristics biologically given by the genes. AH Qualitative The presence of AH in a) Yes Absolute Nominal a patient was b) No frequency Dichotomous considered, if she/he and percentage was previously diagnosed by her/his doctor, if she/he was being treated with antihypertensive drugs, or if the diagnosis was made during hospital admission according to the criteria proposed by the Joint National Committee..sup.7

TABLE-US-00005 Operational Dimension Variable Classification definition Scale Indicator Sociodemographic DM Qualitative The presence of DM in a) Yes Absolute and morbid Nominal a patient was b) No frequency history Dichotomous considered, if she/he and percentage was previously diagnosed by her/his doctor, if she/he was being treated with hypoglycemic drugs, or during the investigation the diagnosis was made according to the criteria of the American Diabetes Association..sup.8 Peripheral Qualitative The set of syndromic, a) Yes Absolute arterial Nominal acute or chronic b) No frequency disease Dichotomous conditions was and percentage considered, which are generally derived from the presence of an occlusive arterial disease that conditions an insufficient blood flow to the limbs .sup.9 Sociodemographic Peripheral Qualitative The set of syndromic, a) Yes Absolute and morbid venous Nominal acute or chronic b) No frequency history disease Dichotomous conditions was and percentage considered, which are generally derived from the presence of an occlusive venous disease that conditions the imbalance established at the microcirculation level between some factors favoring the venous return and other factors hindering the same. Basic Analytical Quantitative Analytical data of Numerical Mean and background biological Continuous hematological, renal and values Standard of the parameters hepatic function Deviation organism's (Leukocytes, Urea, baseline Creatinine, GOT, GPT, GGT) obtained through clinical laboratory methods were considered. Classification Skin ulcer Qualitative It was considered from a) Type 1 Absolute of skin Ordinal the application of a (score10-15) frequency ulcer Polytomous validated instrument a) Type 2 and percentage that typifies ulcers (score16-21) from the result of the a) Type 3 general score (score 22-27) originated from an c) Type 4 assessment by (score 28-40) parameters between 1 and 4 points of macroscopic morphological aspects (appearance, greater extension, depth, amount of exudate, quality of exudate, sphacellated or necrotic tissue, granulatory tissue, edema, pain, surrounding skin) explored at the site of injury.

[0095] Temporary profile Conceptual definition: time was defined as the days between the first and last control photograph.

[0096] Anatomopathological profile Conceptual definition: The preparation of a pathological anatomical report was considered as such, wherein said report was prepared from the microscopic study of tissue obtained from the skin ulcer and obtained by means of culture that describes the characteristics of the global tissue behavior of the ulcers in the patients.

[0097] Microbiological profile Conceptual definition: The preparation of a microbiological report from the study of the tissue culture obtained from the skin ulcer was considered as such.

[0098] Erythematous or epithelial tissue Conceptual definition: pink appearance, shiny, fragile in its beginnings, it indicates that it is in the remodeling phase or by compression or burning that affects the dermis without loss of skin integrity.

[0099] Reddened tissue Conceptual definition: presence of vascularized and fragile granulation tissue indicating that it is in the proliferative phase.

[0100] Pale tissue Conceptual definition: presence of fibrin, an insoluble protein derived from fibrinogen by the action of thrombin and it is pale yellow in color. The pallor of the tissue can also be due to hypoxia (low concentration of oxygen in the tissues) or ischemia (localized deficit of blood in the tissue produced by functional vasoconstriction or obstruction of the vessels).

[0101] Necrotic tissue Conceptual definition: presence of dead, dry, hard and black tissue, although the dead connective tissue may be gray and soft.

[0102] Extension Conceptual definition: it is expressed in the largest diameter or diameter with the greater extension The measuring tools can be a circular template, acetate grid template or a flexible ruler. All of this assisted by a photographic image taking.

[0103] Depth Conceptual definition: in cavitated ulcers there may be muscle, tendon or bone involvement, being sometimes difficult to access. For the measurement thereof a swab is used to take a culture, placing it at the deepest point and measuring up to the upper edge of the wound. When the wound has sacks or lateral folds, its measurement is made according to the pointers of the clock having as base the 12, which corresponds to the head of the person who is being evaluated.

[0104] Exudate Conceptual definition: it is an important part of the wound defense mechanism. The exudate is formed on the surface as a result of fluid loss from small blood vessels, it decreases with healing and disappears with epithelialization.

[0105] Exudate amount

[0106] Rare=1-5 cc

[0107] Moderate=5-10 cc

[0108] Abundant=>10 cc

[0109] Exudate quality [0110] Serous: clear, transparent, yellowish or pink liquid. [0111] Turbid: formed by the mixture of the exudate from the wound healing process, and detritus from the debridement. [0112] Purulent: thick yellowish or greenish liquid, secreted by an inflamed tissue, and composed of serum, leukocytes, dead cells and fat (cholesterol and glucose).

[0113] Sphacellated or necrotic tissue Conceptual definition: pale, hypoxic or ischemic tissue. The presence of necrotic or sphacellated tissue on the wound surface is measured as a percentage.

[0114] Granulation tissue Conceptual definition: red, moist, and fragile connective tissue that fills the wound during the proliferative phase of healing.

[0115] Edema Conceptual definition: it is the excess fluid in the tissues underlying the wound and is measured through finger pressure.

[0116] edema+=<0.3 cm

[0117] edema++=0.3-0.5 cm

[0118] edema+++=>0.5 cm

[0119] Pain Conceptual definition: Analogical scale

TABLE-US-00006   No pain Worst pain   1.sub.——.sub.——.sub.——.sub.——.sub.——.sub.——.sub.——.sub.——4

[0120] Surrounding skin Conceptual definition: the skin near the wound can be modified regarding the cutaneous integrity due to mechanical effects or effects related to the inflammatory process, which can spread into the injury or have consequences. [0121] Healthy skin=Undamaged skin [0122] Peeling=Exfoliation of keratinized cells of variable size; silver, white or tan colored that indicates dryness of the skin and propensity to cracks and fissures. [0123] Erythematous=Epidermis reddened due to mechanical action, pressure, skin friction or irritative dermatitis. It can be accompanied by local heat. [0124] Macerated skin=It shows excoriations (skin lesions due to loss of very superficial substance that only affects the epidermis) and decomposition of tissues by being in contact with a humid environment.

[0125] Information Processing Techniques

[0126] The data was stored and processed automatically in the Statistical Package for Social Sciences (Version 19.0 for Windows). Absolute and percentage frequencies were used as summary measures in the qualitative variables. In the quantitative variables, the mean and standard deviation were used as a summary measure. From the digitalization of the photographic images, the tracking over time was framed and the information obtained was used to design a model that allowed establishing the risk that a patient has of clinical improvement according to the associated probability of Poisson, a confidence level of 90% was established. The statistical significance of the variation in ulcer classification before and after treatment was determined by using the Wilcoxon signed-rank test. A confidence level of 95% was established and any value of p 0.05 was considered significant for the statistician associated with the test. The results were summarized in tables and general explanatory text notes.

[0127] Results and Discussion

[0128] Thirty patients were evaluated, 10 of them were men and 60% of the patients were 58 years and older (Table 1). The time in years of the ulcers and alterations in the solution of continuity of the skin of these patients summed up to 283 years.

[0129] The ulcers were between 1 and 24 square centimeters in area. 73.3% of the patients had an ulcer with an etiology related to venous disease, followed by 13.3% whose etiology was mixed (arterial and venous), although only 46.2% of the patients reported pain, for 56.6% of these patients the pain was intense and exceeded level 6 according to the visual analog scale, and 2 patients reported a pain level of 8 and another 2 patients called the involved pain as the highest level of pain in their lives, qualifying it is with a value of 4. Diabetes mellitus was present in 40% of patients and high blood pressure in 36.6% (Table 2).

[0130] In Table 3 and graph 1 it was reported that most patients showed Type-4 skin ulcers (70%), followed by those with Type-3 skin ulcers (16.6%). It should be noted that there was only one patient with a type-1 ulcer (Table 3). Table 4 reports that after therapeutic intervention a decrease in patients of frequency of Type-4 skin ulcer (40.0%) and Type-3 (23.3%) was obtained, whereas Type-1 skin ulcers (16.6%) increased.

[0131] By conducting the Wilcoxon signed-rank Test (Table 5) it was shown that the regression of skin ulcer severity in the series of patients studied—after the therapeutic intervention, expressed in the reduction of the frequency of Type-4 skin ulcers (70.0% vs. 40.0%; p=0.01; Z=−1.342) and in the increase of Type-1 (3.3% vs. 16.3%; p=0.00; Z=−6.042) is statistically significant.

[0132] The analysis results of the contribution of the therapeutic protocol through the test of Wilcoxon signed-rank Test demonstrated that the applied intervention produced favorable changes in the clinical progression in the series of patients studied. It was evidenced that there was a contribution to the reduction of the injured area without signs of infection and with active borders (Table 5). A value of probability of 0.00 and 0.01 was achieved, which allows rejecting the null hypothesis, favoring the criterion of the clinical characteristics of the skin ulcer independently of the severity degree thereof, and said ulcers have a better evolutionary prognosis after the application of the therapeutic strategy. The model developed to establish a patient's risk of clinical improvement according to the associated probability of Poisson resulted in 0.09. This result shows that a patient suffering from a chronic ulcer of non-active borders, with presence of pain and excessive exudate, obtains control of the exudate and pain relief (directly or indirectly) after receiving the therapeutic care with AB Chitosan Gel, with a probability of 90% (0.09) of clinical improvement of her/his ulcer or affectation in the solution of skin continuity. This finding coincides with the one obtained through the Wilcoxon signed-rank Test.

[0133] These variables are important, since the pain expresses some anomaly in the healing process such as the presence of inflammation with and without infection, and maceration of the perilesional tissue. Pain, considered the fifth vital sign, should be identified and monitored in ulcers. It interferes with the closure of ulcers and wounds due to a direct decrease in blood flow. The exudate is also important, since it hinders the activity of growth factors, thus delaying the closure. Epithelialization takes place from the edges mainly, and it is dependent on an extracellular matrix that allows its migration, so it is a result of the above conditions.

[0134] In the present research, it is highlighted that the therapeutic application of chitosan AB gel did not promote infectious processes or alteration of renal and hepatic function (Table 6).

[0135] The support of the histopathological studies was relevant to confirm the clinical progression. Each case was evaluated in 3 instants: at the beginning, intermediate moment, and end of the application protocol. It was observed: [0136] a. At the beginning: The overall description of this phase would correspond to: “Tissue fragments showing abundant changes of exudative character, with polymorphonuclear predominance and frequent pyocytes that extensively infiltrate the samples, replacing their traditional architecture with accentuated signs of tissue lysis, foci of recent hemorrhage and signs of ancient hemorrhage. Rare vascular formations of capillary character and preserved structure are observed. Devitalized material of fibrinoleucocyte and serohematic character is observed on surface cover”. [0137] b. Intermediate moment: The overall description of this phase would correspond to: [0138] “Tissue fragments showing signs of connective-vascular proliferation of reparative character, characterized by the installation of congestive capillary network, chronic inflammatory infiltration of predominantly lymphoplasmacytic complemented with less polymorphonuclear component. No devitalized material or signs of microorganisms are observed. [0139] c. End of the protocol: The overall description of this phase would correspond to: [0140] “Tissue fragments showing notorious signs of maturation of vascular connective proliferation, with regression of inflammatory infiltration and capillary weft. Reinforcement of collagen fibers at the interstitial level.

[0141] Regarding the microbiological cultures carried out, the report of Staphylococcus aureus was obtained as the most frequent microorganism, followed by Staphylococcus epidermidis, Streptococcus spp, and coliform bacteria, which is consistent with reported findings for this kind of lesions and concomitant pathology, which is of interest, since polymicrobial infections can affect physiopathological aspects, which generates that this group of microorganisms complicates the injury recovery and healing. Indeed, cultures taken once the application of the gel has started were persistently negative.

[0142] The findings obtained in the current research could be explained by the cicatricial and bactericidal properties of the therapeutic product, which has the capacity to cause hyperplasia and increased keratinization. In the particular case of fibroblasts and endothelial cells, an increase in the production of cell surface proteins or fibronectin is observed, which provides the substrate substance required for the growth and differentiation of the epidermis. The results of this study prove the feasibility of using the product.

[0143] The revision of current literature does not show evidence of the use of the therapeutic proposal applied in this series of cases; that is the reason why it is considered a novel contribution in the attention of skin ulcer, which represents a serious problem of national and world public health. However, the results obtained coincide with the ones reported in similar studies.

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[0165] b) Yih, T. C.; Wei, C. (2010) Nanomedicine in cancer treatment. Nanotechnol. Biol.Med. 1, 191-192 [0166] 22. Bourne, (http://www.bourneresearch.com) [0167] 23. Chang, D, Yang, J y Zhao, Y. Chin. Med. Equip. 4: 26-32 (2004). [0168] 24. Cohen, M S, Stern, J M, Vanni, A J, Kelley, R S, Baumgart, R. Field, D, Libertino, J A y Summerhayes, J C Surg. Infect. 8: 397-402 (2007). [0169] 25. Panyman, J y Labhasetwar, V. Adv. Drug Delivery Rev. 55:3329-3347 (2003). [0170] 26. Elechiguerra, J. L., Burt, J., Morones, J R, Camacho-Bragado, A., Gao, X., Lara, H V. Y Yacaman, J. Nanobiotechnol. 3: 6 (2005). [0171] 27. Zhang, Y., Sun, J. Chin. J. Med. Instrum. 31: 35-38 (2007). [0172] 28. Lee, H., Park, H., Lee, Y M, Kim, K. y Park, S B. Chem. Commun. 28:2959-2961 (2007). [0173] 29. Iglesias Gonzalez R. Resumen de las recomendaciones de la American Diabetes Association (ADA) 2014 para la práctica clínica en el manejo de la diabetes mellitus. Diabetes Care. 2014;37: S14-80. [0174] 30. Serrano Hernando F J, Martín Conejero A. Rev Esp Cardiol. 2007; 60(9):969-82. [0175] 31. Vázquez-Hernandez I, Acevedo-Peña M. Prevalencia de insuficiencia venosa periférica en el personal de enfermería. Enfermería Universitaria. 2016; 13(3):166-170 [0176] 32. Suthar, Manish, et al. Treatment of chronic non-healing ulcers using autologous platelet rich plasma: a case series. Journal of biomedical science. 2017; 24(1):16. [0177] 33. Nelson, T. G., et al. Cutaneous ulcers in a returning traveller: a rare case of imported diphtheria in the UK. Clinical and experimental dermatology. 2016; 41(1):57-59. [0178] 34. Lewis, Hal A., et al. “Methotrexate-induced cutaneous ulceration in 3 nonpsoriatic patients: Report of a rare side effect.” JAAD case reports. 2017; 3(3): 236-239.

CONCLUSIONS

[0179] Chronic skin ulcers are a relevant health problem worldwide, it is estimated that 1% to 2% of the population will suffer the consequences thereof during their lifetime. In Chile, the available statistics show that approximately 160,000 patients suffer from some type of wound or ulcer, which represents a public health problem. Chronic ulcer is understood as “a skin injury maintained over time, involving loss of the epidermis, part of the dermis and even hypodermis”; accordingly, this group includes venous ulcers, ischemic ulcers, diabetic foot, pressure ulcers, burns and infected surgical wounds.

[0180] Chronic skin ulcers provide favorable conditions for microbial multiplication due to the loss of skin integrity, tissue hypoxia and contamination of exposed tissues, which hinder the healing thereof further to the increase in antibiotic resistance observed in frequently detected strains at least for usual antibiotic carriers. The above-mentioned arguments motivate the interest in knowing active principles of innovative use, which facilitate both the application, and stable and persistent effect on injured tissues, overlapping mechanisms of bacterial resistance, eventually enriched by antibiotics of known use.

[0181] Thus, the gel variants provided for this study have allowed observing an objectively favorable clinical progression, whose speed of recovery is greater than the one obtained with other therapeutic resources until the moment of receiving the application of the gel in an environment of high asepsis.

[0182] Accordingly, the character of the gel variants applied as a therapeutic agent showed that, [0183] it is an agent that allows providing high levels of asepsis to the tissue injury along with histopathological neoangiogenesis, regressive changes in the cellular inflammatory infiltrate with appearance of fibroblastic response of a cicatricial nature and signs of complementary re-epithelialization. [0184] it is an agent that tends towards safe, well-tolerated, satisfactory healing mechanisms providing benefits superior to the ones achieved with other therapeutic resources used in frequent polyclinic care. [0185] it has features of ambulatory and autonomous application by the patients. As long as it can be complemented with eventual profile of massive access and accessible cost, it would be included in a potential group of profitable therapeutic resources and of high demand, which should be studied in the near future.

TABLE-US-00007 TABLE 1 Distribution of participants according to age and sex (n = 30) Sex Male Female Age groups (years old) No. % No. % 18-37 years old 1 3.3 38-57 years old 1 3.3 2 6.6 ≥58 8 26.6 18 60.0 Total 10 33.3 20 66.6

TABLE-US-00008 TABLE 2 Frequency of morbid history in patients (n = 30) Morbid history No. % Arterial hypertension 11 36.6 Diabetes Mellitus 12 40.0 Etiology Peripheral arterial disease 22 73.3 Peripheral venous disease 3 10.0 Peripheral arterial disease 4 13.3 Peripheral venous disease (mixed)

TABLE-US-00009 TABLE 3 Frequency in skin-ulcer patients according to the score obtained in the classification algorithm before the therapeutic protocol Classification of (n = 30) skin ulcer No. % Type 1 1 3.3 Type 2 3 10.0 Type 3 5 16.6 Type 4 21 70.0

TABLE-US-00010 TABLE 5 Analysis of skin ulcer progression before and after treatment Therapeutic protocol Before After Classification N = 30 n = 30 Value Value of skin ulcer No. % No. % p* Z.sup.¥ Type 1 1 3.3 5 16.6 0.00 −6.042 Type 2 3 10.0 6 20.0 0.00 −3.842 Type 3 5 16.6 7 23.3 0.01 −3.624 Type 4 21 70.0 12 40.0 0.01 −1.342 *Probability value, Wilcoxon signed-rank test. 95% confidence interval (p ≤ 0.05). .sup.¥Value of the distribution of the standard, random and normal variable, Wilcoxon signed-rank test. 95% confidence interval (p ≤ 0.05).

TABLE-US-00011 TABLE 6 Progression of renal, hepatic and hematological function in the studied patients (n = 30) Mean/Standard Deviation Analytical parameters Day 0 Day 42 Leukocytes (cell/mm.sup.3) 11.678 ± 2    8.677 ± 2   Urea (mg/dL) 163 ± 11 121 ± 10 Creatinine (mg/dL)  2.7 ± 0.5  2.0 ± 0.5 GOT (U/I) .sup. 62 ± 0.5 42 ± 5 GPT (U/I) 51 ± 2 43 ± 2 GGT (U/I) .sup. 49 ± 3.5 .sup. 51 ± 3.5

[0186] Histopathological Progression

[0187] Material and Method: [0188] 1. Biopsy was obtained through punch modality, 2 mm in diameter, and 2-3 mm in depth. [0189] 2. Three biopsies were taken from each patient on days 1, 21, and 42. Each punch was immersed in 10% buffered formalin (25 cc). [0190] 3. Each sample was processed within 24 hours of the collection thereof. [0191] 4. Hematoxylin-eosin, Van Giesson and Giemsa stains were done for each inclusion.

[0192] Findings: [0193] 2. From the initial sample: [0194] a. In all cases, evaluable tissue material was observed. [0195] b. In 41.3% of the cases, [0196] i. superficial fibrinoleukocyte material (devitalized, purulent-type) was observed in more than 50% of the material evaluated. [0197] ii. In 24.1% of the cases, it corresponded to more than 75% of the material evaluated [0198] c. In 100% of the cases abundant inflammatory infiltration was observed, predominantly polymorphonuclear. [0199] d. In 27.5% of the cases, serum-leukocytary, and superficial hematic material (scabby) was observed. [0200] e. 10.3% of the cases showed signs of subepithelial sclerosis. [0201] f. 82.7% of the cases showed signs of deficient vascularization, for the clinical progression over time. [0202] g. 93.1% had intense clinical signs of an exudative nature, at least in a focal manner. [0203] h. 17.2% showed paratropic deposit of some substance, hemosiderine mainly. [0204] i. 48.2% of the cases examined showed microgranular hyperbasophilic accumulations of the bacterial colony type. In 6.8% of the cases, suggestive images of spores and hyphae were observed. [0205] j. The overall description of this phase would correspond to: “Tissue fragments showing abundant changes of exudative character, with polymorphonuclear predominance and frequent pyocytes that extensively infiltrate the samples, replacing their traditional architecture with accentuated signs of tissue lysis, foci of recent hemorrhage and signs of ancient hemorrhage. Rare vascular formations of capillary character and preserved structure are observed. Devitalized material of fibrinoleucocyte and serohematic character is observed on surface cover”. [0206] k. The overall diagnostic conclusion for this phase was: “Solution of tegumentary continuity with intense associated, active and chronic inflammatory process, whose diagnostic spectrum includes: diabetic foot ulcer, pustular folliculitis and fistulous tract, among others. [0207] 3. From the intermediate sample: [0208] a. In all cases, evaluable tissue material was observed. [0209] b. In 86.2% of the cases signs of variable intensity of fibroblastic proliferation of reparative character were observed. [0210] c. In 27.5% of the cases signs of peripheral, immature and incipient re-epithelization were observed (edges of the ulcer). [0211] d. In 65.5% of the cases, [0212] i. inflammatory infiltration of predominantly mononuclear, lymphoplasmacytic of chronic type was observed in more than 50% of the evaluated material. [0213] ii. In 41.3% of the cases, it corresponded to more than 75% of the material evaluated [0214] e. 27.5% of the cases showed signs of subepithelial sclerosis. [0215] f. In 86.2% of the cases recovery of vascular pattern, and signs of capillary and endothelial proliferation were observed, which showed signs of mild to moderate edema (neoangiogenesis). [0216] g. 93.1% of the cases did not show signs of superficial fibrinoleucocyte material, or serohematic material (superficial and scabby). [0217] h. No hyperbasophilic microgranular accumulations, such as bacterial colonies or images suggesting spores and hyphae were observed in any of the cases examined. [0218] i. The overall description of this phase would correspond to: “Tissue fragments showing signs of connective-vascular proliferation of reparative character, characterized by the installation of congestive capillary network, chronic inflammatory infiltration of predominantly lymphoplasmacytic complemented with less polymorphonuclear component. No devitalized material or signs of microorganisms are observed. [0219] j. The overall diagnostic conclusion for this phase was: “Solution of tegumentary continuity, with development of granulated, congested tissue. There are no signs of infection. Incipient peripheral signs of re-epithelization, involving incomplete cell differentiation”. [0220] 4. From the final sample: [0221] a. In all cases, evaluable tissue material was observed. [0222] b. 96.5% of the cases showed signs of regression of inflammatory infiltration located at the perivascular level mostly. [0223] c. In 93.1% of cases, no superficial fibrinoleucocyte material (devitalized purulent-type) or superficial hematic material (scabby type) was observed. [0224] d. 100% of the cases showed signs of reduplication of collagen fibers in interstitial matrix. [0225] e. 100% of the cases showed signs of capillary weft. [0226] f. No hyperbasophilic microgranular accumulations, such as bacterial colonies or images suggesting spores and hyphae were observed in any of the cases examined. [0227] g. The overall description of this phase would correspond to: “Tissue fragments showing notorious signs of maturation of vascular connective proliferation, with regression of inflammatory infiltration and capillary weft. Reinforcement of collagen fibers at the interstitial level. [0228] h. The overall diagnostic conclusion for this phase was: “Solution of tegumentary continuity exhibiting scar tissue changes. Signs of re-epithelialization showing adequate tissue differentiation. No microorganisms are observed”.