WO2022036466A1 - Nanoformulation for treating and/or preventing wounds or ulcers that are infected or have critical colonisation, which includes copper nanoparticles and collagen-derived peptides - Google Patents

Abstract

The present invention describes a nanoformulation for treating and/or preventing wounds or ulcers that are infected or have critical colonisation, which includes copper nanoparticles and collagen-derived peptides, and to the applications of the nanoformulation as an antimicrobial and/or as a product that stimulates healing. Also described are products for treating and/or preventing infected wounds or ulcers that include the nanoformulation, in the form of a dressing or topical-use formulations, such as creams, waxes, gels and sprays.

Description

NANO FORMULATION FOR THE TREATMENT AND/OR PREVENTION OF WOUNDS OR ULCERS INFECTED OR WITH CRITICAL COLONIZATION, WHICH INCLUDES COPPER NANOPARTICLES AND PEPTIDES DERIVED FROM COLLAGEN.

SCOPE

The present invention is framed in the field of health, more specifically in the area of treatment and prevention of wounds or ulcers infected or with critical colonization, providing a biotechnological product developed based on a nano formulation that includes peptides derived from collagen in addition to copper nanoparticles. The biotechnological product can be applied directly to the wounded skin surface, or incorporated homogeneously into a matrix, configuring a dressing with defined concentrations of peptides derived from collagen and copper nanoparticles, facilitating the release of copper ions when applied to the tissue or the lesion, exerting its antimicrobial and healing-stimulating action in situ.

STATE OF THE ART

Infected wounds or ulcers affect 1% of the population, mainly affecting older adults. Venous ulcers represent about 80% of all these wounds with an incidence of 2 to 5 cases per thousand people per year and losses of 2 million working days. In the US alone, this injury affects 6.5 million people with a cost of $25 billion dollars annually. In Chile, 170,000 patients have infected wounds or ulcers and management is aimed at venous ulcers, diabetic foot, hypertensive and pressure ulcers.

These injuries will continue to increase mainly in older adults due to the projection of survival and its association with endogenous factors such as vascular diseases, hypertension and Diabetes Mellitus. In addition, the amount of bacteria present in the wound is related to the delay or non-healing of ulcers, where the prevalent pathogens include both Gram positives such as S. aureus and Streptococcus spp, and Gram negatives such as E. coli, Proteus spp, P. aeruginosa and Acinetobacter spp. The lack of effective care results in prolonged periods of incapacity for work, expensive treatments, repeated hospitalizations and surgeries that can lead to amputation, disability and death. Treatment with broad-spectrum antibacterials generates an increase in resistant bacteria. Due to the complexity of the treatment, advanced wound management protocols include dressings with silver, which is today the product of choice for this treatment.

The antimicrobial activity of copper, as well as its action in the regulation of various biological processes associated with healing, makes it possible to improve the treatment of infected wounds or ulcers. This improvement in treatment translates into a high cure rate. Within these biological processes, it has been established that copper stimulates the formation of blood vessels, a process also known as angiogenesis.

Although the antimicrobial activity of copper ions (Cu ⁺² ) as well as their lower toxicity compared to silver has been well documented, only recent studies have shown that copper salts have antimicrobial activity in direct topical application, as well as also indirectly through a gauze impregnated with copper salts.

It was the inventors of the present invention who, in previous studies, managed to show that after applying a gauze with the defined concentration of the copper acetate salt, in an in vivo proof of concept, it is possible to treat patients with infected wounds or ulcers . Specifically, it was evidenced that on days 5 and 10 of treatment with a gauze impregnated with copper salts; there was a decrease in the average bacterial load of 96.7% and 99.5%, respectively, as well as a higher speed of healing, with an average value of 67% on the tenth day of treatment. On the other hand, patients treated with nanocrystalline silver-based dressings only show an average bacterial decrease of 81.3% and an average healing of 35% on day 10 of treatment.

At a global level, statistical data show that currently 6.7 billion people in the world have infected wounds or ulcers. Of this group of people and according to figures from the QMS, 1% (67 million people) show lesions such as chronic ulcer, distributed in venous ulcers, ischemic ulcers, diabetic foot, pressure ulcers, burns and infected surgical wounds , Come in others. For its part, a subgroup of these injuries estimated between 20 to 51% (13.4 to 33.5 million people) correspond to already infected wounds. The solution for managing this type of infected wound requires at least seven cures (one per day) with a particular dressing; and where the unit price range for the dressing considered the industry standard (ie: silver dressing) varies from 10.4 to 16.6 dollars. Although the silver dressing has been the most widely used product for bacterial control in infected wounds or ulcers, it shows important disadvantages in terms of: development of bacterial resistance, unwanted staining of the skin, and greater toxicity compared to copper. .

In the state of the art, there is US patent US5591711A by Masayoshi Koyama and other inventors dated January 7, 1997, which discloses a pharmaceutical composition based on a tripeptide with the sequence L-Lysyl-Glycyl-L-Histidine or complexes of said tripeptide with ions metals, such as copper (II) ions, as an active ingredient for treating wounds. It follows from the document that the therapeutic effect of the composition is given by the tripeptide L-Lysyl-Glycyl-L-Histidine, in particular, its examples show its activity to promote the proliferation of fibroblasts. The document fails to disclose whether the copper (II) ions would have any therapeutic effect, much less if they are provided from a metallic nanoparticle.

On the other hand, the also US patent US5554375A by Loren R. Pickart of September 10, 1996 discloses compositions comprising peptone digests (including collagen) complexed with metal salts (including Cu (II)). Although the compositions are provided to treat topical wounds, among other skin defects; the ionic transition metal that can be copper corresponds to the therapeutic agent, and the addition of protein digests by enzymatic degradation (ie: mixtures of polypeptides of indefinite sequence) should provide greater stability and resistance to degradation after sterilization of the mixture, avoiding the use of high doses of the therapeutic agent. However, this document does not specify that Cu (II) be provided in the form of metallic nanoparticles, nor how to stabilize this material in a nano formulation that contains it.

For its part, the scientific review "Recent advancements in biopolymer and metal nanoparticle-based materials in diabetic wound healing management" by Veena Vijayakumar and collaborators (2019), disclose a compilation of advances in the area of materials based on nanoparticles for wound treatment in diabetics. This review is very interesting regarding the use of metallic nanoparticles, and their combination with other materials to achieve composite materials that improve the therapeutic activity of the metal. Specifically, it is highlighted that the use of copper nanoparticles faces complex challenges during their application and the need to control their stability when used together with biocompatible stabilizers, such as chitosan.

According to the above information, none of the cited state-of-the-art documents manages to anticipate, much less suggest, a stable nano formulation, nor a dressing or cover that incorporates it, which includes a combination of metallic copper nanoparticles and short derivative peptides. of collagen, which manages to solve the technical problem of providing an innovative and competitive alternative to adequately attend to the treatment of wounds.

Thanks to this innovative nano formulation, the present invention has the advantage of providing a greater antimicrobial activity of copper and a better action in the regulation of various biological processes associated with healing, by stabilizing the copper nanoparticles. With all of the above, the nano formulation will improve the treatment of wounds or ulcers infected by both gram-positive and gram-negative agents, in mono-microbial and poly-microbial infections. This improvement in treatment will result in a high percentage of healing due to the greater and faster antimicrobial action of copper ions and their stimulation in accelerating the healing process.

Additionally, because the nano formulation also includes short collagen peptides that stabilize the copper nanoparticles; Providing the nano formulation impregnated in a polyester-rayon matrix presents greater flexibility than the matrices commonly used in silver dressings. The nano formulation can be incorporated into the matrix by direct inoculation with a pipette, vaporization with an atomizer and dyeing by immersion, vaporization being the method that allows delivering better performance in the release of copper ions, microbiological activity and healing evidenced by histology. Furthermore, the physical property of flexibility is advantageously recommended by professionals in the health area who work in the treatment of wounds.

Notwithstanding the foregoing, the nano formulation can be incorporated into products with other formats, so that it can be provided in formulations for topical use such as creams, waxes, gels, aerosols (i.e.: spray) or other equivalents.

Finally, and as an advantage of economic sustainability and production of the dressings of the invention; the production cost of this innovative product with copper-carrying nanoparticles will not be higher than silver dressings, being competitive in the global market, due to the absence of alternatives to the current treatment with silver dressings.

DESCRIPTION OF THE FIGURES

Figure 1. (A) TEM images of the synthesized copper nanoparticles. (B) HAADF-STEM image of the synthesized copper nanoparticles.

Figure 2. Graph of the UV-Vis spectrum of 0.2 mM CUCI2 2H2O (dotted line), 0.2 mM CuNP (black line), in arbitrary units of absorbance (a.u.) versus wavelength (nm).

Figure 3. Graph of the total bacterial strains compared with their controls and studied dressings. Analysis with Anova and Bonferroni tests (***) = p < 0.0001 (**) = p < 0.001.

Figure 4. (A) Graph of Staphylococcus aureus bacterial strains compared to their controls and studied dressings. Analysis with Anova and Bonferroni test (***) = p < 0.0001 (*) = p < 0.05 (B) Graph of the bacterial strains of Enterococcus faecalis compared with their controls and studied dressings. Analysis with Anova and Bonferroni tests (***) = p < 0.0001 (**) = p < 0.001.

DESCRIPTION OF THE INVENTION

The first objective of the present invention is to provide a new nano formulation, with antimicrobial and healing-stimulating properties, comprising copper nanoparticles (also called CuNP) and peptides derived from collagen (also called CLPs or CMPs), for the treatment and / or prevention of infected or critically colonized wounds or ulcers. In embodiments of the invention, said nano formulation comprises a concentration of at least 1,000 pg/ml of copper, in a solvent that is suitable or compatible with topical applications. Within the suitable or compatible solvents, the solvent can be selected from the group formed by physiological serum, distilled water, ascorbic acid solutions, and mixtures thereof, buffered at pH 3.

In particular embodiments of the invention, the nano formulation comprises copper concentrations between 1,000 and 4,000 pg/ml, with 2,000 pg/ml being the most preferred concentration.

In certain embodiments, the nano formulation comprises nanoparticles between 1 to 12 nm in diameter, averaging between 4 and 6 nm in diameter.

On the other hand, the peptides derived from collagen that are included in the nano formulation of the invention are found in concentrations between 1 and 50 pM, and more preferably in concentrations of 30 pM.

The peptides used in the nano formulation of the invention are structurally characterized by being short chains containing between 6 to 7 amino acids, and more preferably 7 amino acids. Peptides have variable sequences, with an amino acid selected from the group consisting of histidine (His) or cysteine (Cys), at their carboxyl terminus.

For purposes of the present invention, some short-chain CLPs have either the sequences aai-(aa) _n -His or aai-(aa) _n -Cys, where n represents the number of amino acids below in the sequence and that varies between 4 and 5, aai is an amino acid selected from the group formed by proline (Pro), hydroxyproline (Hyp) or other residues derived from proline, at its amino terminal end. In particular embodiments of the invention, it is preferred that some short-chain CLPs also include at least one amino acid selected from the group consisting of proline, hydroxyproline or other residues derived from proline, within the internal amino acids of the chain, that is, any of amino acids (aa) _n . The nano formulation of the invention is useful for combating pathogens that are commonly found in infected wounds or ulcers, thanks to its bactericidal and bacteriostatic action. The nano formulation of the invention provides protection against bacteria such as Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas fluorecens, Acinetobacter baumanii, Acinetobacter iwoffi, Klebsiella oxytoca, Morganella morganii, Providencia rettgeri, Enterobacter sakazakii, Enterobacter cloacae, Proteus my rabbi lis, among you will hear.

A second aspect of the present invention involves a simple and direct process for obtaining a dressing or cover, in addition to other types of formulations for topical use, to be applied to wounds, which generally comprises:

A) Mix the CuNPs and the CLPs to obtain the stable nano formulation,

B) Impregnate a suitable matrix with the nano formulation, to obtain an impregnated dressing or cover, and

C) Topically apply the dressing or cover on a wound.

Regarding step B) the impregnation of the nano formulation in a matrix can be achieved by any means available in the state of the art, such as by: absorption by direct inoculation, absorption by spray dispersion, or immersion dyeing.

On the other hand, the suitable matrix is selected from the group formed by gauze, polyester rayon, textile cellulose fibers, among others; because these matrices provide the necessary flexibility for their subsequent application on a wound, which allows them to adapt to the surface of the skin and its particular irregularities of the extremity.

Equivalently, with respect to stage B), in the case of other types of formulations for topical use, such as creams, waxes, gels, spray, among others; the stable nano formulation is also mixed or incorporated with excipients acceptable for topical use, suitable for each type of application.

And finally, a third aspect of the invention is precisely to provide the dressing or cover impregnated with the nano formulation, as a versatile and flexible product, with applications on clean skin wounds, colonized skin wounds, critically contaminated skin wounds or infected skin wounds. APPLICATION EXAMPLES

The following examples show in an illustrative but non-limiting way the obtaining of nano formulations of the invention, and the corresponding tests to evaluate the improved properties in terms of its application as a product for the treatment and/or prevention of infected or colonized ulcers or ulcers. review.

For example, the nano formulation included a mixture of copper nanoparticles with an average diameter of 6 nm and a circular morphology with a concentration of 2,000 pg/ml, together with collagen peptides stabilized with ascorbic acid. The nanoparticles were synthesized according to the following methodology.

CuNP synthesis

25 mL of a 0.2 M CuC^FLO solution was prepared and heated to 80°C with stirring. Subsequently, 25 mL of 0.4 M L-ascorbic acid was added dropwise. The mixture was kept stirring at 80 °C for 12 h. The mixture was cooled to room temperature and centrifuged at 8,000 rpm for 15 min, before washing with milli-Q water. The supernatant was diluted to 50 mL in a volumetric flask. Obtaining spherical nanoparticles with an average diameter of approximately 6 nm, as shown in the transmission electron microscopy (TEM) images of Figure 1A. In addition, a Scanning Transmission Electron Microscopy analysis was carried out with the Wide Angle Annular Dark Field (HAADF-STEM) methodology, where it can be seen from Figure 1 B that the d-space of 0.212 nm it agrees with the (111) plane of the face-centered cubic (fcc) phase of metallic copper Cu(0). The CuNP solution (0.1 M) has a shelf life of up to 2 months.

Characterization of CuNP

The characterization of the nanoparticles was carried out by UV-Vis spectroscopy on reagent and product samples in plastic microcuvettes. Figure 2 presents in the dotted line, the spectrum of CuCL^FLO (0.2 mM) with an absorption peak at 230 nm (Cu-Cu interaction) and a broad peak at 810 nm (Cu-FLO interaction). The CuNP Spectrum (0.2 mM) is presented in the black line, with maximum absorption peaks at 220 nm, 246 nm and 300 nm.

CuNP synthesis associated with CLPs

The previously synthesized CuNPs were mixed and tested with different concentrations of 6 different peptides to perform stabilization.

The busy peptides, similar to collagen, were grouped into those ending in histidine or cysteine. Table 1 presents the amino acid sequence from amino to carboxy terminus of the short peptides and their internal reference names.

Table 1: Sequence (N->C) of peptide groups Histidine and Cysteine

The peptides were designed and their synthesis commissioned at Biomatik, and they were mixed with the CuNPs at different concentrations varying between 1 and 50 pM, using a pH 3 citric acid-citrate buffer, in order to protect the peptides from denaturation and prevent oxidation of nanoparticles.

In vitro activity assays of CuNP-CLPs nano formulations

Microbiological tests of bacterial inhibition halos were carried out to measure the activity of the new nano formulations with peptides and CuNP at pH 3. At this pH, the experiments carried out allow establishing that there is a favorable CuNP-peptide interaction, where the peptide chains allow generating a stabilization greater than copper.

The best results in terms of CuNP stability, from this type of experiments resulted in the nano formulations called P1, P2, P3 and P4, as detailed below:

P1: 2,000 pg/mL CuNP + 30 pM Ce peptide.

P2: 2,000 pg/mL CuNP + 30 pM APACBACCC peptide mix. P3: 2,000 pg/mL CuNP + 2.5 M citric acid-citrate buffer pH 3 + 30 pM Cc peptide.

P4: 2,000 pg/mL CuNP + 2.5 M citric acid-citrate buffer pH 3 + 30 pM APACBACCC peptide mixture.

To prepare 1 L of any of the previous nano formulations with a copper concentration of 2,000 pg/mL, the following were taken: i) 0.1 M solution of copper nanoparticles: 315 mL.

i) Milli-Q water: 314 mL. iii) Citric acid-sodium citrate buffer solution 2.5 M pH 3: 371 mL. iv) Peptide stock solution or peptide mixture (APACBACCC OR CC) 68.78 mM: 46 pL.

The best results obtained from the microbiological tests in terms of MIC (minimum inhibitory concentration) were obtained with the nano formulation P3, as shown in Table 2.

Table 2. MIC result (in pg/ml units) of 4 metallic copper nano formulations with different collagen peptides.

To determine the MIC of the copper nanoparticles, from a mother solution of these nanoparticles at a concentration of 4,000 pg/ml, serial dilutions were prepared in a range of 2,000 pg/ml up to 200 pg/ml in Müeller Hinton pH 7 broth. .0 using a microplate. Subsequently, 100 pl of bacterial inoculum was added to each well with its corresponding dilution. The bacterial inoculum was prepared in 3 ml of sterile physiological serum, in such a way as to obtain an optical density of 0.2, reading at a wavelength of 600 nm in a spectrophotometer.

The reading of the results was carried out after 24 hours of incubation of the microplate at 37°C shaking at 200 rpm by visual reading, considering as MIC the lowest concentration in which a significant inhibition of bacterial growth was observed (>90% ). A growth control containing 100 μl of Müeller Hinton broth (without copper nanoparticles) and 100 μl of bacterial inoculum was used, in addition to a sterility control containing only 200 μl of Müeller Hinton broth without bacterial inoculum.

In addition, the determination of the minimum bactericidal concentration (MBC) was carried out by seeding, on a Müeller Hinton agar plate, 50 pl of each dilution, from the MIC up to 2,000 pg/ml. MBC was considered to be that dilution in which no bacterial growth was observed on the agar plate after 24 hours of incubation at 37°C.

Among the nano formulations P2 and P3, tested against 30 strains of bacteria isolated from infected chronic ulcers, it was established that the nano formulation P3 presented better antimicrobial activity with CBM values close to MIC, also showing a broad spectrum of action, both against cocaceous bacteria gram positive, enterobacteria and non-fermenting gram negative bacilli as shown in Table 3.

Table 3: Results showing the average MIC and MBC in 30 strains of bacteria isolated from chronic wound infection with nano formulations P2 and P3.

In vitro dressing activity assays Based on the previous results, the P3 nano formulation was selected to impregnate dressings, which consist of a 10x10 cm polyester-rayon gauze. Three impregnation methods were used:

1. Prototype 1 dressing (AP1): Absorption by direct inoculation: Each dressing was impregnated with 4 mL of P3 (2,000 pg/ml in copper), completely absorbing this volume. Then, they were dried for 5 days in an oven at 30°C in individual petri dishes.

2. Prototype 2 Dressing (AP2): Absorption by spray dispersion: Each dressing was sprayed with 3.5 mL of P3 (2,000 pg/mL on copper). Then, they were dried in individual petri dishes at 37°C for 4 days.

3. Prototype Dressing 3 (AP3): Immersion staining: Each dressing was immersed in P3 solution (2,000 pg/ml in copper) for 2 minutes. Then, they were dried in individual petri dishes, turning the dressing every so often to prevent it from adhering to the individual petri dish at 37°C for 4 days.

The antimicrobial activity of the 3 prototype dressings and the silver dressing was quantitatively evaluated on 30 bacterial strains isolated from infected wounds or ulcers, compared to controls.

Figure 3 shows that prototype dressing 3 was more efficient than the silver dressing and prototype dressing 2 as efficient as the silver dressing.

On the other hand, in tests by groups of individual bacteria, the results showed that the three prototype dressings of the invention are effective in all bacterial groups, and also showed high efficiency in certain groups of bacteria, confirming their broad spectrum of action. For illustrative purposes only, AP3 was more efficient than the silver dressing against Staphylococcus aureus and Enterococcus faecalis, as can be seen from Figures 4A and 4B, respectively.

Notwithstanding the foregoing, in subsequent trials it was possible to improve the performance of other dressings, AP1 and AP2, by direct inoculation repetitions or by adding moisture to activate, so that they are at least as effective as the silver dressing. Wound dressing tests

In an in vivo proof-of-concept study with 10 patients with infected wounds or ulcers, clinical, microbiological and histological aspects of the AP2 and AP3 prototype dressings were tested, both with the same nano formulation and in a polyester-rayon matrix at a concentration of 2,000 µg/ml. (Group A = AP2 spray impregnated and Group B = AP3 dip dyed).

Clinically, the patients in both groups (A and B) showed significant clinical improvement, recovery of the color of the lesion bed, with very good healing.

Microbiologically, there was no difference in antimicrobial activity between patients in both groups.

Histologically, before starting treatment (day 1), the patients in both groups presented lesions with a large necrotic area with the presence of a chronic infiltrate and a high number of lymphocytes. On day 10, the patients in Group A treated with AP2 show a better evolution by reducing their necrotic tissue by 50% vs. those in Group B (AP3), which decreased by less than 10%. Group A showed a greater presence of newly formed vessels, going from an average between days 1 and 10 of 13 to 40 vessels per microscopic field at 100x magnification. Both groups presented a large increase in collagen fibers (>80%), with the difference that the patients in Group A generated thicker collagen fibers than the patients in Group B. Finally, a decrease in chronic inflammation composed of by lower number of plasma cells, lymphocytes and polymorphonuclear-PMN.

This indicates that the prototype dressing that incorporates the selected nano formulation at a concentration of 2,000 pg/ml with an atomizer into the polyester-rayon matrix is the most efficient, because it contributes to microbial control and stimulation of healing. The latter constitutes a differentiating and advantageous factor with respect to the silver dressing, to which isolation of resistant bacteria has already been described, as well as questioning of patient safety due to the toxicity of silver.

Claims

1.- A nano formulation for the treatment and/or prevention of infected wounds or ulcers, CHARACTERIZED because it comprises copper nanoparticles and peptides derived from collagen. 2.- The nano formulation according to claim 1, CHARACTERIZED in that it also comprises a solvent that is suitable or compatible with topical applications, adjusted to a pH of 3. 3. The nano formulation according to claim 1, CHARACTERIZED in that the copper nanoparticles are in a concentration between 1,000 pg/ml and 4,000 pg/ml. 4. The nano formulation according to claim 1, CHARACTERIZED in that the peptides derived from collagen are found in a concentration of 1 pM and 50 pM. 5. The nano formulation according to claim 4, CHARACTERIZED in that the collagen-derived peptides have an extension of between 6 and 7 amino acids, with a residue at the terminal carboxyl end selected from histidine and cysteine. 6. Use of the nano formulation according to claim 1, CHARACTERIZED because it is useful in the preparation of an antimicrobial product. 7. Use of the nano formulation according to claim 1, CHARACTERIZED because it is useful in the preparation of a product that stimulates healing. 8.- Use of the nano formulation according to claim 1, CHARACTERIZED because it is useful for treating and/or preventing infected wounds or ulcers or with critical colonization of at least one of the bacteria selected from the group formed by Staphylococcus aureus, Enterococcus faecalis , Escherichia coli, Pseudomonas aeruginosa, Pseudomonas fluorecens, Acinetobacter baumanii, Acinetobacter iwoffi, Klebsiella oxytoca, Morganella morganii, Providencia rettgeri, Enterobacter sakazakii, Enterobacter cloacae, Proteus mirabilis, among others. 9.- A product for the treatment and/or prevention of infected wounds or ulcers, CHARACTERIZED in that it comprises the nano formulation according to claim 1, impregnated in a matrix, configuring a dressing. 10. The product according to claim 9, CHARACTERIZED in that the matrix is selected from the group consisting of gauze, polyester rayon, textile cellulose fibers, among others. 11.- A product for the treatment and/or prevention of infected wounds or ulcers, CHARACTERIZED because it comprises the nano formulation according to claim 1, mixed or incorporated with suitable excipients for a formulation for topical use. 12. The product according to claim 11, CHARACTERIZED in that the formulation for topical use is selected from creams, waxes, gels, spray, among others.