TOPICAL FORMULATION FOR TREATING SKIN OR MUCOSAL INFECTIONS, PREPARATION METHOD AND USES THEREOF

Abstract

The present disclosure relates to the field of therapeutics for skin and mucosal infections and refers to the use of bromelain either alone or with antimicrobial agents to inhibit, to reduce or to treat biofilms in infections derived from the presence of this pathogenicity mechanism. This application aims to use the enzymatic action of bromelain to destroy the biofilms and allow for the penetration of the antifungals therefore improving their action in the site of infection, treating and reducing symptoms.

The present disclosure may be used in the pharmaceutical field for the treatment of infections, as creams, lotions, gels or vials for local application in the skin or mucosa.

Formulations with the ability to maintain the stability of the enzyme and of the other active components were developed with good characteristics for skin or mucosal application, particularly in the vagina, promoting, in this way, the efficacy and acceptability of the final product.

Claims

1-21. (canceled)

22. The topical formulation comprising a therapeutically effective amount of bromelain enzyme and an adequate excipient for the treatment of skin or mucosal infections.

23. The topical formulation according claim 22, wherein the concentration of bromelain comprises 0.125-10% (w/w).

24. The topical formulation according to claim 22, wherein the concentration of bromelain comprises 0.25-2% (w/w) of bromelain.

25. The topical formulation according to claim 22, wherein the excipients are selected from the group of: propyleneglycol, gelified propyleneglycol, carbopol, polyetheleneglycol, and mixtures thereof.

26. A topical formulation for the treatment of skin infections, comprising: 0.125-10% (w/w) bromelain 70-98% (w/w) triacetin; 0-17% (w/w) propyleneglycol; 0-17% (w/w) de glycerin; and 1-11% (w/w) colloidal silica dioxide.

27. The topical formulation according to claim 26, wherein the concentration of propyleneglycol comprises 7.5-15% (w/w) and wherein the concentration of glycerin comprises 0-5% (w/w).

28. The topical formulations according to claim 26, further comprising an antimicrobial agent.

29. The topical formulations according to claim 28, wherein the concentration of the antimicrobial agent ranges from 0.1 to 10% (w/w).

30. The topical formulations according to claim 28, wherein the antimicrobial agent is selected from the group of: Thymus, Thymbra capitata, Thymus vulgaris, Thymus mastichina, and mixtures thereof.

31. The topical formulations according to claim 30, wherein the concentration of Thymus ranges from 0.1 to 1% (w/w).

32. The topical formulations according to claim 28, wherein the antimicrobial agent has an antifungal activity.

33. The topical formulations according to claim 28, wherein the antimicrobial agent belongs to the azole group.

34. The topical formulations according to claim 32, wherein the concentration of the fungicidal ranges from 0.5-3% (w/w).

35. The topical formulations according to claim 32, wherein the fungicidal is clotrimazole.

36. The topical formulations according to claim 22, further comprising an antibiotic in a concentration ranging from 1 to 10% (w/w).

37. The topical formulations according to claim 36, wherein the antibiotic is selected from the group of: fusidic acid, bacitracin, neomycin, and combinations thereof.

38. A medical device comprising a material having bromelain as an antimicrobial coating.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of the disclosure.

[0052] FIG. 1: Graphical representation of the effect of bromelain concentration upon the biomass of biofilms from Candida spp. strains, wherein (FIG. 1.1) relates to collection strains (ATCC) and clinical strains of C. albicans; (FIG. 1.2) relates to clinical strains of C. glabrata; (FIG. 1.3) relates to clinical strains of C. parapsilosis; (FIG. 1.4) relates to clinical strains of C. tropicalis; (FIG. 1.5) relates to clinical strains of C. guilliermondi; and (FIG. 1.6) relates to clinical strains of C. krusei.

[0053] FIG. 2: Graphical representation of the combination effect of bromelain and the antifungal clotrimazole upon the biomass of biofilms from C. albicans strains.

[0054] FIG. 3: Graphical representation of the combination effect of bromelain and the antifungal clotrimazole upon the metabolic activity of biofilms from C. albicans strains.

[0055] FIG. 5: Image representation obtained through confocal microscopy in a checkerboard assay to evaluate the combination of bromelain and clotrimazole upon AP25A (I) strain.

DETAILED DESCRIPTION

[0056] The present disclosure describes a formulation comprising bromelain in a dosage form appropriate for skin or mucosal application, particularly for vaginal application, which presents several challenges particularly because its rapid inactivation in aqueous media has been described. In fact, the products that are commercially available for oral use and that contain this enzyme are formulated as tablets which anhydrous composition allows for the maintenance of the substance stability throughout time. The present disclosure developed formulations which avoid bromelain degradation allowing for the maintenance of its efficacy in the treatment of skin and mucosal infections.

[0057] The present disclosure also describes the application of bromelain combined with classical antifungal drugs (such as clotrimazole) or with plant extracts, namely the essential oils from Thymbra capitata, Thymus vulgaris and other species from the genus Thymus, or used alone for the treatment of fungal infections characterized by biofilm formation, such as mucocutaneous candidosis. These substances are included in one semisolid formulation of anhydrous base which assures the stability of the enzyme and the dissolution of azoles or the mixture with the essential oils.

[0058] In an embodiment of the present disclosure, it was shown that bromelain alone has the ability to reduce the biomass of the biofilms from Candida spp, in skin or mucosal infections. It was also shown the reduction of biomass and metabolic activity of C. albicans biofilms caused by the combination of bromelain and clotrimazole. The antifungal effect of the essential oils obtained from Thymbra capitata, Thymus vulgaris and other species which belong to the genus Thymus has also been previously shown.

[0059] The described disclosure consists of the application of the association of bromelain to antimicrobials, or bromelain alone towards the inhibition, reduction or treatment of biofilms in bacterial or fungal infections derived from the presence of this pathogenicity mechanism, such as mucocutaneous candidosis, skin infections or catheter-related infections caused by S. aureus.

[0060] Laboratory assays were performed in pre-formed mature biofilms (48 h) in 96 wells microplates, using colorimetric assays described in the literature to quantify the biofilm biomass (Crystal violet assay) and the metabolic activity of cells (XTT reduction assay).

[0061] The effect of bromelain was tested in pre-formed biofilms of 20 Candida spp. strains belonging to 6 species: C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. guilliermondi and C. krusei. Results are presented in FIG. 1, as graphs showing the reduction of biomass of groups of strains according to their species. It was observed that the effect of bromelain alone upon the reduction of the biomass of Candida spp. biofilms occurs in all species, depending on the tested concentration and varying among strains. So, it is observed that the effect is not specific of a certain species under study. For the higher tested concentrations (250-500 μg/mL) the reduction of biofilm biomass was achieved, varying from 40% to 80%, in all tested species and strains.

[0062] The combination effect of bromelain with the antifungal clotrimazole was evaluated through the checkerboard technique described by Roxana G. Vitale, Javier Afeltra and Eric Dannaoui in Antifungal Combinations and their results concerning biomass and metabolic activity reduction are represented in FIGS. 2 and 3, respectively. FIG. 2 shows that the effect of clotrimazole alone in the reduction of biofilm biomass is limited (without bromelain) and that the addition of the lowest tested concentration of bromelain causes a reduction of this parameter up to 80% for the tested C. albicans strains. For higher concentrations of bromelain this reduction even reaches 90%.

[0063] Concerning metabolic activity, FIG. 3 shows that increasing concentrations of clotrimazole cause reductions of activity in a concentration dependent manner that do not exceed 60% for the tested C. albicans strains. When bromelain is added, an increase of activity reduction is observed, dependent on the enzyme concentration (the addition of a 250 μg/mL bromelain solution leads to metabolic activity reductions near 80% for both strains). Previous studies undertaken by our team show that bromelain does not exhibit direct fungistatic or fungicidal activity against planktonic cells of different Candida species, including C. albicans. Thus, the increase in metabolic activity reduction shown for the combination of bromelain and clotrimazole is not due to a direct fungistatic activity of the enzyme, but to a degradation of the biofilm matrix. The degradation of this matrix allows for the penetration of clotrimazole in the biofilm favouring its antimicrobial action.

[0064] The anti-Candida activity of essential oils extracted from plants belonging to the genus Thymus had been previously shown.

[0065] Although bromelain does not present antifungal activity against planktonic cells, in this application the addition of bromelain, without clotrimazole, significantly reduces the metabolic activity, these reductions varying from 24.48% to 49.67% for the AP25A strains and 54.89% to 76.2% for ATCC 10231. The high reduction upon metabolic activity shown for bromelain are not due to a direct antifungal activity but to destruction of the biofilm, leading to the release of cells from the microplate. Following the washing step of microplates, the number of metabolically active cells available to metabolize XTT is reduced, leading to the overall reduction on metabolic activity.

[0066] The images obtained by confocal microscopy, presented in FIG. 4, visually confirm the previously reported biomass reduction. By using calcofluor for the staining of biomass, a notorious effect from the combination of bromelain and clotrimazole throughout the biofilm can be seen, in comparison with control.

[0067] In an embodiment, formulations were developed as gels, deprived from water, for vaginal application of the combination of the antifungal with bromelain or of bromelain alone (table 1). These formulations contain excipients that mix with water presenting the advantage of being easily washable.

[0068] However, the fact that they do not contain water in their composition is a key factor to assure the stability of bromelain whose proteolysis and degradation in aqueous media has been shown in several studies. In an embodiment, one of the basis of formulations is triacetin, an excipient largely used in pharmaceutical, cosmetic and food industries. This excipient has been selected due to its reported safety not only following oral use but also due to its low irritation potential when applied with no dilution, on the skin and mucosa, as an occlusive patch (patch test). Safety studies performed with triacetin after application in the eye have shown that although it induces an ocular irritation it does not cause harm, supporting its safe use. On the other hand, concerning the vaginal route, this excipient is the basis of cream formulations approved to be used in the vagina and endocervical region, in Europe and the United States of America. Another humectant may be added to this excipient, such as propyleneglycol (PPG) or glycerin in concentrations ranging from 0 to 17% w/w. Colloidal silica dioxide (available under the trade mark Aerosil, among others, and used preferentially with 200 porosity) is used as a gelifying agent in concentrations ranging from 1 to 11% w/w allowing a gel to be formed with adequate viscosity and texture for vaginal application. Bromelain in concentrations of 0.125 to 10% w/w and/or the essential oil or the azole antifungal molecule in concentrations ranging from 0.1 to 3% w/w are dissolved or diluted on part of the mixture form by triacetin and PPG/glycerin or on triacetin (in case no PPG or glycerine is added) and mixed with the gel formed by Aerosil dispersion in the remaining vehicle.

TABLE-US-00001 TABLE 1 Examples of anhydrous gels containing bromelain and a natural or synthetic antifungal agent for the treatment of recurrent vulvovaginal candidosis Raw material (name and concentration % w/w) Thymbra capitata Aerosil Propylenoglycol/ essential Formulation Triacetin 200 Glycerina Bromelain oil Clotrimazole 1 q.s. ad 8 15 1 1 0 2 100 8 15 1 0 1 3 8 0 1 1 0 4 8 0 1 0 1 5 8 0 0.25 1 0 6 8 0 0.25 0.2 0 7 8 7.5 1 1 0 8 6 15 1 1 0 9 6 0 0.25 1 0 10 6 0 1 0 1 11 6 0 0.25 0.17 0 12 4 0 0.25 1 0 13 4 0 1 1 0 14 4 7.5 0.25 1 0 15 4 7.5 0.25 0.17 0

[0069] Other formulations basis may be used to vehicle these agents such as propyleneglycol gelified with carbopol or polycarbophil and polyethyleneglycols.

[0070] Although in the present solution only particular embodiments have been represented and described, those skilled in the art will know how to modify or change the technical characteristics to equivalent ones, depending on the requirements of each situation, without departing from the scope of protection defined by the claims below.

[0071] The embodiments herein described are combinable.

[0072] The following claims set out particular embodiments of the disclosure.