ES2387425B2 - COMPOSITION FOR THE TREATMENT OF BACTERIAL INFECTIONS OF THE SKIN AND MUCOSES - Google Patents

Abstract

Composition for the treatment of bacterial infections of the skin and mucous membranes. # The present invention relates to a composition for the treatment and prophylaxis of bacterial infections of the skin comprising bacteriocin AS-48 in combination with lysozyme. More specifically, these are compositions with the active ingredients dissolved in extracts suitable for applications against Propionibacterium acnes and Staphylococcus aureus, in a stabilized manner in a pharmaceutically acceptable preparation.

Description

COMPOSITION FOR THE TREATMENT OF BACTERIAL INFECTIONS OF

THE SKIN AND MUCOSAS

SECTOR OF THE TECHNIQUE

The present invention is within the pharmaceutical sector, and more specifically in the field of compositions for the treatment of bacterial infections of the skin with antibacterial compounds.

STATE OF THE TECHNIQUE

Healthy human skin is colonized by numerous microorganisms that are responsible for maintaining their natural microbial balance. In fact, its normal microbiota is made up of resident or transient populations of bacteria and various species of fungi (particularly yeasts), some of them very stable. The most common resident microorganisms of the skin are limited to some genera of Gram + bacteria, whose species can represent up to 50%. Among them, those belonging to the Phylum Firmicutes with low G + C content (Streptococcus and Staphylococcus genera) and the Phylun Actinobacteria (Corynebacterium and Propionibacterium genera) stand out. The remaining bacterial groups, including Gram- bacteria, are much more transient since around 70% undergo changes over time and the individual's conditions.

There are, however, a series of bacteria that cause skin infections such as mastitis and acne, among which S. aureus and Propionibacterium acnes, respectively, stand out. Infectious mastitis of the mammary glands is characterized by multiple local and systemic symptoms in nursing mothers. It is mainly caused by S. aureus and Streptococcus species, which are often multidrug-resistant to antibiotics, so antibiotic therapy is, in many cases, unsatisfactory and the problem tends to be recurrent. Its incidence ranges from 3-33% of them, and determines a high percentage of cessation of breastfeeding. On the other hand, common acne (acne vulgaris) is an infection of the skin, particularly in those areas with higher density of sebaceous follicles. It is the most common skin infection among humans, due to changes in the hair follicles and associated sebaceous glands, which occur mainly at puberty (85% of adolescents suffer this problem to some degree), such as multifactorial response that includes hormonal, microbiological and immunological mechanisms.

The use of antibacterial peptides produced by bacteria (bacteriocins) against bacterial infections, is being consolidated as an alternative to the lack of chemotherapeutic agents, due to antibiotic resistance. Bacteriocins are today in the spotlight of novel studies on their application against common pathogens in clinical practice, so it is already a growing trend in the field of pharmaceutical compositions and in antibacterial treatment methods (reviewed by Montalban-Lopez , M., Sanchez-Hidalgo, M., Valdivia, M., MartinezBueno, M., and Maqueda, M. 2011. Are bacteriocins underexploited? Novel applications for ancient antimicrobials. Curr. Pharm. Biotechnol. 12: 1205-1220) . Examples include existing patents protecting the use of antibiotic peptides (W09957281, W02004082701, US5635594, CA2453112) or bacteriocins produced by bacteria (Mutacin CA2268744 and S6521596, gallidermin CA 1338310 (C) -1996-05-07 and Enterocin L50A / B produced by E. faecalis SL-5 which has been registered as Lactopadt ™ Acne as a cosmetic ingredient (INCI) (CTFA file No. 9082) by Cell Biotech Co. You (KOSDAQ) with the aim of using it for cosmetic purposes , in the upper layers of the skin).

Compositions that integrate bacteriocins have the advantage of preventing the development of resistance by pathogens that colonize and infect the skin. For this reason there are some publications on the ability of some bacteriocins to inhibit P. acnes, reducing the inflammatory lesions caused by this bacterium. Such is the case of those produced by Lactococcus sp. HY 449 or Enterococcus faecalis SL-5 discussed above. Similarly, the use of bacteriocins, including AS48, has been described against different MRSA strains of Staphylococcus, and / or those producing mastitis.

Here we propose the use of bacteriocin AS-48 produced by Enterococcus species, in combination with a potentiating agent, lysozyme, an enzyme that hydrolyzes the cell wall of bacteria, which increases the potency of AS-48 in order to combat pathogenic species that cause skin infections, specifically P. acnes and S. aureus. The possible explanation of this synergy is a multi-barrier effect that occurs because the simultaneous application, at moderate doses, of several factors that act at different levels, is much more effective than the application of only one, which, when acting at a single level, it requires a higher dose (Leistner, L. 2000. Basic aspects of food preservation by hurdle technology. Int. J. Food Microbiol. 55: 181-186).

The bacteriocin AS-48 is a circular protein of ribosomal synthesis consisting of 70 natural amino acids. Its nature is strongly amphipathic which allows it to adapt to different environmental conditions. Thus, in aqueous solutions, AS-48 adopts a dimeric form (DF-I) in which it exposes hydrophilic domains, but this association changes in hydrophobic environments such as the case of the skin or the presence of cell membranes, adopting a new dimeric form. (DF-II) in which hydrophobic domains are exposed, which undoubtedly facilitates their insertion into strongly negatively charged membranes as occurs in the case of bacteria, but without activity against eukaryotic cells.

AS-48 has been recognized as one of the bacteriocins with the greatest potential, due to its broad spectrum of action against the bacterial membranes of most Gram + bacteria tested and some Gram- bacteria. It favors its activity and therefore its possible biotechnological applications, the great stability that AS-48 presents in wide ranges of pH, temperature and surfactant treatments, due to its structural characteristics (a circular protein), which justifies the success of its use in food bioconservation.

Lysozyme, also called 1,4 N-acetyl muramidase, is a hydrolytic enzyme that specifically catalyzes the hydrolysis of beta 1,4 junctions between N-acetylmuramic acid and N-acetyl-D-glucosamine residues of bacterial peptidoglycan, a majority compound of the bacterial cell wall, in particular of Gram-positive bacteria. This enzyme lacks protease, kinase, lipase, amylase or phosphatase activity and is found naturally in numerous secretions (tears, mucus, saliva and milk) and in large quantities in egg white, which is its source of use for use. industrial (Sigma).

The application of multiple barriers in the control of microorganisms can have an additive or even synergistic inhibitory effect, especially if they act on different targets of the microorganism. This is the case we describe in which AS-48 forms pores in the cell membrane, and said activity is favored by the action of lysozyme that, by partially hydrolyzing the cell wall, facilitates the access of AS-48 to its target, the membrane bacterial cytoplasmic.

Currently there is a great interest in the application of bacteriocins alone or in combination with other inhibitors and also with physical or chemical agents that act synergistically against pathogenic and / or altering bacteria, allowing in some cases to extend their spectrum of action to Gram bacteria -negative. Thus, nisin has been used together with other bacteriocins and with chemical preservatives (nitrites) for the control of Clostrídíum. The high pressures have been combined with various bacteriocins and strains that produce them (nisin, lacticin 3147, lacticin 481, AS-48, TAB 57, pediocin AcH and enterokines 1, AYB) in different foods for the control of the altering microbiota and pathogenic. It has also been proven that nisin and lysozyme act synergistically in the control of Lactobacillus and S. aureus and that their spectrum can be extended to Gram bacteria by the addition of chelating agents. Similarly, AS-48 has been successfully combined with other antibacterials, including lysozyme, for the elimination of foodborne pathogens.

It is important to mention the existence of treatments that could be mistakenly considered as naturopathic or homeopathic medicine, such as some homemade applications used to combat acne through egg white (due to its richness in lysozyme) as well as with yogurt, limestone or honey, To name a few. However, these treatments are usually as harmless as they are ineffective for the elimination of a bacterium such as P. acnes that is difficult to access because it is located in the sebaceous glands.

OBJECT OF THE INVENTION

The object of the present invention is a composition comprising AS-48 in combination with lysozyme for the treatment and prophylaxis of bacterial infections of the skin and mucous membranes. In addition, this composition allows the P. acnes and S. aureus batteries to be completely eliminated.

More specifically, the use of these compositions is described, with the active ingredients dissolved in suitable extracts, against P. acnes and S. aureus, in a stabilized manner in a pharmaceutically acceptable preparation.

According to in vitro activity tests of the active ingredients object of this invention, dissolved in neutral gel or in aqueous solutions, or emulsified in a glycerin-based cream, the most appropriate vehicle for activity is the cream, followed by Neutral gel and aqueous solution, both equally.

These compositions allow the control of bacteria, especially Staphylococcus species involved in infections and superinfections of the skin, as well as P. acnes, responsible for inflammatory lesions (acne vulgaris), which often lack adequate antibiotic treatment due to Resistance has developed.

The compositions will preferably be used by topical application, two or more times a day, being formulated in cream, gel and / or aqueous solution.

The proposed invention has the following advantages:

•

The inhibitory activity of AS-48 is increased by a factor of 10 in the presence of lysozyme

•

The compositions have not produced, in any of the cases tested in vivo, irritation (allergies, erythema) on the skin, despite having been used topically on healthy people for many days.

•

The preparations do not suffer decrease or loss of activity during storage at different temperatures, or by interaction of the active molecules with cosmetic compounds (emulsifiers and / or lipid components

or aqueous) nor by antagonism between the different agents employed.

•

It has been found that AS-48 alone or in combination with lysozyme lacks hemolytic activity.

•

The absence of cytotoxicity on the CCD18 cell line of fibroblasts (from normal human colon) and on the epithelial line of the human mammary gland MCF 10A has also been confirmed.

•

AS-48 has been selected as an antibacterial agent for skin infections due to its biochemical and structural characteristics. The amphipathic character of AS-48 and its particular mechanism of action favored by the different conformations that AS-48 adopts depending on the environment in which it is located, facilitates its access to the different layers of the skin where the staphylococci are found and also to the sebaceous follicles clogged by excess sebum, where P. acnes is found.

•

Due to the structural characteristics that AS-48 presents, its antibacterial activity and stability is far superior to that of most of the bacteriocins described against the proposed pathogens, including L50A / B that has been registered as Lactopadt ™ Acne.

DESCRIPTION OF THE FIGURES

The concentration of an antibacterial agent necessary to completely eliminate a bacterium is called the "minimum bactericidal concentration" (CMB). This CMB is expressed as a function of the logarithm of the resulting colony forming units (Iog of UFCs), after incubation of the bacterium in the presence of antibacterial compounds at different concentrations, either jointly and / or separately.

Figures 1 and 2 show the minimum bactericidal concentration (CMB) of AS-48 tested alone (fig. 1) And in the presence of lysozyme (fig. 2) against P. acnes over time (144 h) in a laboratory culture at 37 ° C. Figure 3 shows the synergistic effect of lysozyme and AS-48 on the removal of S. aureus CECT976 in a laboratory culture (BHI at 37 ° C).

Figure 1.- Determination of the CMB of AS-48 against P. acnes in BHI. The viability of P. acnes is expressed in CFU / ml.

Figure 2.- Determination of the CMB of AS-48 combined with 4 mg / ml lysozyme against P. acnes in BHI. The viability of P. acnes is expressed in CFU / ml.

Figure 3.- Determination of the CMB of AS-48 (10 ... tg / ml) in combination with lysozyme (8 mg / ml) against S. aureus CECT 976 in BHI medium at 37 ° C. The viability of S. aureus is expressed as a log of the CFU / ml.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes compositions comprising the bacteriocin AS48, a very stable molecule that possesses a broad antibacterial activity, in combination with lysozyme, an enzyme with muramidase activity, which significantly enhances the activity of the first, for the control of Propionibacterium acnes , Staphylococcus aureus and other bacteria involved in skin infections.

Conditions caused by bacterial infections that can be treated with the compositions of the present invention include lesions caused by P. acnes (acne vulgaris) and dermal infections and / or superinfections due to S. aureus, a bacterium that is also The most important infectious agent in patients suffering from atopic eczema.

The active compound AS-48 significantly increases its activity in the presence of lysozyme, while lysozyme, by itself, has no activity under the test conditions).

The slnerglco effect of this joint application makes it possible to significantly reduce the necessary dose of AS-48 to obtain the same inhibitory effect, both "in vivo" and "in vitro" in the control of infections by said pathogens.

The compositions of the present invention, whose lack of toxicity has been confirmed, are preferably formulated for topical applications. The compositions contain the active ingredients and have the most convenient physical-chemical properties for topical application, considering the characteristics of the infections to be treated.

Given the behavior of AS-48 in the different formulations, and the different levels of sensitivity of the bacteria tested, it is considered that, in the treatment against S. aureus, it is more appropriate to use a fatty base such as petroleum jelly based cream , for application at the concentrations indicated below.

In the case of P. acnes a formulation with fatty compounds is contraindicated because it would affect the increase of fat in the skin, so it is recommended to use neutral gel or aqueous solution, at the concentrations set forth below.

EMBODIMENTS OF THE INVENTION

The bacteriocin used in the compositions and methods of the present invention is AS-48 obtained from supernatants of the UGR10 probiotic strain of Enterococcus faecalis isolated from cheeses (Cebrián, R., Baños, A, Valdivia, E., Pérez-Pulido, R , Martínez-Bueno, M, Maqueda M. 2012. Characterization of functional, safety, and probiotic properties of Enterococcus faecalis UGRA10, a new AS-48-producer strain. Food Microbiol. 30, 59-67).

As a culture medium of the producing strain, waste from the dairy industry will be used under the conditions previously optimized in our laboratory.

The lysozyme used is commercial and is obtained from egg white (Sigma L6876).

Formulations / compositions developed

Composition against S. aureus Cream: emulsion of fused petroleum jelly with antibacterial agents in a proportion between 100 and 200 ... tg AS-48 and 8 mg of lysozyme per g of cream. The emulsion is carried out using a homogenizer.

Composition against P. acnes Neutral gel added of the antibacterial agents in a proportion between 10 and 20 ... tg of AS-48 and 4 or 8 mg of lysozyme (Sigma) per g of gel. The dissolution will be carried out using a homogenizer.

Aqueous solution of antibacterial agents added in proportion between 10 and

20 I-1g of AS-48 and 4 or 8 mg of lysozyme (Sigma) per ml. The mixture will be homogenized

well before use.

5 Comparative data on the efficacy of AS-48 alone or in synergistic combination with

lysozyme

The figures show the activity of AS-48 tested at different

concentrations, alone or in combination with Iisozyme vs. P. acnes (Figures 1 and 2)

10 And in front of S. aureus (figure 3)

Concentration \ Time Oh 3h 6h 24 h 48 h 96 h 144 h Control 4.30 3.84 4.42 5.09 7.26 7.09 7.86

0.01 ~ lm / ml 4.30 3.51 3.48 4.87 3.16 4.46 5.58

0.1¡tm / ml 4.30 3.46 3.22 2.44 2.51 1,039 2.96

1.0¡tm / ml 4.30 2.78 2.59 2.12 2.31 0.30 0.0

5.0¡tm / ml 4.30 2.32 1.43 1.81 0.84 °

10.0¡tm / ml 4.30 1.18 0.57 0.39

° ° °

Table 1.-Growth of P. acnes (CFU / ml) in BHI over 144 h of incubation in anaerobiosis in the presence of different concentrations of AS-48

Concentration \ Time Oh 3h 6h 24 h 48 h 96 h 144 h Control 4.30 3.84 4.42 5.09 7.26 7.09 7.86

AS-48

4.30 3.51 3.48 4.87 3.16 4.46 5.58 0.01 MT / ml AS-48

4.30 3.46 3.22 2.44 2.51 1.04 2.96 0.1 MT / ml Lysozyme

4.30 3.48 3.30 4.53 7.55 6.50 7.24 4 mg / ml

Lysozyme 4 mg / ml 4.30 3.75 3.71 1.89 0.30 0.30 0.15

+ AS-48 0.01¡tm / ml Lysozyme 4 mg / ml

+ 4.30 3.23 2.41 1.20 O O

AS-48 °

0.1¡tm / ml

Table 2.-Growth of P. acnes (CFU / ml) in BHI over 144 h of incubation in anaerobiosis in the presence of AS-48, lysozyme or combinations of both

Table 1 provides the CMB data against P. acnes of different

Compositions containing increasing concentrations of AS-48 (from 0.01 to 10 µg / ml) and in Table 2 those of the CMB of AS-48 combined with lysozyme (4 mg / ml) against the same bacterium. Controls without active agents and with lysozyme separately are also exposed.

•

The results associated with Table 1 are presented in Figure 1 and show that in a closed system such as a laboratory culture, a concentration of 10 µg / ml of AS-48 is required to remove P. acnes in 48 h .

Lower concentrations, such as 5 or 1 µg / ml of AS-48 require 96 h or 144 h respectively, to exert the same effect.

•

However, the activity of AS-48 (Figure 2) is increased by a factor of 10 in the presence of lysozyme (4 mg / ml), with 0.1 µg / ml of AS-48 being sufficient to eliminate P. acnes in 48 h. It is also observed that lysozyme completely lacks activity under the test conditions.

Concentration \ Time Oh 2h 4h 6h 8h 14 h 18 h 22 h 24 h

Control 6.18 6.79 7.84 8.20 8.99 9.08 9.07 9.27 9.39

Lysozyme 8 mg / ml 6.18 6.48 7.67 8.48 8.47 9.14 9.14 9.14 8.77 AS-48 10 J.lm / ml 6.18 3.81 3.18 2.36 4.35 7.71 7.44 8.16 8.63

Lysozyme 8 mg / ml 6.46 4.07 3.13 4.59 3.41 NO NO NO 9.06 +

AS-48 5 J.lm / ml Lysozyme 8 mg / ml 6.18 3.69 2.65 1.89 1.78 O O O O

+ AS-48 10 J.lm / ml

Table 3. Growth of S. aureus (CFU / ml) in BHI over 24 h of incubation in the presence of AS-48, lysozyme or combinations of both

Table 3 shows the results obtained against S. aureus, with AS-48 alone and added lysozyme (8 mg / ml).

• As can be seen in Figure 3, 10¡tg / ml of AS-48 are not able to eliminate the bacteria, while the addition of lysozyme (8 mg / ml) to the

same concentration of AS-48, achieves its total elimination in 14 h, thus confirming the synergistic effect between both active compounds, in laboratory conditions These results also show that S. aureus is less

5 sensitive that P. acnes to this bacteriocin.

It is considered that, since they are topical applications, the formulations, which will be different for both bacteria, will be prepared by increasing the concentrations of the active ingredients, in accordance with the following:

• Given the confirmed safety of the preparations and according to the efficacy of the vehicles used, which has been previously confirmed in activity tests of the active ingredients in vitro, it is preferable to increase the concentration of at least 1 mg / ml AS-48 in neutral preparations

15 or aqueous (due to the lower efficacy of these and the difficulty of accessing the bacteria) and in the composition based on petroleum jelly, in order to ensure its effectiveness in vivo against Propionibacterium acnes and Staphylococcus aureus, respectively.

• In the case of lysozyme successfully used at a rate of 4 mg / ml against P.

20 acnes, it is preferable to increase its concentration to double (8 mg / ml or 8 mg / g). In the case of Staphylococcus aureus, the concentration tested (8 mg / ml or 8 mg / g) is maintained due to the optimal results obtained.

Claims (12)

one.

Use of a composition comprising bacteriocin AS-48 and lysozyme for the preparation of a medicament for the treatment of bacterial infections of the skin and mucous membranes.

2.

Use according to claim 1 wherein the bacterial infections of the skin and mucous membranes are caused by Propionibacterium acnes or Staphylococcus aureus.

3.

Use according to claim 2 wherein the amount of bacteriocin AS-48 is at least 10 Ilg / ml and the bacterial infection is caused by P. acnes.

Four.

Use according to claim 2 wherein the amount of bacteriocin AS-48 is at least 100 Ilg / ml and the bacterial infection is caused by S. aureus.

5.

Use according to any one of claims 1 to 3 wherein the amount of lysozyme is at least 4 mg / ml.

6.

Use according to any one of claims 1 to 4 wherein the amount of lysozyme is at least 8 mg / ml.

7.

Use according to any one of claims 1, 2, 3, 5 or 6 wherein the medicament is a neutral gel and the bacterial infection is caused by P. acnes.

8.

Use according to any one of claims 1, 2, 3, 5 or 6 wherein the medicament is an aqueous solution and the bacterial infection is caused by P. acnes.

9.

Use according to claim 7 or 8 wherein the amount of bacteriocin AS-48 is at least 1 mg / ml.

10.

Use according to any one of claims 1, 2, 4 or 6 in which the medicament is a topical cream and the bacterial infection is caused by S.

aureus

eleven.

Use according to claim 10 wherein the topical cream is a stable emulsion of fused petroleum jelly fused with the bacteriocin AS-48 and lysozyme.

12.

Use according to claim 10 or 11 wherein the amount of bacteriocin AS-48 is at least 1 mg / ml.